E 2030

Five disks from each group were used to observe the bacterial biofilm via scanning electron microscopy (SEM). The disks were gently rinsed with distilled water and fixed in 2.5% glutaraldehyde (Tianjin Fuchen Chemical Reagents Factory, Tianjin, China) at 4 °C overnight. Specimens were then dehydrated in ascending ethanol series, followed by desiccation with a lyophilizer (Hitachi E-2030, Tokyo, Japan). After being mounted on aluminum stubs and sputter-coated (Hitachi E-1045; Hitachi, Tokyo, Japan), the specimens were observed with SEM (FE-SEM 4800; Hitachi, Tokyo, Japan).

Floc structure of the bioaggregate sample was evaluated by a scanning electron microscope42 . The bioflocculation samples were fixed with 2.5% glutaraldehyde for 1.5 h in a 4 °C refrigerator. These samples were gently washed with phosphate buffer solution and then dehydrated by successive passages through 50%, 70%, 80%, 90%, and 100% ethanol. Each rinsing and dehydrating step took 10 min. The samples were refreeze dried (Hitachi E-2030, Japan) for 4 h, subsequently coated with gold powder by Sputter Coater (Hitachi E-1010, Japan) and finally attached on to the microscope supports with silver glue. Scanning electron microscope images were taken at 5 kV using an SEM (Hitachi S-3400N, Japan).