Continuous two-stage lactate-based fermentative hydrogen production from cheese whey with co-production of short-chain carboxylates

Fermentative hydrogen production from cheese whey (CW) is often hindered by the dominance of lactic acid bacteria (LAB), which suppress hydrogen-producing populations and redirect carbon flux toward lactate accumulation. Lactate-driven dark fermentation (LD-DF) offers an alternative approach by converting lactate into hydrogen and short-chain organic acids (SCOAs) through syntrophic interactions between LAB and lactate-oxidizing bacteria. In this study, a continuous two-stage LD-DF system was evaluated, in which lactic fermentation (R1) and hydrogenogenic lactate conversion (R2) were spatially decoupled to enhance process performance through microbial specialization. In R1, operated without pH control, homofermentative metabolism predominated, producing 35-55 g lactate/L, which accounted for more than 80% of total soluble acids, confirming its role as a primary lactate fermentation stage. In R2, maintained at pH 6.3, maximum hydrogen productivity reached 6.7 NL H 2 /L-d (50-58% H 2 in the off-gas), accompanied by the production of butyrate (3-6 g/L), lactate (15-19 g/L), and propionate (8 g/L) as the dominant SCOAs. Microbial community analysis revealed Lactobacillus dominance in R1 and a LAB-rich consortium in R2, with limited enrichment of Clostridium sensu stricto. The results demonstrate, for the first time, that two-stage LD-DF improves hydrogen productivity from CW. However, they also highlight the need for targeted strategies to stimulate lactate-oxidizing hydrogen-producing populations for improved hydrogen production efficiency.