Microbiome mechanisms of composite carbon sources for enhancing denitrification and reducing N₂O emissions

Biological nitrogen removal in wastewater treatment plants （WWTPs） is commonly limited by insufficient influent carbon sources， thus making external carbon addition an important strategy for enhancing denitrification. However， conventional single carbon sources often fail to meet the metabolic demands of complex microbial communities， resulting in reduced nitrogen removal efficiency and stability. Composite carbon sources， by providing multiple electron donors， can improve metabolic cooperation among microorganisms， yet their underlying microbial mechanisms are still insufficiently understood. In this study， activated sludge from a municipal wastewater treatment plant 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 the microbial community structure and functional gene expression under composite carbon source conditions. The results showed that， compared with sodium acetate as the single carbon source， the composite carbon source system increased the denitrification rate from （6.822±0.141） mg/（L·h） to （8.370±0.186） mg/（L·h）， 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， thereby enhancing the supply of electron donors and improving denitrification efficiency. These findings indicate that composite carbon sources synergistically enhance denitrification performance by regulating microbial community structure and functional gene expression， providing a theoretical basis for carbon source optimization in WWTPs.