Novel insights into electron transfer mechanisms in mixotrophic denitrification: A potential quorum sensing-mediated electron acquisition strategy

Mixotrophic denitrification has recently emerged as a promising approach for nitrogen removal from low carbon-to-nitrogen (C/N) ratio wastewater, yet the intrinsic mechanisms underlying electron transfer and interspecies interactions are not fully understood. In this study, mixotrophic systems driven by different biomass-inorganic mineral composites were established. Compared to the Control (68.83% and 0.43 NO₃⁻-N L⁻¹ h⁻¹), the mixotrophic denitrification systems exhibited both superior nitrogen removal (76.80%–93.72%) and higher denitrification rates, ranging from 0.60 to 1.05 mg NO₃⁻-N L⁻¹ h⁻¹. Electrochemical analysis demonstrated efficient extracellular electron transfer (EET) across all mixotrophic systems. This enhanced EET in the sulfur-based system (BS⁰) was attributed to the resilient sulfur cycle and secretion of abundant extracellular polymeric substances (EPS). In contrast, the introduction of iron minerals (in BS⁰P and BS⁰S) triggered a change in electron acquisition strategy, whereby microbes orchestrated by quorum sensing (QS) preferentially utilized thermodynamically more favorable Fe oxidation over S oxidation. QS promoted the expression of outer-membrane cytochrome genes (e.g., mtrABC), enhanced siderophore biosynthesis, and downregulated energy-intensive pili synthesis. Meanwhile, the iron sulfur-based systems developed EPS with stronger adhesion properties, optimizing the cell-mineral interface for electron acquisition. Microbial evidence further revealed the tightly cooperative consortia with strongly connected networks in iron sulfur-based systems, which served as an essential ecological framework for the QS-mediated strategy and provided a necessary interface for electron transfer. This study offers novel insights into the QS-involved electron acquisition strategy and provides theoretical support for the development and application of efficient mixotrophic denitrification processes for low C/N ratio wastewater treatment.