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Analyses of chemical composition on bacteria surface treated in atmospheric pressure plasmas
Analyses of Chemical Composition on Bacteria Surface Treated in Atmospheric Pressure Plasmas 大気圧プラズマで処理した細菌表面における化学組成解析 Shohei Yoshida, Tadashi Fukuda, Kazuo Takahashi, Takuya Urayama*, Shinji Aoki** 吉田昇平，福田匡，高橋和生，浦山卓也*，青木慎二** Department of Electronics, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 京都工芸繊維大学大学院工芸科学研究科電子システム工学専攻，〒606-8585 京都市左京区松ヶ崎 * Adtec Europe Ltd., London **Adtec Plasma Technology Co.,Ltd, 6-10, 5-chome Hikino-cho, Fukuyama City, Hiroshima, 721-0942, Japan （株）アドテックプラズマテクノロジー，〒721-0942 広島県福山市引野町五丁目6番10号 The sterilization mechanism was analyzed in atmospheric plasmas. It was confirmed that the cell wall of G.stearothermophilus exposed to plasmas was elapsed observing with scanning electron microscope (SEM). From the x-ray photoelectron spectroscopy (XPS) spectra, the content of -CH 2 -CH 2 - bond decreased, and then the signal from O 1S increased with plasma exposure. Analyses of SEM photographs and XPS spectra would mean that the cell wall of bacteria was oxidized and damaged with plasma exposure. 1. Introduction We have been studied about sterilization mechanism of bacteria, investigating the cellulose and bacteria on paper strip exposed to plasmas. Cellulose composed of pyranose rings was a model material of peptidglycan corresponding to major content of bacteria cell wall. Exposing plasmas to cellulose caused pyranose rings to be opened and ring chains to be cleaved (Fig. 1). The changes of chemical bond composition in pyranose rings correspond to damages of sugar chain on the bacteria cell wall, which results in killing bacteria. Besides we confirmed that it was possible to sterilize bacteria on paper strip with in our atmospheric plasmas setup. In this study we observe bacteria (G.stearothermophilu ATCC 7953) on Si substrate coated by Au using scanning electron microscope (SEM). For analysis of chemical composition on bacteria surface we make use of x-ray photoelectron spectroscopy (XPS) and evaluate how bacteria Fig.1 Schematic for changing of pyranose ring structure. surface changes by exposed to plasmas. In all experiments, bacteria are treated with not only plasmas but also heatgun to be investigated the effect of heat from plasmas. 2. Experiment Figure 2 shows the schematic of the experimental setup. Atmospheric pressure plasmas were generated by applying 2.45 GHz microwave power to a metal rod as an antenna in the plasma torch. A stub tuner was used for matching the microwave power. Ar gas was introduced to the torch. Samples of bacteria (G.stearothermophilus ATCC 7953) on Si substrate deposited Au (55 mm 2 ) was treated with Ar gas flow rate of 3.5 slm, plasma exposure time of 10 min, distance from antenna to sample of 10 mm, and microwave power of 70 W. In order to investigate the effect of heat from plasmas, we treated bacteria with heated air flow (200°C) from heatgun in the experimental conditions same as in case of plasmas. The XPS was employed for chemical analyses on bacteria surface. On the other hand, the effect of plasma on sterilization was evaluated in observation of bacteria using SEM. In addition, we measured optical emission spectra of plasmas to investigate the sterilization mechanism. Opening of pyranose ring Cleaving of pyranose ring chain OH N 2 Ar O 2 Fig.2 Schematic of setup for atmospheric plasmas. Fig.3 SEM photograph of bacteria. 3. Results and Discussions 3.1 SEM photograph of bacteria Figure 3 shows bacteria exposed plasmas. The bacterium of left side in Fig. 3 has an ellipsoidal shape and looks like to be partly ruptured. One of right side in Fig. 3 looks like to be destroyed completely. Thus we can understand that burst of cell wall or cell membrane happens in the Fig. 3. The one treated with heatgun did not change in SEM observation. 3.2 XPS spectra of bacteria Figure 4 shows XPS spectra on the bacteria surface. The original (pre-treatment) surface is known to have several chemical bond components of (-C(=O)-NH- or –O-C-O-), -C-OH, -CH 2 -CH 2 -, and -C-C- (Fig. 4(a)), since the predominant constituents of bacteria surface are hydrocarbon followed by polysaccharides and peptides [1]. After the plasma treatment, the chemical bond of -CH 2 -CH 2 - disappeared on the surface (Fig. 4(b)). It seems that hydrogen was disorbed from the cell wall, and the wall was oxdized and damaged in the plasma treatment. We could not find any changes in the XPS spectra of bacteria treated with heatgun. 3.3 Optical emission spectrum Figure 5 shows optical emission spectrum from plasma discharge. The signals from OH, N 2 , and O were obserbed as well as that from Ar. Several species coming from atmospheric may be candidate for reactants on the cell wall. Fig.4 XPS spectra on the bacteria surface. Fig.5 Optical emission spectrum in the plasma (Ar: 7 slm, Power: 50 W). 4. Summary We analysed the sterilization mechanism with SEM photograph, XPS spectra of bacteria (G.stearothermophilu), and Optical emission spectrum of plasma. The cell wall of bacteria could be ruptured by plasma exposure. The chemical bond of -CH 2 -CH 2 - disappeared and O-content increased. These cahnges in chemical bond compositions corresponded to oxidization and damages of the cell wall. On the other hand, the effect of heat from plasmas did not related to rupture of bacteria. In plasmas, several species of excited Ar and those from atmosphere may be candidate for the reactants of sterilization. References [1] Jess J. Ojeda, Mara E. Romero-Gonzlez, Robert G. Edyvean, and Steven A Banwart ‘Characterization of the Cell Surface and Cell Wall Chemistry of Drinking Water Bacteria by Combining XPS, FTIR Spectroscopy, Modeling, Potentiometric Titrations’ Langmuir 4032-4040, 24 (8) 2008 Ar Plasma Torch Silica Tube Bacteria 2.45 GHz Tuner . ring chains to be cleaved (Fig. 1). The changes of chemical bond composition in pyranose rings correspond to damages of sugar chain on the bacteria cell wall, which results in killing bacteria. . surface. On the other hand, the effect of plasma on sterilization was evaluated in observation of bacteria using SEM. In addition, we measured optical emission spectra of plasmas to investigate. from plasmas, we treated bacteria with heated air flow (200°C) from heatgun in the experimental conditions same as in case of plasmas. The XPS was employed for chemical analyses on bacteria surface.