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In this study, the optimal conditions of production of bioflocculant by the culture of Pseudomonas alcaligenes PS-25 were examined, using brewery wastewater to replace glucose as carbon source and energy source in the culture medium. Results showed that the COD concentration in brewery wastewater favorable for the production of the bioflocculant was 5000 mgÂ·L-1, and an optimal culture condition of inoculum size of 5%(v/v), 30Â°C, initial pH 6.0 and shaking speed of 160 rÂ·min-1, under the optimal culture conditions, the highest flocculating activity achieved for Kaolin suspension was 96.8% and 2.89 g bioflocculant per liter broth was obtained after 72 h cultivation. The bioflocculant produced using brewery wastewater substrate possesses flocculating activity comparable or superior to that of bioflocculant produced using conventional substrate. The bioflocculant was mainly composed of sugar (86.5%) and protein (11.2%), and an average molecular weight of 3.56 Ã 106 Da. Fourier transform infrared (FTIR) spectra showed that hydroxyl and carboxylate groups were present in the bioflocculant molecules. The bioflocculant was effective in flocculating some soluble reactive dyes in aqueous solution, reactive Light-Yellow K-4G and reactive Turquoise Blue KN-G with a decolorization efficiency of 92.1 and 90.5%, respectively, using 25 mL of the flocculant in 500 mL of 100 mgÂ·L-1 dye solution. The decolorization efficiency was dependent on the flocculant dosage and solution pH. The hydroxyl and carboxylate groups in the bioflocculant molecule are believed to play an important role in flocculation from the viewpoint of electrostatic interaction.