Biogas production from the microalgae-bacteria flocs (MaB-Flocs) in batch reactors was conducted in this study. A batch test was performed to determine optimum inoculums that were taken from a running biogas plant (BP), a municipal wastewater treatment plant (MWTP) and a river sediment (RS). The maximum biogas yield (304.83 +/- 4.23 mL per gram volatile solids introduced (mL g-VS (1))) was obtained with the inoculum from MWTP. Subsequently, the effect of substrate-inoculum (S/I) ratios, temperature and pre-treatment methods on fermentative biogas and methane production was investigated. The optimum S/I ratios and incubation temperature were determined as 0.2 g VSsubstrate/g VSinoculum and 37 +/- 1 degrees C, respectively. The results of the CH4 fermentation show that the methane yields could be increased from 216.72 +/- 3.52 mL CH4 g-VS (1) to 271.34 +/- 6.65 mL g-VS (1) by using enzymatic pre-treatment at the S/I ratio of 0.2 g VSsubstrate/g VSinoculum and mesophilic conditions. (C) 2015 Elsevier Ltd. All rights reserved.