Co-treatment with free nitrous acid and calcium peroxide regulates microbiome and metabolic functions of acidogenesis and methanogenesis in sludge anaerobic digestion

Wasted activated sludge (WAS) is a promising feedstock for carbon management because of its abundance and carbon-neutral features. Currently, the goal is to maximize the energy in WAS and avoid secondary toxic effects or accumulation of harmful substances in the environment. Chemical pretreatment is an effective strategy for enhancing WAS disintegration and production of short chain fatty acids (SCFAs). However, the role of pretreatment in shaping the core microbiome and functional metabolism of anaerobic microorganisms remains obscure. Here, the mechanisms of SCFA synthesis and microbiome response to free nitrous acid (FNA) and calcium peroxide (CaO₂) co-treatment during sludge anaerobic digestion (AD) were investigated. The combination of FNA and CaO₂ enriched acidogenic Macellibacteroides, Petrimonas, and Sedimentibacter to a relative abundance of 15.0%, 10.3%, and 7.3%, respectively, resulting in an apparent increase in SCFA production. Metagenome analysis indicated that FNA + CaO₂ co-treatment facilitated glycolysis, phosphate acetyltransferase-acetate kinase pathway, amino acid metabolism, and acetate transport, but inhibited CO₂ reduction and common pathway of methanogenesis compared with the untreated control. This work provides theoretical insights into the functional activity and interaction of microorganisms with ecological factors.