Elucidation of Cell Killing Mechanism by Comparative Analysis of Photoreactions on Different Types of Bacteria S. Swetha, Maheshwari Kumari Singh, K. U. Minchitha and R. Geetha Balakrishna* Center for Nanobiosciences, Jain University, Ramanagaram, Bangalore 562112, India Received 25 September 2011, accepted 28 November 2011, DOI: 10.1111/j.1751-1097.2011.01057.x ABSTRACT The mechanism of biocidal action of nano titania on Escherichia coli and Staphylococcus aureus has been evaluated by various biochemical techniques like lipid peroxidation, hydrolysis of orthonitrophenol b-D-galactopyranoside, estimation of protein– amino acid and bacterial nucleic acids leakage into solution, in addition to morphology studies by electron microscopy (TEM and SEM) and K + ion leakage by inductively coupled plasma optical emission spectrometry. The active anatase phase of nano titania has been synthesized by sol-gel and pulverization tech- niques to obtain particle sizes averaging around 11 nm. The nano semiconductor with a bandgap of 3.2 eV responds well to the UV source to liberate reactive oxygen species (ROS). Gram negative bacteria easily succumb to the ROS at a faster rate than gram-positive bacteria with an observable difference in the mode of attack. The use of analytical techniques revealed the release of peroxidized lipid (26 nmol mL )1 ) and protein content (370 lg mL )1 ) with a K + ion concentration of 22 000 ppb on complete destruction of E. coli. INTRODUCTION Precise manipulation of matter at the nanoscale is expected to tailor the properties and functionalities of nanomaterials for environmental applications. Photocatalytic performance has been known to be dependent on several variables such as preparation method, particle size, reactive surface area and adsorption characteristics (1–6). Attempts have been made in the present study to improve the efficiency of titania photocatalyst through modified synthesis. Nanostructured titania semiconductor has been well estab- lished as an excellent and efficient photocatalyst for degrada- tion and inhibition of numerous toxic and pathogenic contaminants (1,7–12). Generally, it is believed that nano- materials in such semiconductor-mediated photocatalyzed inactivations involve the generation of electron hole pairs (11,12). Due to the strong oxidizing power of photogenerated holes and reducing power of electrons to produce superoxide for dioxygen, a very active free radical species called reactive oxygen species (ROS) are generated (13). The ROS released from the catalyst have been proposed to be the major cause for biocidal action in such reactions. Matsunaga et al. proposed the direct oxidation of coenzymes in TiO 2 -treated cells (14), while Kiwi and Nadtochenko have reported the degradation of main cell wall structure components at the TiO 2 interface by ATR-FTIR, during such catalyzed irradiations (15). Although a wealth of information has been amassed, which demonstrates the efficacy of biocidal actions of TiO 2 catalyst, there is lack of definite evidence regarding the mode of attack of TiO 2 nanoparticles on microorganisms. Most of such studies have demonstrated the conventional standard plate count (SPC) method for the measurement of the extent of inactivation (16–18). To evaluate the effect of TiO 2 on cell membrane integrity and to understand the mechanism behind such inactivations, the present article investigates the various biochemical techniques to measure the extent of lipid peroxidation, protein–amino acid leakage, release of b-galac- tosidase and bacterial nucleic acids into solution, in addition to analysis of morphology studies by electron microscopy (TEM and SEM) and K + ion leakage by inductively coupled plasma (ICP) optical emission spectrometry. The study also presents the biocidal influence on two different types of bacteria, the gram-negative bacteria Escherichia coli and the gram-positive bacteria Staphylococcus aureus, in order to fully evaluate its possible use as a bactericidal material. Differences in suscep- tibility to photocatalyst between the two were expected to a certain extent and hence had to be investigated individually in depth to substantiate the key and secondary targets involved in a cell death of bacteria in general. E. coli is predominantly constructed from tightly packed liposaccharide of 2–7 nm thick wall, which provides an effective permeability barrier (19,20). Cell wall (15–80 nm thick) of S. aureus consists of 3–20 times more homogeneous peptide inter bridges, which stabilizes the cytoplasmic membrane to aid the retention of shape and integrity (19,20). Because of this, it readily acquires multi drug resistance. Hence, study of such strains which are particularly resistant to antibiotic treatment along with an indicator organism like E. coli was important. MATERIALS AND METHODS Synthesis. Fine grain powders of anatase form of nano TiO 2 (n-TiO 2 ) were prepared by gel to crystalline conversion method using TiCl 4 (21). The dried crystals were pulverized using high energy ball mill (SPEX 8000M Mixer ⁄ Mill) equipped with tungsten carbide vial for an optimum time of 90 min. The milled powder was subjected to annealing at 600°C for 6 h to get anatase phase of n-TiO 2 . Bacterial strain and culture conditions. An ATCC 25922 E. coli and 25923 S. aureus cultures were obtained from St. John’s medical college *Corresponding author email: geethabalakrishna@yahoo.co.in (R. Geetha Balakrishna) Ó 2011 Wiley Periodicals, Inc. Photochemistry and Photobiology Ó 2011 The American Society of Photobiology 0031-8655/12 Photochemistry and Photobiology, 2012, 88: 414–422 414