Enhancing butanol fermentation via improved interspecies electron transfer in a Clostridium syntrophic co-culture using a biochar-methyl viologen composite

Anaerobic fermentation suffers from unavoidable carbon loss due to pyruvate decarboxylation during glycolysis, imposing a stoichiometric upper limit on carbon recovery and product yield. Anaerobic non-photosynthetic mixotrophy (ANPM) using syntrophic Clostridium co-cultures enables the re-assimilation of CO2 and reducing equivalents through the metabolic coupling of sugar fermentation and gas-fixing metabolism; however, its efficiency remains constrained by inefficient, contact-dependent interspecies electron transfer. Here, we report a hybrid biochar-methyl viologen (BM) composite designed to function as a synthetic electron transfer interface to enhance microbial cooperation in a C. acetobutylicum–C. ljungdahlii co-culture system. The BM composite is designed to combine conductive pathways and interfacial redox activity, providing a biocompatible scaffold for microbial attachment and indirect electron exchange without continuous cell–cell contact. The redox mediator is effectively confined within the composite, exhibiting minimal leaching (<0.002% over 7 days). The BM-assisted system achieved a butanol yield of 0.39 g g−1, the highest reported to date for fermentative butanol production, with carbon recovery reaching 90.32%. Butanol titers reached 17.07 ± 0.06 g L−1 at 37 °C and 19.99 ± 0.16 g L−1 at 25 °C, representing substantial improvements over conventional fermentation. This work demonstrates that engineered conductive-redox materials can overcome carbon loss in fermentation, providing a practical strategy for carbon-efficient and high-yield biofuel production.