Novel Insights into the Long-Term Thiosulfate Pretreatment for Enhanced Short-Chain Fatty Acids Production from Sludge Anaerobic Fermentation: Organics Transformation, Electron Transfer, and Microbial Cooperation

Thiosulfate-assisted anaerobic fermentation (AF) effectively converts waste activated sludge into high-value products (e.g., short-chain fatty acids (SCFAs)). However, the roles of thiosulfate in organics transformation, electron transfer, and microbial interactions within AF systems are not fully understood, especially under long-term operations. In this study, an 88 day long-term experiment was conducted to address this knowledge gap. The results indicated an average SCFA yield of 3625.1 mg COD/L and an acetate proportion of 49.4% with a thiosulfate dosage of 600 mg S/L. Model organic degradation tests revealed that thiosulfate functioned as an electron acceptor, facilitating NAD⁺/NADH transformation, stimulating the expression of protein complexes like cytochrome c to enhance electron transport, and lowering thermodynamic barriers of propionate and butyrate to acetate (ΔG1_propionate = −335.0 kJ/mol; ΔG2_butyrate = −113.8 kJ/mol). Molecular ecological networks analysis showed that thiosulfate strengthened cooperative relationships among biomarkers of hydrolytic bacteria (i.e., Proteiniphilum, UBA5851), acidogenic bacteria (i.e., UBA4179), and sulfur reducers (i.e., JAEUSI01). Functional gene analysis using random forest confirmed that thiosulfate upregulated the expression of key genes (e.g., 2-oxoacid ferredoxin oxidoreductase) associated with electron transfer and acidogenic metabolism. This study deepens our understanding of thiosulfate, facilitating electron transfer and strengthening microbial cooperation within AF systems.