While knowing the amylolysis mechanism is important to effectively decompose corn starch fed into an anaerobic digestor, the objective of this study was to detect the activities and locations of alpha-amylase in a continuous reactor and batch cultures. In the continuous reactor operated at 35 degrees C, the greatest cell-bound alpha-amylase activity was found to be 4.7 CU mL(-1) at hydraulic retention time (HRT) = 9 h, while the greatest volumetric hydrogen production rate (r(H2)) was observed at HRT = 3 has 61 mmol L-1 day(-1). In the batch tests, the cell-bound alpha-amylase activities increased when the carbohydrate concentration decreased, and no significant reducing sugar accumulation was found in the serum bottles. By examining the specific hydrogen production rate (q(H2)) against different corn starch concentrations, the half-saturation constant (K-Sta) and the maximum q(H2) were regressed to be 0.47 g L-1 and 6 mmol g-VSS-1 d(-1), respectively. The electronic microscopic images showed that the microbes could colonize on the starch granules without the disturbance of any floc-like materials. Conclusively, by excluding the methanogens and floc matrix, the secreted alpha-amylases are predominately bound on the cell surfaces and enabled the microbial cells favorably attach on large substrates for hydrolysis under the mesophilic condition. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.