Effect of iron-loaded biochar on medium-chain fatty acid production from waste activated sludge.

Conductive materials can enhance medium-chain fatty acid (MCFA) production from waste activated sludge (WAS), but the influence of their addition timing remains unclear. Here, a biochar-supported zero-valent iron composite (ZVI/BC) was added either before acidification (the AC group) or before chain elongation (CE; the CE group) in a two-stage anaerobic fermentation system. Adding ZVI/BC before acidification yielded the highest caproate production of 10.0  g/L as chemical oxygen demand, 64.5 % and 119.5 % higher than those in the control and CE groups, respectively. This advantage was accompanied by marked extracellular polymeric substance (EPS) restructuring, including divergent changes in soluble chemical oxygen demand and total organic carbon in the tightly bound fraction and enrichment of soluble microbial byproduct-like and humic-like substances. Microbial analysis further showed that early ZVI/BC addition was associated with a distinct community, with higher relative abundances of Firmicutes and the genera Romboutsia, Paraclostridium, and Clostridium_sensu_stricto in the AC group. Meanwhile, cytochrome c and reduced nicotinamide adenine dinucleotide increased by 10.5 % and 14.0 %, suggesting endpoint changes in redox-related components and intracellular reducing-power availability. Functional prediction indicated higher acetyl coenzyme A-related precursor potential in the AC group. Overall, adding ZVI/BC before acidification was more effective than adding it before CE, providing a practical strategy to improve caproate production from WAS.