Au–Pd Nanoalloy-Catalyzed Intracellular Reducing Power Regeneration to Boost the Biohydrogen Production in a Biohybrid System

Efficient coenzyme regeneration in biohybrids can help overcome the challenge of insufficient reducing power in biohydrogen production, but the performance of biohybrids is often hampered by light-dependent and inefficient photoelectron transmembrane transfer. Here, we present an intracellular hybrid system composed of gold–palladium nanoalloys and Clostridium butyricum, which demonstrates efficient dark-catalyzed coenzyme regeneration, thereby enhancing hydrogen production capabilities. By utilizing triethanolamine (TEOA) as the electron donor, the hybrid system achieved a maximum hydrogen production of 2.14 mol of H₂·mol^–1 glucose, resulting in a remarkable increase of 47.37%. The Au–Pd nanoalloy regenerated intracellular NADH through chemical catalysis with TEOA as the electron donor, which was confirmed by increased reducing power levels and pronounced peak currents. Consequently, the hybrid system had a higher reducing power level, which enhanced the hydrogen-producing activity of the pyruvate formate-lyase (PFL) and NADH-ferredoxin oxidoreductase (NFOR) pathways. The PFL pathway oxidizes pyruvate, while the NFOR pathway directly oxidizes NADH. Pyruvate, a substrate required for hydrogen production in the PFL pathway, is generated through a combination of glucose phosphate transfer and phosphoenolpyruvate (PEP) dephosphorylation. This study offers theoretical guidance for the development of a dark hybrid system of nanocatalysts and microbes that can effectively produce biohydrogen and be used for other applications.