A bio-nonbio hybrid for synergistic nitrogen removal and N₂O mitigation in aquaculture wastewater

Conventional heterotrophic denitrification is often associated with high energy consumption and substantial N₂O emissions, limiting the sustainability of biological nitrogen removal. In this study, a bio-nonbio hybrid was developed by integrating Comamonas testosteroni FJ17 with biologically-derived carbon quantum dots (bio-CQDs) wherein bio-CQDs act as regulatory molecule, to achieve synergistic denitrification and greenhouse gas mitigation in aquaculture wastewater. GC-MS and nitrogen mass balance analyses demonstrated that bio-CQDs reduced N₂O emissions by 21.12%. Mechanistic analysis revealed that bio-CQDs facilitated extracellular electron transfer by reducing charge transfer resistance, enhancing the activity of the electron transport chain, and increasing cytochrome c levels, thereby enhancing electron transfer from donors to acceptors. This process further stimulated cellular energy metabolism and the secretion of extracellular polymers (EPS), triggering the formation of protein-rich matrix structures, and enhanced microbial aggregation, which in turn accelerating electron transfer and facilitating the reduction of N₂O to N₂. Under continuous-flow reactor operation (HRT = 16 h, DO = 3.0 mg·L^-1), the hybrid consistently achieved Class IA effluent standards using real aquaculture wastewater. Overall, this study elucidates the dual regulatory and adaptive response role of bio-CQDs in microbial denitrification and provides a scalable, low-carbon strategy for energy-efficient nitrogen removal and greenhouse gas emission reduction.