*3.3. Determination of MDA to Study the Membrane Lipid Peroxidation*

Time dependent generation of MDA (a key biomarker of membrane lipid peroxidation) for *E. coli* and *S. aureus* subjected to photocatalytic disinfection under their respective optimum catalyst concentration and temperature of 35 ◦C is shown in Figure 6a,b. Earlier experiments had shown complete disinfection at 60 and 90 min, respectively, for *E. coli* and *S. aureus* (Figure 3a,b). Hence, a similar correlative result can be inferred from the above mentioned figure. It is quite evident that maximum generation of MDA is observed after 75 min i.e., 0.03 nmol/mg cell dry weight and 90 min i.e., 0.0375 nmol/mg cell dry weight for *E. coli* and *S. aureus* respectively, which indicates cell membrane disintegration resulting in disinfection. Slight elevation in MDA production is seen within the first 30 min, which may be attributed to a loss of membrane integrity due to the action of shear stress produced on the microbial cells due to the continuous stirring conditions [28]. Additionally, the misbalance of ionic potential may also play a role in loss of membrane integrity leading to lipid peroxidation. It may also be noted that after the reported disinfection time, a decline in MDA concentration has been initiated. After a threshold level of MDA is generated in the photocatalytic system, it is expected to be mineralized being an organic compound itself [18,29]. When the microbial cells were exposed to sunlight without the presence of photocatalysts, less than even 0.01 nmol/mg cell dry weight generation was observed in both the microbes as shown in Figure 6a,b. However, the effect of Ag@ZnO on microbial cells without the presence of light is also found to be non-substantial, where the concentration was less, as 0.005 nmol/mg cell dry weight were quantified for both the test microbes. It is proposed that generation of ROS (such as OH• radical) in the photocatalytic process may lead to peroxidation of the cell membrane peptidoglycan layer and membrane proteins, followed by decomposition of cellular components and cellular disintegration [16–19,29], as ROS mainly (•OH) hit unsaturated membrane lipids to make lipid radicals. This, in the presence of oxygen is expected to give a lipid peroxyl radical capable of abstracting hydrogen from an adjacent unsaturated lipid and produce a lipid hydroperoxide and a lipid radical. This series of reactions continues until all the membrane unsaturated lipids are destroyed and malondialdehyde (a stable by-product of membrane lipid peroxidation) is subsequently produced. MDA generation patterns suggest that lipid peroxidation in *E. coli* maintains a uniform rate while a sporadic rate occurs for *S. aureus*, thus suggesting a higher robustness of the latter in comparison to the former [24].

**Figure 6.** Lipid peroxidation kinetics of (**a**) *E. coli* and (**b**) *S. aureus* cells subjected to solar-photocatalysis in presence of 2 mg/L and 3 mg/L Ag@ZnO 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).
