Facilitating Intracellular Electron Bifurcation by Mediating Flavin-Based Extracellular and Transmembrane Electron...

Pyrogenic carbon is considered an enhancer to H₂-yielding dark fermentation (DF), but little is known about how it regulates extracellular electron transfer (EET) and influences transmembrane respiratory chains and intracellular metabolisms. This study addressed these knowledge gaps and demonstrated that wood waste pyrogenic carbon (biochar) could significantly improve the DF performance; e.g., addition of pyrogenic carbon produced by pyrolysis at 800 °C (PC800) increased H₂ yield by 369.7%. Biochemical quantification, electrochemical analysis, and electron respiratory chain inhibition tests revealed that PC800 promoted the extracellular flavin-based electron transfer process and further activated the acceleration of the transmembrane electron transfer. Comparative metagenome/metatranscriptome analyses indicated that the flavin-containing Rnf complex was the potential transmembrane respiratory enzyme associated with PC800-mediated EET. Based on NADH/NAD⁺ circulation, the promoted Rnf complex could stimulate the functions of the electron bifurcating Etf/Bcd complex and startup of glycolysis. The promoted Etf/Bcd could further contribute to balance the NADH/NAD⁺ level for glycolytic reactions and meanwhile provide reduced ferredoxin for group A1 [FeFe]-hydrogenases. This proton-energy-linked mechanism could achieve coupling production of ATP and H₂. This study verified the important roles of pyrogenic carbon in mediating EET and transmembrane/intracellular pathways and revealed the crucial roles of electron bifurcation in DF for hydrogen production.