C045a047a

α-FeOOH catalyst was synthesized following a method developed by Sui et al. [22 ]. A solution of ferric nitrate (Fe(NO₃)₃·9H₂O, 0.25 M) was added to a sodium hydroxide solution (NaOH, 5 N); the pH was adjusted to >12, and the solution was allowed to stand for 48 h. The mixture was filtered through a 0.45-µm filter (C045A047A, Advantec, Tokyo, Japan) to collect the precipitates. The precipitates were then rinsed with distilled water until the effluent remained at a neutral pH. Then, the precipitate was dried at 120 °C for 24 h before being used in the experiments. The catalysts were characterized using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) on a Hitachi S-3000N system. The results are shown in Figure 2. The Brunauer–Emmett–Teller (BET, Beckman Coulter SA3100) surface area of the α-FeOOH catalyst was 61.9 m²·g⁻¹, as determined by N₂ physisorption at 77 K.

Water samples were collected from WLR, Pingtung, Taiwan, which is polluted with domestic and agricultural wastewaters. Water samples were collected in 25-L polyethylene tanks and stored in a refrigerator at 4 °C for less than 7 days. Samples were warmed back to the experimental operating temperature (20 °C) before being used and were passed through a 0.45-µm filter (C045A047A, Advantec, Japan) to remove coarse suspended and colloidal solids.

Isolation and purification of milk sEVs were carried out as described previously [15 (link),16 (link)] with slight modifications. Briefly, after removing the milk fat by centrifugation at 2000× g for 20 min using a centrifuge (MX-307, Tomy Seiko, Tokyo, Japan), defatted milk was preheated at 37 °C for 10 min. For efficient isolation of milk sEVs, acetic acid was added (finally 1%), and casein was removed by centrifugation at 5000× g for 20 min. The whey was filtrated by using 1.0, 0.45, and 0.2 μm pore-size filters (GA-100, C045A047A, and C020A047A, Advantec, Tokyo, Japan). Further, milk sEVs were concentrated from the whey by ultracentrifugation (UC) at 100,000× g at 4 °C for 1 h using a P42A angle rotor (Hitachi Koki, Tokyo, Japan) in a Himac CP80WX ultracentrifuge (Hitachi Koki). The supernatant was discarded and the bottom pellet was resuspended with phosphate-buffered saline (PBS) up to 10 mL into a 13PET tube (Hitachi Koki). The UC was carried out twice again at 100,000× g at 4 °C for 1 h using a P40ST swing rotor (Hitachi Koki) and the milk sEVs pellet was resuspended with 100 µL of PBS for further use.