Regulation of biochar-to-iron ratio on synergistic enhancement mechanisms of biochar-iron composites for chain elongation and elucidation of electron transfer pathways

Medium-chain fatty acids, as high-value bio-based chemicals, have their biosynthesis efficiency limited by low electron transfer efficiency in chain elongation reactions and performance bottlenecks of functional materials. In this study, biochar-supported nanoscale zero-valent iron composites with different mass ratios of biochar to iron were prepared to systematically investigate their enhancing effects on CCE reactions in an ethanol-acetate system, and the core mechanisms were analyzed by combining metagenomics and electron transfer inhibitor experiments. The results showed that the composite with a biochar-to-iron ratio of exhibited optimal performance at a dosage of 10 g/L, with caproate production reaching 11787.3 mg/Land a carbon conversion rate of 76.1%, which were 101.5% and 38.3% higher than those of the control group,respectively, significantly outperforming single BC or nZVI. Material characterization indicated that it formed a stable interface through Fe-O-C bonds, inhibited nZVI agglomeration, and enhanced redox activity .Metagenomic analysis revealed that this composite significantly enriched functional bacteria of the phylum Bacillota , upregulated key genes involved in acetyl-CoA generation and reverse oxidation pathways, and enhanced the expression of genes related to cytochrome c, e-pili, and electron transfer flavoproteins. After innovatively introducing 2-mercaptoethanol to inhibit endogenous electron transfer,it could recover caproate production by 25.6%, directly confirming its core role in alleviating metabolic blockage by mediating electron transfer. This study is the first to clarify the synergistic mechanism of biochar-to-iron ratio regulation on CCE, providing experimental basis for the rational design of high-efficiency biochar-iron composites and the regulation of electron transfer in medium-chain fatty acid biosynthesis.