2.4. Quantitative analysis of microbial cluster size
Fifty microliter mixed sludge medium were collected from each reactor at 0, 3, 9, 12, and 24 h after inoculation, respectively, and were then analyzed. We employed 3-Deazaneplanocin A optical microscope system (Olympus BX41, Japan) to analyze microbial clusters sizes via image acquisition, and ten slides of each sample were recorded for size analysis. Microbial cluster size was represented by its projected area on the slide  and .
2.5. Extraction and analysis of EPS
2.6. Surface charge
The surface charge of microbial clusters was determined using colloid titration modified from Mikkelsen . Polybrene and polyvinyl sulfate potassium salt (PVSK) were used as positive and negative colloidal reagents, respectively. First, 5.0 mL of 0.001 N polybrene was added to 100.0 mL diluted sample (containing 1.0 mL mixed sludge). The excess polybrene was back titrated with 0.0005 N PVSK to a community simplification colourimetric endpoint indicated by toluidine blue. Equal volumes of polybrene in centrifuged supernatant without microbial clusters were used as blanks.