This work explores the production of biohydrogen from brewery wastewater using as inoculum a culture produced by natural fermentation of synthetic wastewater and Klebsiella pneumoniae isolated from the environment. Klebsiella pneumoniae showed good performance as inoculum, as evaluated using assays of between 9 and 16 cycles, with durations of 12 and 24 h, carbohydrate concentrations from 2.79 to 7.22 g L-1, and applied volumetric organic loads from 2.6 to 12.6 g carbohydrate L-1 day(-1). The best results were achieved with applied volumetric organic loads of 12.6 g carbohydrate L-1 day(-1) and cycle length of 12 h, resulting in mean volumetric productivity of 0.88 L H-2 L-1 day(-1), maximum molar flow of 10.80 mmol H-2 h(-1), and mean yield of 0.70 mol H-2 mol(-1) glucose consumed. The biogas H-2 content was between 18 and 42%, while the mean organic compounds removal and carbohydrate conversion efficiencies were 23 and 81%, respectively. The inoculum produced by natural fermentation was not viable. (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.