Sterilization, alkaline-thermal, and acid-thermal treatments were applied to different suspended sludge solids (SSS) concentrations and the pre-treated sludge was used as raw material for bioflocculant-producing bacteria R3 to produce bioflocculant. After 60 h of fermentation, three forms of bioflocculant (broth, capsular, and slime) were extracted, and maximum broth bioflocculant of 2.9 and 4.1 g L-1 were produced in sterilized and alkaline-thermal treated sludge as compared to that of 1.8 g L-1 in acid-thermal treated sludge. Higher bioflocculant quantity was produced in SS of 15, 25, and 35 g L-1 compared to that produced in SS of 45, 55, and 65 g L-1. Bioflocculant combined with 0.5 g Ca2+ in 1.0 L kaolin suspension acted as conditioning agent, and maximum flocculating activity of 94.5% and 92.8% was achieved using broth and slime bioflocculant, respectively. The results demonstrated that wastewater sludge could be used as sources to prepare bioflocculants. (C) 2013 Elsevier Ltd. All rights reserved.