Soy-Derived Agroindustrial Byproducts as Sustainable Substrates for Biohydrogen Production via Dark Fermentation

The valorization of byproducts from biofuel production is crucial in advancing sustainable energy solutions. Particularly in biodiesel processes, byproducts such as glycerin, soapstock, and wastewater with high Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD) contain rich organic content suitable for biological conversion. Among these, soybean molasses and soapstock stand out as cosubstrates for dark fermentation to produce biohydrogen. This study aimed to obtain a new microbial consortium from the sludge of an anaerobic reactor thermally pretreated at 80 °C for 60 min and its use in bioH₂ production in a culture medium composed of soybean molasses diluted in a pretreated lysogoma. All experiments were conducted at bench scale under batch conditions. The physicochemical parameters (soybean molasses concentration, pH, and temperature) were optimized using Central Composite Rotatable Design (CCRD) and response surface analysis. The profiles of bioH₂ production, sugar consumption, and volatile fatty acid production were evaluated through kinetic analysis under optimal conditions. The kinetic analysis of the points 0, 26, and 54 h was also used to verify the consortium's behavior throughout dark-fermentation. <i>Clostridium butyricum</i> was the predominant species in the initial consortium and throughout fermentation, indicating the success of the inoculum's thermal pretreatment method, which resulted in its enrichment with spores of this microorganism, widely used in dark-fermentation trials. Sucrose was consumed as the preferred sugar, yielding acetic and butyric acids as fermentation byproducts. The maximum bioH₂ production was achieved after 48 h of fermentation, resulting in a gas composed of H₂ (57.14%) and CO₂ (42.86%), culminating in a yield of 1.6 L of bioH₂/L of culture medium. These findings underscore the viability of soybean byproducts as sustainable feedstocks for biological hydrogen production, contributing to circular bioeconomy strategies and renewable energy development.