In situ assembly of the humic acid-protein conductive network facilitates chain elongation for medium-chain fatty acids anaerobic production from waste activated sludge

Biosynthesis of medium-chain fatty acids (MCFAs) from waste activated sludge (WAS) is primarily limited by intracellular reductive stress (NADH accumulation) and energy shortages. This work demonstrates that humic acid (HA), functioning as a redox mediator, effectively enhances the carbon chain elongation (CE) process. Optimal HA supplementation (1000 mg/L) increased the peak MCFAs yield by 98.3%, driving a fundamental shift in the dominant product spectrum from short-chain fatty acids (SCFAs) to MCFAs. Combined metagenomic and electrochemical analyses reveal that this enhancement originates from HA-mediated spatial and metabolic integration across multiple scales. Macroscopically, HA complexes with proteins to construct a conductive biopolymer network. Functioning as a highly efficient extracellular electron sink, this network significantly accelerates transmembrane electron discharge to consume excess intracellular electrons. This rapid electron extrusion alleviates reductive stress and relieves product feedback inhibition on dehydrogenases, concurrently inducing an elevated cellular energy charge (ATP surge). Subsequently, feedback regulation driven by this high-energy state suppresses the competitive acetogenic branch (Pta-ackA pathway), effectively preventing carbon loss. Dominated by the highly enriched CE taxon Candidatus_Microthrix, the microbial consortium exhibits a robust metabolic potential to channel carbon into synergistic RBO and FAB pathways. This metabolic shift, fueled by abundant precursors and energy, effectively circumvents acidic toxicity by rapidly consuming SCFAs. These findings elucidate the critical role of HA in reshaping microbial redox homeostasis, providing a robust mechanistic foundation for high-value carbon recovery engineering from complex solid wastes.