pH-dependent medium-chain fatty acid synthesis in waste activated sludge fermentation: Metabolic pathway regulation.

Transforming waste activated sludge (WAS) into medium-chain fatty acids (MCFAs) via chain elongation (CE) technology is sustainable, yet pH effects on this process are poorly understood. In this study, semi-continuous flow experiments demonstrated that WAS degradation was highest under alkaline pH (10) but unsuitable for CE. Continuous output of MCFAs indicated that CE could be successfully performed under acidic pH (5) and neutral pH (7). Moreover, neutral pH optimized MCFAs production, achieving higher MCFAs yield (8.9 ± 1.2 g COD/L), MCFAs selectivity (51.2 ± 7.3%), and WAS degradation (25.4 ± 0.4%) than acidic pH. Further metagenomic and metatranscriptomic analysis revealed that the reverse β-oxidation cycle was the primary CE pathway. The absence of CE-related microorganisms and enzymes under alkaline pH hindered MCFAs synthesis, while under acidic pH, carboxylate accumulation may reduce cellular protection capabilities and affect energy metabolism, thereby inhibiting anaerobic fermentation. Conversely, neutral pH enhanced amino acid and butyrate metabolic pathways, facilitating WAS degradation and SCFAs production, providing precursor substrates for MCFAs production. Additionally, neutral pH promoted the enrichment and activity of CE-related microorganisms and enzymes, contributing to the accumulation of high-concentration MCFAs. Notably, Clostridium_kluyveri and Sporanaerobacter_acetigenes were key CE-functional bacteria at neutral pH.