Abstract
Clostridium pasteurianum DSM525, Clostridium beijerinckii DSM791, Clostridium acetobutylicum DSM792, and Clostridium intestinale DSM6191 were cultivated using sugar beet raw molasses (RM) as the sole nutrient source to evaluate their growth performance and capacity for simultaneous bio-H 2 and total bio-alcohol production. Fermentation was carried out anaerobically for 168 h at RM concentrations of 4%, 8%, and 10% (w/v). Along with biomass formation, the lowering of pH (from 7.0 to ~ 4.1) and oxidation-reduction potential (ORP) (values attaining − 560 ± 10 mV) were stated. Analysis of total carbohydrate (TCs) consumption, volatile solids (VSs) degradation, and fermentation end-product profiles confirmed efficient bacterial substrate utilization and active metabolism on RM. 4% (w/v) RM provided the most favorable balance between yield and rate of the bio-H 2 and bio-alcohol production. C. pasteurianum achieved a maximum bio-H 2 yield of 46 mmol L −1 g − 1 TCs (3162 ± 10 mL L −1 g − 1 TCs) and 68 mmol L −1 g − 1 VSs at 4% (w/v) RM, while the highest bio-alcohol production (82 ± 3 mmol L −1 g − 1 TCs ) was observed at 8% (w/v) RM. C. pasteurianum and C. beijerinckii also demonstrated enhanced growth (OD 600 ≈ 1.6), as well as the most significant decline in ORP. Results demonstrate that RM without chemical pretreatment is an effective substrate for sustainable bioenergy production. Strain-specific behavior reflects distinct regulatory strategies governing solventogenesis, while dynamic ORP changes highlight the critical role of redox control in maximizing simultaneous H 2 and alcohol production. These findings emphasize the importance of selecting appropriate strains, substrates, and monitoring redox conditions to optimize bioenergy yields in dark fermentation systems.