## 2 Materials and Methods  
### Lab-Scale MBR Operation for Evaluating Permeability by Two-Stage Backwashing  
Lab-scale MBR was operated to evaluate the two-stage backwashing protocol. MBR sludge was sampled from a local wastewater treatment plant. The initial mixed liquor suspended solid (MLSS) of the MBR sludge was 5900 mg/L. After the sludge adapted to synthetic wastewater for over 30 days, the MLSS of the sludge was approximately 6200 mg/L. The composition of the synthetic wastewater is shown in Table 2. A hollow fiber membrane module, composed of PVDF and manufactured by Toray, Japan, was reassembled to fabricate a lab-scale module. The detailed specifications of the membrane are as follows: a pore size of 0.01 \(\upmu\)m, a maximum transmembrane pressure of 100 kPa, and a maximum NaOCl exposure as Cl\({}_{2}\) of 1,000,000 mg/L\(\cdot\)h. The nominal surface area of the lab-scale membrane module was 87.96 cm\({}^{2}\). Two modules were submerged in each reactor (i.e., total membrane surface area in each reactor was 175.92 cm\({}^{2}\)) and permeation pumps were operated under a constant flux condition of 20 L/m\({}^{2}\)/h. The nominal volume of the MBR tank was 3 L, and the operating conditions of MBR were HRT 8.38 h, temperature 24 \({}^{\circ}\)C \(\pm\) 1, SRT 30 days, and aeration 1 L/min, respectively. The transmembrane pressure (TMP) of each module was monitored by a digital pressure gauge, and the module was withdrawn when the TMP reached 80 kPa. Two-stage backwashing with various concentrations of NaOCl was performed on the withdrawn module. A 1000 mg/L CDA stock (in 10% ethanol carrier) was diluted to 300 nM with distilled water (DW). Sodium hypochlorite was diluted to each concentration with DW after the concentration of sodium hypochlorite stock solution was confirmed by DPD-free chlorine reagent (Hach, USA). Each withdrawn module was immersed in 1700 mL of DW, and backwashing was performed by CDA 300 nM solution at a flux of 30 L/m\({}^{2}\)/h for 30 min. The optimal concentration and contact time of CDA were based on previous studies [27, 28, 31]. After the CDA backwashing had finished, NaOCl backwashing was performed at the same flux and duration. The concentration range of NaOCl is determined based on dosages of applications on membrane bioreactors in practice and literature reviews [11]. The concentration of NaOCl was 0 (i.e., DW), 100, 200, 300, 400, 500 mg/L, respectively. Each backwashed membrane module was gently rinsed with DW and returned to the bioreactor. When the TMP of the membrane module reached 80 kPa five times, the operation was terminated.  
\begin{table}
\begin{tabular}{c c c} \hline
**Protocols** & **Advantages** & **Limitations** \\ \hline Hydraulic backwashing & Low cost, easy to apply, chemical-free & Cannot remove irreversible fouling \\ \hline Chemical enhanced backwashing & Low cost, superior cleaning efficiency & Can cause damage to membrane and microorganism \\ \hline Two-stage backwashing & Superior cleaning efficiency, reduced & \\  & dosage of NaOCl, more sustainable & Complex to apply, additional cost \\ \hline \end{tabular}
\end{table}
Table 1: Comparison of advantages and limitations of various backwashing protocols.