Three crystal structures of titanium dioxide are mainly reported: low-temperature form tetragonal anatase (a-TiO₂), high-temperature stable tetragonal rutile (r-TiO₂) and middle temperature orthorhombic brookite (b-TiO₂) [1]. The first a-TiO₂ and third b-TiO₂ transform easily into r-TiO₂ at higher temperatures than 1188 K and 923 K, respectively [2]; these temperatures depend on their particle and crystallite sizes, purity and synthetic process conditions [3]. Among them, r-TiO₂ has been widely used in the industry that is textiles, electronics, wastewater treatment, and catalysis [4] [5] [6]. Recently, TiO₂ nanoparticles (TiO₂ NPS), have been attracting much attention due to their functional and physicochemical properties, such as, a white pigment and a personal skin care product (due to its brightness and high refractive index with high safety margin), and bactericidal agents (photocatalytic properties and its induced antimicrobial activity under ultraviolet (UV) radiation or visible light) [7] [8] [9]. Up to now, many papers and reviews concerning about toxicity mechanism have been published from the viewpoints of reactive oxygen species (ROS) [10] - [17], that is hydroxyl radical ·OH and superoxide anion O 2 − which are generated by hole-electron pairs in the valence and conduction bands of TiO₂, respectively. Their bactericidal mechanism has been explained by introducing bio-cell wall damage and lipid peroxidation of membrane, and TiO₂ NPS’s adherence to intercellular organelles and biological macro molecules [17] [18] [19] [20] [21].

The authors have been studying metal oxide powders which can reveal strong antimicrobial activity in the shade for long time. Biocompatible zinc oxide ZnO also has been interested due to its microbial toxicity. However, its activity is a little lower than TiO₂ NPS. Recently, ZnO powders have been prepared by hydrothermal treatment in zinc nitrate aqueous solution with a concentration of 3 mol·L⁻¹ at 443 K for 2.52 × 10⁴ s, followed by re-oxidation heating at 873 K for 3.6 × 10³ s in air. And then it has been cleared that thus obtained ZnO powders can reveal strong antibacterial activity even under dark conditions [22] [23] [24]. During investigation of antibacterial ZnO, the authors found that 1) among commercially available TiO₂ powders, some a-TiO₂ could submit a small amount of ROS in the dark, however, r-TiO₂ did not, and furthermore, 2) among a-TiO₂, ROS were submitted from a-TiO₂ which contained a trace amount of potassium K and phosphorus P as impurities.

The effects of potassium K doping on physicochemical properties of TiO₂, such as their crystallinity, surface areas S_A, solubility, photocatalytic activity, and band gap E_g, were studied by several researchers [25] [26] [27] [28]. Their results were summarized as follows; K-solubility was a very low around 0.2 at%, therefore, E_g was not changed and photocatalytic activity was decreased under visible light conditions. Hao et al. [29] studied the antibacterial activity of K-doped TiO₂ powders, which were prepared using molten potassium nitrate KNO₃ and metal Ti under various calcination temperatures. The obtained samples consisted of a-TiO₂ as well as metal Ti. Doped (2.6 - 10.6 at% K) TiO₂ samples showed a photocatalytic activity under visible light conditions, despite having a small change in the band gap (3.14 eV), contradicting a previous study [27].They assumed that K⁺ ions decreased E_g of a-TiO₂ and the doped samples killed bacteria under visible light conditions. Under dark conditions, the samples also showed some antibacterial activity.

Yu et al. [30] studied phosphorus-doped TiO₂ powders and reported their microstructures; phosphate P doping inhibited the grain growth during calcination, significantly increasing S_A, and decreased the pore size. About the values of E_g, opposite results were reported, E_g became larger [30] and smaller [31] [32] than those of undoped one. This might be explained in terms of a different source of P and way of synthesis method. Shi et al. [32] investigated the bonding of P in the crystal structure and reported that P was only present as P⁵⁺, and only Ti-O-P bonds were detected. This substitution consequently led to a charge imbalance, which needs to be compensated. The compensation was proved through the decrease in oxygen vacancies. It is known that decreasing oxygen vacancies improved photocatalytic activity, because oxygen vacancies acted as recombination centres for electron-hole pairs [33] [34]. In addition, they also observed an increase in the photocatalytic activity with increasing the P content. Jin et al. [35] found that P doping increased hydroxyl radical emission from the surface, which was also favourable in terms of photocatalytic activity.

Up to now, there is no report on the generation of ROS from a-TiO₂ NPS under dark conditions. Then, we started to investigate the relationship between the amount of ROS and the contents of K, P, and their combined doping, from the viewpoint of the microstructure and physicochemical properties. Finally, we found that anatase (a-TiO₂) powders which could submit a lot of ROS even in the dark; the amounts of ROS were much higher than our previous antibacterial ZnO, in addition, this powder could be prepared with much simpler process. The present paper treats the physicochemical properties in relation with the microbial toxicity of thus prepared anatase (a-TiO₂) as a function of impurity contents and its doping method.