A novel hydrolase biomanufacturing-driven strategy for boosting production of volatile fatty acids and vivianite in iron-rich waste activated sludge fermentation

Efficient sludge pretreatment methods that minimize reliance on costly chemical or energy inputs have garnered significant attention, as waste-activated sludge (WAS) disposal occupied nearly 50% of operational expenses in wastewater treatment plants (WWTPs) which displayed a poor sustainability in current practices. In this study, a novel strategy utilizing bio-manufactured hydrolases (primarily protease and α -amylase, produced by Aspergillus oryzae based on waste molasses cultivation) was proposed to enhance the solubilization of iron-rich WAS by releasing biodegradable organic matters (a net SCOD of 840 ± 14 mg/L release after 8-h) and disrupting extracellular polymeric substances (EPS) via loosing EPS proteins structure and increase hydrophilicity. The VFAs production reached a peak level of 4380 ± 24.6 mg COD/L, accompanied by an optimal orthophosphate release of 7.79 ± 0.31 mg/L through fermentation enhanced by enzymatic pretreatment. Moreover, the relative fraction of P in vivianite, assessed as non-apatite inorganic phosphorous (NAIP), increased by 10.12%. The bio-manufactured hydrolases not only enhanced overall microbial diversity, but also enriched key microbial populations–including hydrolyzing bacteria (e.g. Chloroflexi and Actinobacteria ), major acidogens (e.g. Petrimonas , Jeotgalibaca , Proteiniclasticum , and Macellibacteroides ), and iron-reducing genera (i.e. Ercella and Desulfovibrio ). Furthermore, this strategy upregulated the relative abundance of functional genes related to carbohydrate and amino acid metabolism, and reduced the competitive interference of Ca 2 + ions on soluble phosphorus availability by favoring Fe(II)-P complexation, thereby synergistically enhancing volatile fatty acids (VFAs) production and vivianite formation. This study presents an efficient, economically favorable pretreatment strategy to enhance the recovery of carbon (C) and phosphorus (P) from iron-rich WAS, demonstrating strong application sustainability. • Hydrolase was bio-manufactured by inoculating Aspergillus oryzae using waste molasses. • Hydrolase triggered solubilization of iron-rich WAS via loosing protein structure of EPS. • Hydrolase boosted microbial metabolic capability for VFAs production. • Ercella and Desulfovibrio contributed to Fe(III) reduction for vivianite formation.