This study aimed to investigate the characteristics of hydrogen production by mixed cultures using Laminaria japonica hydrolysates. The hydrolysates of L. japonica were prepared by pretreatment methods, including heat (100 degrees C or 121 degrees C) and acid (HCl or H2SO4) pretreatments. The mixed cultures could produce hydrogen using L japonica as a substrate, with the highest cumulative hydrogen production of 825 +/- 14 mL/L from HCl-pretreated L japonica. High-throughput sequencing of the 16S rRNA gene revealed that the microbial community in the hydrolysate of HCl-pretreated L japonica was the most diverse among all the samples, with a Shannon diversity index of 5.253. The mixed culture from HCl-pretreated L japonica and those from heat-pretreated (100 degrees C and 121 degrees C) L japonica occupied different regions in a principal component analysis (PCA) plot. The dominant population in the hydrolysate of HCI-pretreated L japonica was represented by hydrogen-producing bacteria, Clostridium spp. and Bacillus spp. The results suggested that L japonica was an optimal feedstock for hydrogen production. The acid (HCl) pretreatment method could effectively enhance the hydrogen production from L. japonica. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.