Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor

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 between 3.2 and 11.6 L_H2/L-d. In contrast, relatively high OLR (58.8 and 88.2 g_Lactose/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH₂⁺), which theoretically consumed up to 5.72 L_H2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 L_H2/L-d) but the H₂ yield dropped to a minimum of 0.74 mol_H2/mol_Hexose at OLR = 58.8 g_Lactose/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.