Antibacterial activity of P25-coated Petri dishes
The results expressed as the logarithm of the surviving bacterial fraction (E. coli, S. aureus, P. putida and L. innocua) obtained at different times of exposure are reported in Figure 3. The initial experimental conditions (e.g., exposure time and temperature) were established with the E. coli strain because E. coli was the first bacterium to be tested during antibacterial trials.
The results obtained with E. coli (Figure 3a) showed a low fraction of surviving bacteria in BN and TN samples at 300 min of exposure and no surviving bacteria were observed in any of the exposed Petri dishes (TE and BE). This antibacterial activity could be due to the experimental stress conditions for E. coli associated with a long exposure (300 min), such as resuspension in deionised water, light exposure during the sterile operations and UV irradiation.
At 180 min of exposure, good antibacterial activity of TiO2 was observed. In fact, the bacterial viability in TE Petri dish was low, whereas a slight reduction of the bacterial level was observed in BE Petri dishes due to the E. coli sensitivity to UV radiation. A reduction of bacterial density was also observed in Petri dishes that were not exposed to UV (BN and TN) but at low levels. These results confirm that 180 min of UV exposure is the UV exposure time required to demonstrate the antibacterial activity of UV-activated TiO2-coated surfaces with E. coli. In fact, at this time of UV exposure, a statistically significant difference between the survival ratio of TE and the other Petri dishes was observed (p < 0.05). Therefore, 180 min was used as the starting exposure time for the trials with the other bacteria.
During the 180 min of exposure, the temperature in a non-TiO2-coated Petri dish was monitored to evaluate the effect of this parameter during UV irradiation because in some studies, an increase in temperature was reported during exposure (Vohra et al. 2005). The temperature reached 22.8°C after 30 min of exposure, showing a decrease in temperature compared with the beginning of the experiment (23.9°C). This trend may be related to the ventilation system used in the exposure chamber that allowed air exchange while avoiding temperature increase. After 180 min of the exposure, the temperature was 23.8°C. The results showed that the temperature ranged between 22.8 and 23.8°C with a variation of 1°C, which most likely would not influence the bacterial survival data during the experiment.
The survival curve of S. aureus at 180, 60 and 30 min of exposure is reported in Figure 3b. At 180 min of exposure, a reduction of S. aureus survival was observed in all of the Petri dishes. The bacterial death observed at this time of exposure could most likely be related to the different stress conditions during the experiment (e.g., resuspension in deionised water and light exposure during the sterile operations), including the long exposure to UV-A radiation. However, the effect of these stress conditions was less evident at 30 and 60 min of exposure, particularly in TN and BN samples. Although low survival was observed in TE dishes at both 30 and 60 min of exposure, the best antibacterial effect in S. aureus related to the TiO2 treatment was at 60 min of exposure. This finding was confirmed by ANOVA, which showed a statistically significant difference between the antibacterial activity obtained in TE and the other experimental conditions at this time of exposure (p < 0.001).
Regarding the survival of P. putida at different times of exposure (180, 60 and 30 min) (Figure 3c), the highest antibacterial effect of TiO2 treatment was obtained at 30 min of exposure, at which a statistically significant difference was observed between TE and the other Petri dishes (p < 0.05). Considering the survival data obtained in the BE dishes, a bactericidal effect associated with UV exposure was also observed at 180 and 60 min. Moreover, at 30 and 60 min of exposure, in BN samples a proliferation of P. putida was observed. Both of these results highlight a particular resistance of this bacterium under the experimental conditions, most likely due to the environmental origin of Pseudomonadales and of this strain, which was isolated from soil.
The survival curve of L. innocua is reported in Figure 3d. As observed for the other bacteria in this study, L. innocua appeared to be sensitive to UV starting at 60 min of exposure (BE and TE). Additionally, a reduction of bacterial load was observed for the BN dish at 180 min of exposure, when the experimental stress conditions caused the reduction of living bacteria. Considering the combined UV-TiO2 effect, a lower decrease in L. innocua load was observed at 30 min of exposure compared with S. aureus and P. putida (TE dishes). Moreover, at this time of exposure, no statistically significant difference was observed (p > 0.05) between TE and the other experimental conditions; therefore, a further exposure time of 20 min was evaluated for this strain. Under this experimental condition, the difference between TE and the other Petri dishes was statistically significant (p < 0.001).
Antibacterial activity of TiO2-coated ceramic tiles
The described experimental protocol was tested with TiO2-coated ceramic tiles to evaluate the antibacterial activity of a real industrial material. The UV exposure times determined by the data obtained in the previous trials with TiO2-coated Petri dishes for each bacteria investigated were used during the experiments with the ceramic tiles (180, 60, 30 and 20 min for E. coli, S. aureus, P. putida and L. innocua, respectively). In particular, experimental conditions that highlighted the antibacterial effect of TE Petri dishes were selected. These conditions were associated to a statistically significant difference between the antibacterial activity of TE and the other Petri dishes (TN, BN and BE) (p < 0.05 or p < 0.001).
The survival data of E. coli, S. aureus, P. putida and L. innocua are reported in Figure 4 and compared with the results obtained in the Petri dishes at the same UV exposure time. Considering the data obtained with TiO2-coated ceramic tiles, a statistically significant reduction (p < 0.05 for E. coli, S. aureus and P. putida; p < 0.001 for L. innocua) of bacterial concentration was observed for all of the microorganisms exposed to UV irradiation (TE) compared with non-UV-exposed tiles and controls (BN, BE, TN). Moreover, in general, a slight reduction was revealed in the BE tiles. As observed in the Petri dishes, this trend was most likely related to the antibacterial effect of UV radiation. A low reduction of bacterial survival or proliferation was observed in the BN and TN tiles. Comparing the survival data obtained with ceramic tiles and Petri dishes, no clear difference was observed between the antibacterial effect of the P25-coated Petri dishes versus the treated tiles, except for P. putida.
Evaluation of photocatalytic activity
All of the UV-exposed samples demonstrated first-order degradation kinetics with an exponential decay of the MB concentration, given by the following equation:
where C(t) is the MB concentration at the time t (mol m-3), t is the time (s) and k is the exponential decay constant (s-1). The constant k was calculated for each sample with a non-linear regression of the experimentally measured concentrations and equation (1).
The surface-specific photocatalytic activity of each sample was expressed as the degradation rate D at a predetermined reference concentration according to the following:
(2)
where D(C
R
) is the degradation rate at the concentration C
R
(mol m-2 s-1), k is the exponential decay constant, C
R
is the reference concentration (mol m-3), V is the solution volume in the batch reactor (m3) and A is the photocatalytic sample exposed surface (m2).
The results, expressed as degradation rate at 1 mmol m-3 MB (1 μmol l-1), are reported in Figure 5. A repeated test on the treated tile demonstrated a ± 5% repeatability error in the resulting D value. The degradation of MB in the UV-exposed solution without sample was comparable to the decay measured with the untreated tile sample. The MB degradation of non-exposed TiO2-coated and blank samples (TN and BN) was negligible because in the absence of UV, the MB solution is stable, which was also observed in the presence of TiO2.
The results showed the higher specific photochemical activity of the P25 Petri sample under these experimental conditions, which was greater than the activity of the treated tile by an order of magnitude. Interestingly, the higher photocatalytic reactivity of P25 samples was not reflected in the antibacterial measurements. In fact, except for P. putida, no difference was observed in the antibacterial effect of the P25-coated Petri dishes compared with the treated tiles.