A novel strategy to discontinuously increase the biomass concentration in a continuous stirred-tank reactor was evaluated to enhance the performance of dark fermentation. Different concentrations of biomass were evaluated at organic loading rates (OLR) ranging from 90 to 160 g lactose/L-d with a hydraulic retention time (HRT) of 6 h. The study revealed that the discontinuous increase of biomass enhanced the hydrogen (H-2) production rates and carboxylic acids concentrations by 19-25% and 8-23%, respectively. In particular, a maximum H-2 production rate of 30.8 L H-2/L-d with carboxylic acids concentration of 20 g/L was reached at an OLR of 138 g lactose/L-d with a biomass concentration of 15 g volatile suspended solids/L. The analysis of microbial communities showed the co-dominance of Clostridium and lactic acid bacteria. Overall, the discontinuous increase of biomass was an effective strategy to improve the performance of suspended-biomass reactors operated at high OLR and low HRT. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.