Metabolic and ecological responses of denitrifying consortia to different carbon source strategies under fluctuating C/N conditions.

Frequent fluctuations in the carbon-to-nitrogen (C/N) ratio of urban wastewater influent can undermine denitrification performance, posing challenges for stable nitrogen removal. Although supplying additional carbon sources is a recognized strategy to mitigate these issues, the underlying microbial interactions and metabolic reconfigurations triggered by changing C/N ratios remain incompletely understood. Here, we employed methanol, glycerol, sodium acetate, and glucose in long-term denitrification reactors and integrated denitrification kinetics, 16S rRNA gene amplicon sequencing, metagenomic binning, and metabolic modeling to elucidate how these systems respond to a declining C/N ratio. Our results show that lower C/N ratios diminished denitrification efficiency in all treatments, with each carbon source eliciting distinct shifts in microbial assemblages. Fluctuations in the C/N ratio determine the extent of directional selection of microbial communities based on carbon source metabolism and induce significant changes in non-dominant microorganisms. Throughout the process, the synthesis potential of PHA is closely linked to the system's ability to withstand fluctuations. Notably, metabolic modeling indicated that heightened tricarboxylic acid (TCA) cycle activity in the methanol- and glucose-fed communities was associated with suboptimal nitrogen removal. These findings offer novel insights into the metabolic and ecological mechanisms governing carbon source-driven denitrification under fluctuating C/N conditions, providing a valuable framework for optimizing nitrogen removal in urban wastewater treatment systems.