Unlocking the hidden carbon pool: Refractory organic matter drives superior chain elongation in sludge alkaline fermentation liquid.

Converting waste activated sludge (WAS) into medium-chain fatty acids (MCFAs) via chain elongation (CE) offers a promising route for sludge valorization. In two-stage sludge CE systems, primary fermentation is typically optimized to maximize the short-chain fatty acid (SCFA) pool for downstream MCFA production; however, whether retained refractory dissolved and undissolved organic matter (rDOM and rUOM) also contributes to CE remains unclear. Here, we evaluated the roles of rDOM and rUOM in ethanol-driven CE using sludge alkaline fermentation liquid (SAFL) and thermal-alkaline pretreatment fermentation liquid (STAPFL) as feedstocks. Although SAFL contained fewer SCFAs after primary fermentation than STAPFL (3.25 vs. 3.60 g COD/L), it yielded 44% more MCFAs during CE (11.56 vs. 8.03 g COD/L). Integrated physicochemical and molecular analyses indicated that this advantage arose from greater retention of refractory organics during primary alkaline fermentation and their continued mobilization during downstream CE. Filtration experiments and COD-based estimation indicated a much greater total apparent COD contribution from retained rUOM and rDOM in SAFL than in STAPFL (2.16 vs. 0.15 g COD/L). FT-ICR-MS and metagenomic analyses further suggested compositional transformation of retained refractory organics and stronger functional potential for coordinated hydrolysis, acidogenesis, and CE in SAFL, which together supported continued precursor supply and higher MCFA production. These results indicate that downstream MCFA production in real sludge fermentation liquids depends not only on the initial soluble SCFA pool, but also on the continued mobilization of retained refractory carbon during CE. This study advances understanding of retained refractory carbon utilization during ethanol-driven CE in two-stage sludge fermentation for MCFA production.