TiO2 광 촉매에 의한 박테리아 살균효과에 관한 연구

김병훈, 김세영*, 김도환*, 유선균, 김태영, 박춘순**,박병건**, 조동련, 조성용

전남대학교 공업기술연구소, 환경공학과*, 광주여자대학교 환경보건학과**

A Study on Bactericidal effect of TiO2 Photocatalyst

B. H. Kim, S. Y. Kim*, D. H. Kim*, S. K. Yoo, T. Y. Kim, C. S. Park**,  B. G, Park**, D. L. Cho, S. Y. Cho

Engineering Research Institute, Chonnam National University

*  Dept. of Environmental Engineering, Chonnam National University

** Dept. of Environmental and health, Gwangju Women University

Introduction

Since the discovery of Photoinduced water cleavage on titanium dioxide(TiO2) electrodes by Fujishima and Honda in early 1970s[1], TiO2 photocatalysts have attracted great attention as alternative materials to aids in the purification of water and air[2-8].

TiO2 photocatalysts generate strong oxidizing power when illuminated with UV light with wavelength of less than 385nm. With holes and hydroxyl radical(OH●) generated in the valence band, and electrons and superoxide ion(O2-) generated in the conduction band , illuminated TiO2 photocatalysts can decompose and mineralize organic compounds by participating in a series of oxidation reactions leading to carbon dioxide.

The reactive oxygen species(ROS)generated by the TiO2 photocatalytic reactions cause various damages to living organisms. In 1985, Matsunaga and coworkers reported for the first time the microbiocidal effect of TiO2 photocatalytic reactions[9].

Since then, research work on TiO2 photocatalytic killing has been intensively conducted on a wide spectrum of organisms including viruses, bacteria, fungi, algae, and cancer cells.

The direct oxidation of coenzyme A(CoA) that inhibited cell respiration and subsequently caused cell death was proposed as the first killing mode by Matsunaga et al.[9]

A second killing mode was therefore proposed by Saito et al.: bacterial death was caused by a significant disorder in cell permeability and the decomposition of the cell wall[10].

In this study, we investigated the bactericidal effect and cell death kinetics with  Salmonella choleraesuis and Vibrio parahaemolyticus.

Material and Method

Bacterial culture

The organisms used were Salmonella choleraesuis subsp. KCCM 11806 and Vibrio parahaemolyticus KCCM 11965. They were grown aerobically in 300ml conical glass flasks containing 50ml of typtic soy broth(Merck, Germany) at 37℃ on a rotary shaker for 24hr. In case of  V. parahaemolyticus, natural salt 3%(w/v) was added to culture medium.  The speed of the shaker was set at 150rpm. S. choleraesuis subsp. cells were harvested by centrifuging at 4000× g for 15 min, washed twice in sterile phosphate-buffered saline(PBS, pH 7.2), and resuspended in PBS or sterile deionized water. In case of  V. parahaemolyticus, washed twice in sterile deionized water added natural salt 3%(w/v).

The cell concentration was determined by a viable count procedure on typtic soy agar(Merck, Germany) plates after serial dilutions of the culture in PBS.

Photocatalytic reaction

The photocatalyst used in this study was TiO2(anatase, Yakurt pure chemical Co, osaka, Japan)particle.

Washed cells(approximately 107 cfu ml-1) were resuspended in deionized water. TiO2 0.18g were added to 87 mm petri dish  containing 18 ml of sterilized deionized water and 2ml of washed cells. The TiO2-cell mixtures was placed on a magnetic stir plate with continuous stirring, and illuminated with two 20W UV-A(TL 20W/05, Philips. Co, Holland) light from above.

Cell viability assay

The cell viability was examined by the viable count procedure. The TiO2-cell mixture was exposed to UV-A light with continuous stirring.   S. choleraesuis subsp. and V. arahaemolyticus suspension without TiO2 were illuminated as a control, and the reaction of TiO2-cell mixture in the dark was also carried out. Samples were taken at 30 min intervals for 120 min in triplicates. The viable count was performed on typtic soy agar plates after serial dilutions of sample in PBS. All plates were incubated at 37℃ for 24 h.

Fig. 1 shows TiO2 bactericidal experiment apparatus.

Result and Discussion

The viability of TiO2-treated cells was determined by colony counting after 24hr, 37℃ of incubation.

The survival curve in Fig. 2 shows that S. choleraesuis subsp cells (approximately 107 cfu/ml) underwent illumination for 30min in the presence of 10mg/ml TiO2 particle, more than 92% of the cells lost their viability. However the viability is remarkably lower than that without TiO2 particle, in which UV light cause 8% of the cells lost their viability. Complete killing was achieved after 60min of illumination. The cells in the dark does not affect the survival ratio.

Also we investigated survival ratio of V. arahaemolyticus cells,  it showed tendency as same as S. choleraesuis subsp cells

Fig. 1 Systems for illumination: A)  UV           lamp,  B) Petri dish, C) Magnetic             stirrer,  and D) supporter.

Acknowledgement

A Korea Research Foundation Grant(KRF-YOO-290)supported this research.

References

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