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 $\alpha$-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 soluble chemical oxygen demand (COD) of 840 ± 14 mg/L release after 8 h] and disrupting extracellular polymeric substances (EPS) via loosing EPS proteins structure and increase hydrophilicity. The volatile fatty acids (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 such 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 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, with demonstrating a strong application sustainability.