A Novel Resazurin-Loaded Electroactive Hydrogel Enhanced Methane Production in Anaerobic Digestion by Triggering Dual Electron Transfer Pathways and Metabolic Regulation

Anaerobic digestion (AD) of waste-activated sludge (WAS) is regarded as an attractive strategy for simultaneously achieving WAS minimization and bioresource recovery, but the unsatisfactory extracellular electron transfer (EET) efficiency and poor syntrophic metabolism limit the biosynthesis of methane. In this study, a novel resazurin-loaded conductive hydrogel (RZ/PANI) was prepared to play the role of a "biological capacitor" and as an electron shuttle to facilitate electron exchange among microorganisms. The accumulative methane yield was significantly increased by 141% after adding RZ/PANI. Analysis of the variations in the key enzyme activities, electron transfer system activities, and concentrations of intermediates (e.g., fatty acids and cytochrome C, which play an important role in cellular respiration) revealed that RZ/PANI could enhance acidification and methanogenesis via strengthening both direct interspecies electron transfer (DIET) and mediated interspecies electron transfer (MIET) processes involved in intracellular respiration, NAD+/NADH conversion, and ATP synthesis, but had limited effects on the solubilization and hydrolysis stages. Theoretical calculations demonstrated that PZ/PANI primarily improved the thermodynamic efficiency related to DIET-based methanogenesis and sustained much higher electron transfer fluxes of both DIET and MIET pathways. Microbial analysis demonstrated that the abundance of acid-producing bacteria and electroactive methanogens as well as key functional gene prediction in fatty acid oxidation and hydrogenotrophic and acetotrophic metabolism were significantly enhanced by RZ/PANI due to the establishment of concurrent direct interspecies electron transfer and mediated electron transfer. This study provided an effective and economically attractive strategy to stimulate multiple electron transfer behaviors between fermentative bacteria and methanogens for enhancing AD efficiency and to guide the rational design of EET-related electroactive materials.