Microbiome Mechanisms of Composite Carbon Sources for Enhancing Denitrification and Reducing N2O Emissions
Biological nitrogen removal in wastewater treatment plants (WWTPs) is often limited by insufficient influent carbon sources, necessitating external carbon addition to enhance denitrification. Conventional single carbon sources, such as sodium acetate, frequently fail to meet the metabolic demands of complex microbial communities, compromising nitrogen removal efficiency and stability. Composite carbon sources, by providing multiple electron donors, can improve metabolic cooperation among microorganisms, yet their underlying microbial mechanisms remain insufficiently understood. In this study, activated sludge from a municipal WWTP was used to investigate the microbial mechanisms of composite carbon sources during denitrification. Batch denitrification experiments were conducted in combination with metagenomic and metatranscriptomic analyses to systematically characterize microbial community structure and functional gene expression under different carbon source conditions. Results showed that, compared with sodium acetate as the single carbon source, the composite carbon source system (sodium acetate: sodium succinate: ethanol = 2:1:3) increased the denitrification rate from (6.822 ± 0.141) mg/(L·h) to (8.370 ± 0.186) mg/(L·h), representing a 22.7% improvement, while reducing N2O accumulation by approximately 55%. Metagenomic analysis revealed that Ottowia, Rubrivivax, Thauera, and Zoogloea were the dominant denitrifying genera. Metatranscriptomic results further demonstrated that the composite carbon sources significantly upregulated the transcription of key denitrification genes, with nirS, norB, and nosZ increasing by 37.8%, 27.4%, and 48.6%, respectively. In addition, the composite carbon sources promoted complementary carbon metabolic strategies among different microbial communities, enhancing electron donor supply and improving denitrification efficiency. These findings indicate that composite carbon sources synergistically enhance denitrification performance through regulation of functional gene transcription in complex microbial communities, providing a theoretical basis for carbon source optimization in WWTPs.