In this study, mixed and pure bacterial cultures are inoculated in a dark-fermentation process for hydrogen production from Pistia stratiotes hydrolysate. The maximum hydrogen yield (HY) of 2.3 mol(H2)/mol(glucose) (energy yield 0.3 kJ/g(DW)) is obtained at pH 5.5, fermentation time 6.7 h, substrate-to-biomass ratio (S/X) 0.5 g(COD)/g(VSS), and 25 degrees C using a mixed culture system. The plots of HY versus pH and time result in quadratic linear concave-up curves, whereas the effects of S/X and temperature on HY cause quadratic concave-up and flat curves, respectively. Fluorescent in situ hybridization (FISH) indicates that the mixed culture contains multiple H-2-producers including Enterobacteriaceae, Gammaproteobacteria, Betaproteobacteria, and Clostridium histolyticum. Three isolates belonging to Bacillus cereus, Bacillus anthracis, and Enterobacter cloacae are used as pure cultures, achieving HYs of 2.21, 1.10, and 1.97 mol(H2)/mol(glucose), respectively. Accordingly, the mixed culture inoculum subjected to thermal treatment at 100 degrees C for 30 min can deliver higher HYs than pure cultures. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.