Food waste (FW) has been considered a potential carbon reservoir and can be used for high-value volatile fatty acid (VFA) production via anaerobic fermentation. Nevertheless, more work is needed to unravel how to regulate the structural defects of FW and enhance microbial metabolisms for improving VFA production through pretreatment methods. This work investigated the enhancement of VFA production from FW fermentation through enhancing depolymerization and stimulating microbial traits via enzymatic pretreatment. The results exhibited that, after pretreatment, VFA production was improved by 50.1% compared to the unpretreated group, and the concentrations of bioavailable substrates (e.g., reducing sugar and soluble chemical oxygen demand) increased significantly. Further analysis proved that the enzymatic pretreatment could highly reduce the particle sizes of FW by 69.3% and thus promoted the solubilization of substrates and lipids. Meanwhile, the ratio of α-helix/(β-sheet + random coil) in the protein secondary structure of FW decreased from 0.32 to 0.23 after enzymatic pretreatment, indicating that the pretreatment process accelerated the FW depolymerization. Furthermore, the organic substrates after the pretreatment were favorable for enriching the bacteria involved in hydrolysis and acidification (e.g., Olsenella sp. and Sporanaerobacter sp.), upregulating the gene expressions involved in hydrolysis, pyruvate metabolism, and fatty acid biosynthesis (e.g., malZ, PK, accA, and accC), and stimulating the enzymatic activities involved in acetate and butyrate formation (e.g., AK, PTA, BK, and PTB), which was in accordance with the enhanced VFA generation from FW fermentation via enzymatic pretreatment.

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