Biofuels such as n‐butanol have gained significant attention as sustainable alternatives to depleting fossil fuels and as a means to reduce greenhouse gas emissions. Notably, n‐butanol offers several advantages, including a high octane number (96), a calorific value of 33 MJ/kg, low moisture content, an air-to-fuel ratio comparable to gasoline, and an appropriate specific gravity, which together underscore its compatibility and economic viability as a biofuel. Lignocellulosic biomass - sourced from agricultural residues (e.g., corn stover, rice straw), industrial byproducts (e.g., spent grains from brewing and distilleries), and municipal waste (e.g., kitchen waste, paper, wood) - is rich in cellulose and hemicellulose. These polymers can be rendered accessible via pretreatment under acidic or alkaline conditions, followed by enzymatic hydrolysis with a blend of hemicellulases and cellulases that cleaves glycosidic linkages to release fermentable sugars. In this study, metabolically engineered strains of Clostridium tyrobutyricum (∆cat: adhE2, and ∆ack: adhE2) were utilized to ferment hydrolysates derived from various wastes, thereby enhancing the metabolic flux toward n‐butanol production. This innovative waste valorization approach not only contributes to sustainable energy generation but also addresses the growing energy demand and environmental pollution concerns.
