Electro-Stimulated Dual-Species Catalysis Enables CO₂ Fixation Toward Selective 1,4-Butanedioic Acid Biosynthesis

Succinic acid (SA)/1,4-Butanedioic acid is a key platform-chemical with broad industrial relevance, yet its biocatalytic production is constrained by redox imbalance, by-product accumulation, and limited CO₂ sequestration. This study overcomes the above limitations and enhanced the SA production (0.6 g g^-1; 6 g L^-1) by dual-species catalysis using Citrobacter amalonaticus (NCIM 5782, CA) and Bacillus subtilis (BS, NCIM 5781), in a bioelectrocatalytic system. Comprehensive gene-expression-profiling revealed upregulation of phosphoenolpyruvate carboxylase (ppc/PPC) in CA and pyruvate carboxylase (pyc/PYC) in BS, reflecting intensified carboxylation activity via the reductive tricarboxylic acid pathway. Protein/structural modeling/docking of PPC and PYC demonstrated enhanced catalytic-site exposure under electro-fermentative co-culture conditions, correlating with greater carboxylation efficacy. Notably, beyond extracellular-CO₂ fixation, intracellular-CO₂ sequestration is also evident, as indicated by a marked enrichment of H₂ in the biogas produced during dual-species-catalysis compared to monoculture systems. Thermodynamic and electrochemical evaluation indicated greater stability and electron flow in co-culture reactors (R9:ΔG = -42.29 kJ) compared to monocultures. Collectively, this study presents a scalable hybrid bioelectrochemical strategy leveraging species-specific metabolic roles and electron-steering to facilitate high-yield, selective SA production, offering a promising blueprint for sustainable carbon-based biomanufacturing.