Synergistic effects on biohydrogen production from vinasse and molasses co-digestion: Influence of mixture composition on process stability

Organic raw materials processed through anaerobic digestion are increasingly recognized as valuable renewable energy sources. Agroindustrial biomasses like vinasse and sugarcane molasses, rich in carbohydrates, can produce hydrogen via dark fermentation. This study evaluated the co-fermentation of vinasse and molasses for hydrogen production using the Simplex Lattice mixture design. Batch tests were conducted to analyze the interactive effects between vinasse (V) and molasses (M) and to determine mixture compositions for achieving maximum volumetric hydrogen yield (VHY_CODappl) and production rate (VHPR_CODappl) per applied load in terms of chemical oxygen demand (COD_appl). The tested conditions were 100 % vinasse (V100/M0), 75 % vinasse + 25 % molasses (V75/M25), 50 % vinasse + 50 % molasses (V50/M50), 25 % vinasse + 75 % molasses (V25/M75), and 100 % molasses (V0/M100). Results indicated synergistic interactions, though mixtures with ≥75 % vinasse caused process instability. Reactors achieved carbohydrate removal efficiencies of 46.49–74.75 %, COD removal of 13.49–26.53 %, and volatile solids reduction of 41.58–50.93 %. The V50/M50 condition yielded the highest production potential (3113.27 mL-H₂) and production rate (10.07 mL-H₂/h), with maximum VHY_CODappl of 595.63 mL-H₂/g-COD_appl and VHPRCOD_appl of 50.63 mL-H₂/g-COD_appl/d, statistically similar to V25/M75 and V0/M100. Significant quadratic models (p ≤ 0.05) with strong fits (R² = 0.98 for H₂ yield and R² = 0.92 for H₂ production rate) were observed. Butyric acid was the primary metabolite, supporting hydrogen production through this route.