2.4.2. Biofilm characterization
After completion of the biofouling experiments, the bacteria adhered to themembrane surfacewere characterized by confocal laser scanning microscopy (CLSM; FLUOVIEW FV1000, Olympus Corporation, Tokyo, Japan) and image analysis [11]. The membranes were detached from the cell and rinsed thoroughly with aqueous 0.85 wt.% NaCl solution. The adhered bacteria on the membrane surface were then stained using the LIVE/DEAD BacLight Bacterial Viability kit in 0.85 wt.% NaCl solution. The membranes were rinsed again with 0.85 wt.% NaCl solution and immersed in aqueous 2.5 vol.% glutaraldehyde solution for 1 h at 4 °C to fix the adhered bacteria. The membrane surface was observed using CLSM, and the CLSM images were analyzed using the COMSTAT software [23] to calculate the volumeper unit area (μm3/μm2) of the adhered bacteria on the membrane surface.