*3.4. Analysis of Potassium Ion (K+) to Study the Cell Membrane Damage*

Leakage of K+ ions is generally considered as a dominating evidence of compromised cellular integrity. The results obtained through K<sup>+</sup> leakage analysis are in agreement with many previous studies which mention the dysfunction of potassium channels of microorganisms on photocatalytic treatment [18,30–32]. It can be observed that the concentration of K<sup>+</sup> (in ppm) increases with the increase of the reaction time up till a particular time period beyond which the concentration in the reaction environment becomes constant. As shown in Figure 7a,b the maximum K+ estimated after 120 min for *E. coli* and *S. aureus* after photocatalytic disinfection was found to be 575 ppb and 440 ppb, respectively. An interesting observation was made that the time required for complete disinfection for each of the target bacterium, as evaluated from the decreasing CFU count, does not correspond well with the K<sup>+</sup> leakage pattern. This must be because the primary target of photocatalytically produced ROS is membrane lipids. Once the entire membrane of the bacteria is compromised, an increase in K<sup>+</sup> ion is expected. This pattern of K+ release suggests that the increase in the concentration of potassium ion in the reaction environment indicates a steady progress in the photocatalytic disinfection process. Once the entire bacterial death is achieved, it is expected that the total amount of K<sup>+</sup> will be maintained for the remaining reaction phase [33].

**Figure 7.** Leakage of K+ ion from (**a**) *E. coli* and (**b**) *S. aureus* cells subjected to solar-photocatalysis in presence of 2 mg/L and 3 mg/L Ag@ZnO core-shell NPs, respectively. Initial bacteria concentration = <sup>5</sup> <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).

#### *3.5. Stability and Reusability of the Catalyst Post Disinfection*

When the stability of the catalyst in post-reaction condition was investigated using XRD, no alteration in the crystal structure of Ag@ZnO was observed, suggesting its structural stability throughout the process [11,34]. It is known that leaching of material could re-toxify the system and it could also be argued that Ag<sup>+</sup> and Zn2+ ions which are reported to show antimicrobial properties may leach out of the system and hence, may be the actual cause of disinfection. However, the answer to this possibility is already communicated in our previous paper [11], there being no detectable amount of Ag<sup>+</sup> and Zn2+ ions in the system post-disinfection. If the catalyst could be recycled after photocatalytic disinfection then it may be suitable for commercial exploitation of the process. Ag@ZnO core-shell nanoparticles were recycled after the photocatalytic disinfection experiments and used for next batch of bacterial disinfection experiment (after heating at 80 ◦C). As shown in Figure 8, core-shell nanophotocatalyst exhibited insignificant reduction in *E. coli* and *S. aureus* disinfection efficiency, even after three consecutive cycles.

**Figure 8.** Effect of Ag@ZnO core-shell NPs reusability till three rounds of solar-PCD kinetics of (**a**) *E. coli* and (**b**) *S. aureus*. 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).
