Abstract
In this study acid‐hydrolyzed mixed vegetable waste was investigated as a sole carbon source for anaerobic fermentative growth and biohydrogen (H 2 ) production by Escherichia coli . E. coli BW25113 wild type and a septuple mutant lacking key hydrogenase and fermentative enzymes were cultivated during anaerobic conditions using vegetable waste hydrolysates (VWHs) prepared at substrate concentrations of 5%–20% (w/v) and varying dilution levels. Both strains were able to grow anaerobically on VWHs, confirming the effective release of fermentable substrates during acid pretreatment. Maximum biomass formation by the wild‐type strain reached 0.46 g cell dry weight (g CDW) L −1 in undiluted 20% VWH, while the septuple mutant exhibited improved growth only upon dilution, indicating higher sensitivity to hydrolysate derived inhibitory compounds. Fermentable carbon removal efficiency (CRE) and H 2 production were strongly dependent on substrate concentration and dilution level. H 2 production was completely suppressed in undiluted VWHs but was restored upon dilution, with the highest cumulative H 2 production (406 NmL) observed for the septuple mutant grown on 20% VWH at two‐fold dilution. The corresponding H 2 yield reached approximately 2.4 NL H 2 g −1 (consumed) carbohydrate g −1 biomass or 355 NmL per gram initial carbohydrate. These results demonstrate that VWH can support both biomass formation and competitive H 2 production by E. coli , highlighting their potential as a sustainable platform for waste valorization and bioenergy generation.