Innovative application of CaO2 in two-phase anaerobic digestion to enhance methane production from waste activated sludge: Condition optimization and mechanistic insights

Abstract

Calcium peroxide (CaO₂) treatment can effectively overcome the rate-limiting steps in the anaerobic digestion (AD) of sludge, leading to the production of high-quality short-chain fatty acids (SCFAs). However, its application in single-phase AD is limited by the inhibition of the methanogenesis process. In this study, the combination of two-phase AD with CaO₂ treatment was investigated to improve the methane (CH₄) production of waste activated sludge (WAS). The results showed that this synergistic technology not only increased the CH₄ yield and rate but also eliminated the lag phase. Under the same CaO₂ treatment conditions, two-phase AD utilized organic matter more thoroughly in a shorter time than single-phase AD. It was found that 0.1 g/g VSS was the optimal CaO₂ dosage in the two-phase system for CH₄ production. Through metagenomics analysis, two-phase AD enriched Methanothrix (acetoclastic methanogens), Methanobacterium (hydrogenotrophic methanogens), and Candidatus_Methanofastidiosum (methylotrophic methanogens), promoting all four methanogenesis pathways (M00567, M00357, M00356, and M00563). Proteomics analysis revealed that CaO₂ upregulated proteins related to environmental resistance, material transport and CH₄ production, particularly enhancing the expression of proteins such as Mtr, Mcr, and Hdr-Mvh in methanogenic metabolic pathways. This study provides a new application of CaO₂ and develops a promising technology for CH₄ production from sewage sludge.