*3.1. Effect of TiO2 in the Dark*

Figure 2 shows the bacterial concentration of *E. coli* cells after 0 and 24 h of contact with TiO2 nanoparticles (11 mL inoculation of air-dried TiO2 film in Petri dishes). From these data, an increase can be seen in the number of CFUs for control samples (+2.25 ± 0.06 log) and a decrease in the number of CFUs for TiO2 samples (í0.91 ± 0.14 log). The corresponding antibacterial activity, calculated from Equation (1), is 3.22 ± 0.14 log. It is therefore likely that the activity of TiO2 on *E. coli* in the dark is correlated with a growth inhibitory effect as a major pathway and a bactericidal effect as a minor pathway. These results highlight the physical impact on *E. coli* cells induced by contact with TiO2 nanoparticles, without regard to the photocatalytic process. This also agrees with earlier observations by Liu *et al.* [50] and Gogniat *et al.* [38], which showed a loss of bacterial culturability after contact with TiO2 nanoparticles in the dark. A study by de Niederhãusen and Bondi [51] on the self-cleaning of Ag-TiO2-coated ceramic tiles also showed significant antibacterial activity for 24 h in the dark.

Interestingly, we detected no difference in the CFU counts between bacterial suspensions with TiO2 (1 g/L and 10 g/L) and a control bacterial suspension (without TiO2) after direct plating of 2 × 1 mL on TSA and 48 hours' incubation (data not shown). It seems possible that the physical damage sustained is not sufficient to kill bacterial cells when they are growing in a nutrient-rich culture medium. Such a "neutralizing" effect of culture media is current with antiseptic and disinfectant molecules, which highlights the impact of test conditions on efficiency evaluation.
