*3.2. Free Surface Drop Deposit vs. Forced Contact between Bacteria and Nanoparticles*

The antibacterial activities of TiO2 nanoparticles on *E. coli* during the deposited-drop experiment are presented in Figure 3. No activity was detected for samples kept in the dark for 6 h under normal testing conditions (A). The bacterial reduction reached 0.92 ± 0.09 log after 6 h of irradiation (A).

**Figure 3.** Antibacterial activity of TiO2 as a support under UV irradiation (§2.5 W/m2 ): (**A**) standard conditions; (**B**) after application of a transparent plastic film on the inoculum. Mean ± SE, *n* = 3.

Since the bactericidal effect induced by photocatalysis of TiO2 nanoparticles depends on many factors, such as the amount [35,37,52–55] and the crystalline nature [39,55–57] of TiO2, the irradiation time and intensity [35,52,55,58] and the inoculum concentration [42,53,59], it is reasonable to assume that the dispersion of TiO2 particles and bacteria (low probability of contact) and the very low light intensity used in our experiment (2.5 W/m2 in order to avoid any UV-damage) could be major factors explaining the low activity observed after 6 h of irradiation. Various studies from the literature show intensities of over 10 W/m2 and antibacterial activities on *E. coli* easily greater than 3 log after 90 min of irradiation [39,40,42,60].

Following the application of a transparent film (9 cm2 ) onto the inoculum, a significant increase in the antibacterial activity was observed. As shown in Figure 3B, the activity was 3.74 ± 0.1 log after 6 h under UV irradiation and 0.27 ± 0.03 log after 6 h in the dark.

The present findings seem to support the idea that reducing the distance between bacterial cells and TiO2 nanoparticles enhances the photocatalytic disinfection process. This also agrees with earlier research highlighting the importance of the contact between bacterial cells and the surface of TiO2 [39–41,43,44,50,61–63]. In addition to the oxidative stress induced by reactive oxygen species (ROS) on bacterial cells, contact with the TiO2 surface leads to the direct oxidation of cells by photogenerated holes, which also reduces the recombination of charges inside the photocatalyst [38,43,44,64]. Moreover, it has been suggested that direct contact and adsorption on TiO2 nanoparticles cause (I) a loss of membrane integrity [38,50,61] and possibly (II) a process of phagocytosis of the nanoparticles by the cells (the findings of Cai *et al.* [64] must be interpreted with caution in this paper, because they focused on HeLa cells and not bacterial cells.) [64], both leading to the reduction of the number of cultivable cells, if not to cell death. These results also agree with the findings of other studies that have highlighted the major role of surface radicals compared to free radicals in photocatalytic disinfection [40,41,62].
