*3.2. Photocatalytic Disinfection of Target Pathogens*

Figure 3a,b show the photocatalytic disinfection achieved against the target pathogens at different catalyst concentrations. In Figure 3c,d bacterial disinfection is represented in its corresponding log reduction profile and the disinfection pattern is validated through comparison of the obtained profile with the standardized Chick-Watson model [11,20,21]. Figure 3a,b suggest that amongst the concentrations tested, 2 mg/L and 3 mg/L resulted in complete disinfection (6 log reductions) of *E. coli* and *S. aureus*, respectively, in 60 min and 90 min, respectively. It is observed that sunlight alone is not effective for the complete disinfection of the targeted pathogens as only 3 and 2.5 log reductions could be observed for *E. coli* and *S. aureus*, respectively, at 120 min. Experiments conducted under dark conditions did not show any remarkable change in the microbial colony counts, as less than 0.5-log reduction for both the microorganisms was achieved in 120 min (Figure 2c,d). Using the optimum concentration of Ag@ZnO nanoparticles for each of the bacteria for photocatalytic disinfection, comparative sunlight-assisted photocatalytic disinfection activity was evaluated with pure-ZnO and commercial TiO2 (Degussa P25) and the results are shown in Figure 4a,b. Figure 4c,d shows the Chick-Watson disinfection kinetics of *E. coli* and *S. aureus* using different photocatalysts. These results suggest the superior disinfection efficiency of Ag@ZnO nano-photocatalyst compared to the conventional metal oxide systems. An increase in inactivation for both targeted bacteria was observed with the increase in catalyst concentration from 1 to 2 mg/L in *E. coli* and 1 to 3 mg/L in *S. aureus*. With further increase in the catalyst concentration beyond the mentioned range, a deterioration in disinfection rate was obtained for both the targeted microorganisms. With lower concentration of catalyst the amount of reactive oxygen species (ROS) generated is comparatively less. Thus complete disinfection required a longer irradiation time [22]. It is expected that as the

rate of ROS production is slow at lower concentrations of catalyst, and under the initial conditions the microorganisms may activate their molecular resistance mechanisms. Therefore an extended disinfection time period is required for sufficient ROS generation and thus under the constant attack of ROS, bacteria may lose their capability of reactivation. With an increase in catalyst concentration the ROS generation rate increases, which is expected to improve the disinfection rate. Similarly, under the optimal conditions, the rate of ROS generation is maximum and therefore it may be expected that the interaction of the same with bacterial cells is more frequent. This may lead to an enhanced disinfection rate. It is further noticed that with increase in the catalyst concentration, disinfection gets delayed. This is mainly because with the increase in catalyst concentration the turbidity of the system increases, thereby blocking the sunlight irradiation from uniformly reaching the catalyst particles and cells, hence resulting in slower inactivation [23]. The current study involves *E. coli* and *S. aureus* bacteria. The photocatalytic performance of a photocatalyst depends both on its concentration and the irradiation time. *E. coli* was found more sensitive to sunlight-assisted photocatalytic disinfection process than *S. aureus*, as it requires comparatively less catalyst concentration and shorter sunlight irradiation time in comparison to *S. aureus* as evidenced from Figure 3a,b. The difference in susceptibility of both bacterial species to Ag@ZnO nanoparticles can be ascribed to the differences in their cell membrane/wall structures, chemical components, biological shape, and differences in robustness of Gram-positive and Gram-negative bacteria [24].

**Figure 3.** Effect of Ag@ZnO core-shell NPs loading on the solar-PCD kinetics of (**a**) *E. coli* and (**b**) *S. aureus*. Linear fitting plots of PCD kinetics of (**c**) *E. coli* and (**d**) *S. aureus* according to Chick-Watson model. Initial bacteria concentration = 5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL, Temperature = 35 <sup>±</sup> <sup>2</sup> ◦C. Error bars indicate the standard deviation of replicates (*n* = 3).

**Figure 4.** Effect of different catalysts on the solar-PCD kinetics of (**a**) *E. coli* and (**b**) *S. aureus* at a catalyst loading of 2 mg/L and 3 mg/L respectively. Linear fitting plots of PCD kinetics of different catalysts against (**c**) *E. coli* and (**d**) *S. aureus* according to Chick-Watson model at a catalyst loading. Initial bacteria concentration for each experiments = 5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL, Temperature = 35 <sup>±</sup> <sup>2</sup> ◦C. Error bars indicate the standard deviation of replicates (*n* = 3).

From Figure 4, it is observed that Ag@ZnO nanoparticles show enhanced disinfection efficiency for both targeted pathogens in comparison to the classical metal oxide systems (ZnO and TiO2). The expected reason behind the enhanced efficiency may be the positioning of the noble metal (i.e., Ag) in the core and encapsulating it with a ZnO shell. Photocatalytic disinfection involves the excitation of the photocatalyst with light energy greater than or equal to that of the band gap [25]. On excitation the electrons forming the valence band of the metal oxide shuttle to the conduction band, where they are usually accepted by electron acceptors present in the reaction environment. This reduction pathway leads to the formation of ROS which results in killing of microbial cells by damaging their membrane integrity [24,26]. Therefore it leads to subsequent release of the intra-cellular components, which become vulnerable to the ROS attack [26,27]. Figure 5a–d show the effect of temperature on the photocatalytic disinfection of the targeted pathogens. These results show that as the temperature increased, a maximum process efficiency was observed at a reaction temperature of 55 ◦C. It is thus observed that, the rate of disinfection improved as the temperature of the reaction system increased. At 55 ◦C disinfection is achieved within 45 min and 60 min for *E. coli* and *S. aureus*, respectively. The post-disinfection reactivation of the target microbes was monitored for 24 h. None of the microbes showed an6y reactivation thus suggesting cell death due to damage caused by the ROS to both the target pathogens.

**Figure 5.** Effect of different reaction temperature on the solar-PCD kinetics of (**a**) *E. coli* and (**c**) *S. aureus* at a catalyst loading of 2 mg/L and 3 mg/L respectively. Linear fitting plots of PCD kinetics of different reaction temperature against (**b**) *E. coli* and (**d**) *S. aureus* according to Chick-Watson model. Initial bacteria concentration for each experiments = 5 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL, Error bars indicate the standard deviation of replicates (*n* = 3).
