In the present work, the influence of hydraulic retention time (HRT) on dark fermentation metabolism was evaluated through the operation and analysis of a series of four continuous stirred tank reactors (CSTR) at four HRT ranging from 6 h to 24 h. A maximum volumetric hydrogen production rate (VHPR) of 2000 149 mL/L-d corresponding to an H-2 yield of 0.86 moll(H2/)mol(lactose) was observed at 6 h HRT. In depth analysis of metabolite profiles and microbial communities showed that low values of HRT favored the emergence of a community dominated by Clostridiaceae-Lachnospiraceae-Enterobacteriaceae, which performed metabolic pathways co-producing hydrogen. In contrast, long HRT led to the establishment of Sporolactobacillaceae-Streptococcaceae microbial community that outcompeted hydrogen producing bacteria and was responsible of lactate production. Results suggested that these two communities mutually excluded themselves and HRT can act as an operational parameter to control the microbial communities and consequently the related metabolic pathways. (C) 2017 Elsevier Ltd. All rights reserved.