Coordinating substrate accessibility with indigenous microorganisms functionality to enhance medium-chain fatty acid production from waste activated sludge

Efficient medium-chain fatty acid (MCFA) production from waste activated sludge (WAS) depends on both substrate accessibility and microbial conversion. To clarify their roles in chain elongation (CE), three pretreatments were evaluated in a two-stage fermentation system with indigenous microorganisms retained or inactivated. Thermal-alkaline pretreatment achieved the highest MCFA production (4973.9 ± 274.2 mg COD/L) by improving substrate accessibility, although its relatively low selectivity indicated that greater precursor availability did not necessarily favor carbon allocation to MCFA. Retaining indigenous microorganisms increased MCFA production by 35.8-76.9% compared with corresponding autoclaved reactors and altered ethanol consumption and short-chain fatty acid dynamics, suggesting their involvement in intermediate utilization. Community analysis associated effective CE with complementary hydrolytic, acidogenic, and chain-elongating populations, including Macellibacteroides, Romboutsia, Clostridium, and Caproiciproducens, rather than the enrichment of chain-elongating bacteria alone. Metagenomic analysis of the thermal-alkaline system indicated differences in genetic potential for substrate degradation, glycolysis, electron transfer, acetyl-CoA formation, and reverse β-oxidation. These results suggest that coordinating pretreatment-driven substrate supply with indigenous microbial conversion capacity is essential for improving sludge-derived MCFA production.