## 2 Materials and Methods
### Pure Water Permeability and Anti-Fouling Tests  
Ultrafiltration tests were conducted using a pressurized nitrogen gas dead-end cell filtration system (Millipore). The total volume of the cell was 350 mL, with an effective area of 28.26 cm\({}^{2}\). The experiments were performed at room temperature, and the feed was stirred at 110 rpm. Before the test, the membrane was compacted for 30 min at 5 bar with distilled water. The operative pressure was varied from 1 to 5 bar; each test was anticipated by a stabilization period of 10 min. The permeate was collected every 5 min for 1 h, and flux was calculated according to Equation (4):  
\[\text{J}_{\text{w}}=\frac{\text{V}}{\text{A}\times\text{t}} \tag{4}\]  
where J\({}_{\text{w}}\) is the water flux (L m\({}^{-2}\) h\({}^{-1}\)), V is the of permeate volume (L), A is the membrane area (m\({}^{2}\)), and t is the time (h).  
To measure the rejection and anti-fouling efficiency of the membranes, solutions of humic acid (HA) at a concentration of 100 mg L\({}^{-1}\) were employed as feed and the flux (J\({}_{\text{HA}}\)) was calculated again. The physicochemical properties of the HA solution are reported in Table 2.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Concentration** & **P\(\mathbf{H}\)** & **Zeta Potential \(\zeta\)** & **Particle Size in Suspension** \\
**[mg/L]** & **pH** & **[mV]** & **[nm]** \\ \hline
100 & 6 & \(-27.1\pm 0.01\) & 532.7 \(\pm\) 4.5 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Physicochemical Properties of HA solution (determined by Zetasizer Nano ZS90, Nanoparticle Size Analyzer, Tokyo, Japan).  
Then, the fouled membranes were washed with deionized water for 30 min and the recovered flux (J\({}_{w2}\)) was registered in order to evaluate the water permeability decline due to the irreversible fouling.  
Several UF experiments were carried out to study the fouling tendency of CA and PAA/CA membranes and their long-term performance stability. Three cycles of 240 min were performed by altering pure water (120 min) and HA solution (120 min) as feed to investigate the degree of reversibility of the performance of the developed UF membranes.  
In order to characterize the anti-fouling performance of the membrane, some parameters were observed and calculated according to the relative formulas:  
\[\text{FRR }(\%)=\frac{\text{J}_{w2}}{\text{J}_{w}}*100 \tag{5}\]  
\[\text{Rr }(\%)=\frac{(\text{J}_{w2}-\text{J}_{\text{HA}})}{\text{J}_{w}}*100 \tag{6}\]  
\[\text{Rir}(\%)=\frac{(\text{J}_{w}-\text{J}_{w2})}{\text{J}_{w}}*100 \tag{7}\]  
\[\text{Rt }(\%)=\text{Rr}+\text{Rir}=\left(1-\frac{\text{J}_{\text{HA}}}{ \text{J}_{w}}\right)*100 \tag{8}\]  
where FRR (%) was the flux recovery ratio quantifying the antifouling property after a UF cycle, Rr (%) represented the reversible flux decline ratio caused by reversible fouling eliminated by hydraulic cleaning, Rir (%) estimated the irreversible flux decline ratio, and Rt (%) described the flux decline ratio caused by HA fouling [33].  
During the test performed with HA solution, the absorbance of HA was evaluated using spectrophotometer (PerkinElmer, Waltham, MA, USA) through an excitation-emission matrix (EEM). EEM spectroscopy provides information on the emission (Em) profile concurrently with excitation (Ex) wavelength data, creating a 3-D map of fluorescence characteristics [34]. The samples were diluted 1:5 and analyzed in a quartz cuvette with a path length of 10 mm maintained at a constant temperature of 20 \({}^{\circ}\)C. The EEMs were recorded at 10 nm intervals for excitation spectra between 190 and 600 nm and emission spectra between 190 and 600 nm at an integration time of 0.1 s with excitation and emission slit of 5 nm and scan rate of 600 nm min\({}^{-1}\). The HA rejection ratio was calculated employing the following Equation (9):  
\[\text{R}(\%)=\left(1-\frac{\text{C}_{\text{P}}}{\text{C}_{\text{f}}}\right) \times 100 \tag{9}\]  
where C\({}_{\text{P}}\) and C\({}_{\text{f}}\) were the HA concentration of permeate and feed solution, respectively.