Biochar enhance biobutanol production by modulating redox cofactors and allocating reducing power.

Acetone-butanol-ethanol (ABE) fermentation is a promising route for converting carbohydrate-rich feedstocks into biobutanol, but its efficiency is fundamentally constrained by the inefficient intracellular allocation of reducing power. Biochar addition can improve fermentation performance, but the mechanisms underlying this enhancement remain insufficiently resolved. Here, we investigated the fermentation performance, intracellular redox cofactors, and proteomic responses of Clostridium acetobutylicum to corn straw biochar prepared at different pyrolysis temperatures. We found that the pyrolysis temperature of the biochar (optimally 600℃) significantly dictated the enhancement of biobutanol production. Separation of water-soluble organic carbon from bulk biochar showed that the soluble fraction alone did not explain the enhancement. Comparable butanol production in direct-contact and Transwell systems further showed that biochar-cell contact was unnecessary. Additional experiments with acid-pretreated and oxygen-oxidized biochar suggest that extracellular environmental regulation may contribute to the enhanced butanol production. Intracellularly, biochar upregulated enzymes involved in NAD biosynthesis, glycolysis, acid reassimilation, and solventogenesis. This metabolic shift successfully redirected carbon flux and reducing equivalents away from competing pathways, such as hydrogen evolution, and channeled them toward solvent biosynthesis, ultimately increasing biobutanol production by 30%. Based on these mechanistic insights, we propose a compartmentalized biochar unit design for scalable implementation. These findings bridge the fundamental knowledge gap regarding biochar-mediated redox regulation and offer a robust, scalable engineering strategy for optimizing anaerobic bioproduction.