## 3 Results
### Surface Coverage Analysis  
The images of CDA with DW and 100 and 200 mg/L NaOCI specimens are shown in Figure 6, and the COMSTAT analysis result for all conditions is shown in Figure 7. Dead and live cells were observed in the areas of the membranes that are presumed to be directly affected by backwashing. However, the variations in the regions influenced by backwashing were too diverse to be adequately quantified. Therefore, we prioritize areas outside the direct influence of backwashing to analyze the aggregate impact of microbes distributed on the membrane surface. The selected area presented a very low intensity of dead cell dye in all conditions, resulting in a noise level, so the COMSTAT analysis of the CLSM images was conducted for live cells only. Figure 6: CLSM images of two-stage backwashed membrane with CDA 300 nM, (**a**) DW, (**b**) 100 mg/L NaOCI, and (**c**) 200 mg/L NaOCI. The black voids in the green biofilm structure in Figure 6a may indicate that microbes exit the biofilm as they disperse, creating pores in the biofilm [31]. In Figure 6b, the voids in Figure 6a were not observed, instead showing an irregular pattern throughout. On the other hand, in Figure 6c, the voids in Figure 6a and the detached biofilm can be observed simultaneously. This may indicate the mechanism of the two-stage backwashing with CDA and NaOCl. At 100 mg/L, NaOCl spread evenly in the CDA-weakened biofilm and no dispersed marks were visible, but at 200 mg/L, the higher NaOCl concentration caused the biofilm to detach and NaOCl preferentially flowed through the already formed channels, making CEB less effective. The increase in average thickness and biomass with an increment in the NaOCl concentration is shown in Figure 7a,b. This suggests that the NaOCl that passed through the membrane in the backwash spread in the reservoir and acted as oxidation stress. As shown in Figure 7c, the surface-to-biovolume ratio increased with the increment in the NaOCl concentration. The surface-to-biovolume ratio is the proportion of biomass to external contact area, and a larger surface-to-biovolume ratio means more efficient nutrient intake. In nutrient-limited environments, biofilms can morph to increase their surface-to-biovolume ratio. In this research, the increasing surface-to-biovolume ratio in Figure 7c is assumed to be a reaction to recover biofilm after backwashing. The roughness coefficient is represented by a value between 0 and 2 depending on the gap in element thickness compared to the average thickness. As shown in Figure 7d, the roughness coefficient decreased with the increment in the NaOCl concentration. It has been reported that the application of CDA increases the roughness coefficient of biofilm, but the influence of CDA seems to be neutralized by the increasing NaOCl concentration. The decrease in the roughness coefficient indicates a simplification of the biofilm structure. The decrease in the roughness coefficient and the increase in the surface-to-biovolume ratio may suggest that the remaining biofilm is structurally optimized. The structural stabilization and large surface area may be the mechanisms underlying the rapid regeneration of the biofilm after backwashing [27, 41]. Figure 7: COMATA analysis of CLSM image: (**a**) average thickness, (**b**) biomass, (**c**) surface-to-biovolume ratio, and (**d**) roughness coefficient. The cleaning efficiency might be affected by the pH of the solution. The pH of the backwashing reservoir was observed to range from 7.2 to 8.8, corresponding to NaOCl concentrations varying from 100 to 500 mg/L. These pHs were near to the pKa of HOCl, which is 7.53, where the ratio of HOCl to OCl\({}^{-}\) is equal. Outside of this pKa, the ratio changes significantly. It is widely accepted that HOCl is superior to OCl\({}^{-}\) in terms of germicide. Due to its non-polar structure, HOCl can penetrate the cell membrane of microorganisms and attack cells from the inside and outside, resulting in a low CT value for planktonic microbial organisms.