*3.2. Analysis of Residual Antibacterial Activity of Antibiotic after Photocatalytic Degradation*

Ciprofloxacin, as already discussed, is an antibiotic that belongs to the FQ class of antibiotics. The antibiotics that belong to this group, generally inhibit the growth of several microorganisms via the inhibition of DNA Gyrase, which is a factor is responsible for the division of bacterial cells. ciprofloxacin is active against a wide spectrum of Gram positive and Gram negative bacteria and ciprofloxacin and antibiotics of the FQ group are widely present in wastewaters such as those from hospital, municipal, pharmaceutical industry sources, etc. [1,22,27]. The residues of these antibiotics in the wastewaters generate antibiotic resistant bacteria in the environment, which is a potential major threat to public health.

The current work aims to employ photocatalysis for the successful degradation of the antibiotic ciprofloxacin. After subjecting ciprofloxacin to photocatalytic treatment with Fe-doped ZnO nanoparticles, a confirmatory bacterial inhibition experiment was conducted to check whether the antibiotic was completely degraded in the experimental system using as test organisms *Staphylococcus aureus* and *Escherichia coli* [19]. The results of the experiments (Table 1 and Figures 6 and 7) showed that for both *Staphylococcus aureus* and *Escherichia coli*, ciprofloxacin lost its antibacterial activity after 60 minutes and 75 minutes post-irradiation, respectively. With increasing time, a decreasing

zone of inhibition in both *Staphylococcus aureus* and *Escherichia coli* was evident. The zone of inhibition decreased from 12 mm to 5.5 mm and from 15 mm to 6 mm in the case of *Staphylococcus aureus* and *Escherichia coli* in 60 min and 75 min post-irradiation, respectively.


**Table 1.** Shows residual antibiotic activity of the antibiotic ciprofloxacin after photocatalytic degradation with Fe-doped ZnO nanoparticles against *Staphylococcus aureus* and *Escherichia coli*.

SEM stands for standard error of mean, calculated from the standard deviation, PCD-photocatalytic degradation, DC-dark control, PL-photolysis, *n* (number of replicates) = 6. Zone of inhibition (ZOI) = total zone (including the disc)—diameter of the disc (6 mm). The well diffusion assays were performed in accordance with the Clinical & Laboratory Standards Institute (CLSI) Guidelines. No ZOI have been observed from the solvent controls i.e., with distilled water, no contaminating bacteria were found to grow around the treated samples when poured without the test bacteria. Experimental Conditions: catalyst concentration 150 mg/L, pH 9, antibiotic concentration 10 mg/L and temperature 30 ◦C.

**Figure 6.** Residual antibiotic activity of the antibiotic ciprofloxacin after photocatalytic degradation with Fe-doped ZnO nanoparticles against *Staphylococcus aureus*. Yellow markings denote the time points at which sampling has been done. (**a**), (**b**), (**c**), (**d**) denotes the zone of inhibition shown by the antibiotic slurry collected at different time intervals.

**Figure 7.** Residual antibiotic activity of the antibiotic ciprofloxacin after photocatalytic degradation with Fe Doped ZnO nanoparticles against *Escherichia coli*. Yellow marking denotes the time points at which sampling has been done. (**a**), (**b**), (**c**), (**d**), (**e**), (**f**) denotes the zone of inhibition shown by the antibiotic slurry collected at different time intervals.

It can be seen that *Escherichia coli*, a Gram negative organism, shows susceptibility to ciprofloxacin that has been collected 75 minutes post-irradiation, which is slightly less than that of *Staphylococcus aureus* (sample collected 60 minutes post-irradiation), before completely showing zero susceptibility in both cases. As a Gram negative microorganism *Escherichia coli* has a weak cell wall that is made up of lipopolysaccharides [28,29]. Therefore it is easy for a disinfecting agent to penetrate its cellular defenses. compared to *Staphylococcus aureus*, which is Gram positive. In the case of the light control and dark control, antibacterial activity was not lost even after 120 min for both *Escherichia coli* and *Staphylococcus aureus*. There was little decrease in the zone of inhibition parameters and it clearly signified that ciprofloxacin was still present in the case of experimental controls, suggesting that both the photocatalyst (Fe-doped ZnO) and sunlight are indispensable in the degradation process.
