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The hydrogen (H₂) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H₂ or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H₂ production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7–44.1 g_Lactose/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H₂ yield of 2.14 mol_H2/mol_Hexose obtained at 29.4 g_Lactose/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was …