Fe(III)-dependent Nrf activity determines nitrate reduction partitioning in nitrate-reducing communities

Identifying the factors that affect the nitrate reduction partitioning between dissimilatory nitrate reduction to ammonium (DNRA) and denitrification is crucial for mitigating nitrogen loss in ecosystems. Conventionally, the nutrient status of the environment (e.g., the carbon-to-nitrogen ratio) is recognized as the key determinant of nitrogen conversion pathways. Here, we report that the availability of Fe(III) regulates the nitrate reduction partitioning in Geobacter metallireducens and Alcaligenes faecalis co-culture. We controlled the availability of Fe(III) in the coculture medium and tracked nitrogen conversion dynamics and community composition. The results demonstrated that the coculture performed DNRA, contributed mainly by G. metallireducens under Fe(III)-replete conditions, while performing interspecies synergistic denitrification between both species under Fe(III)-depleted conditions. Nitrate/nitrite reductase activity calculations and mutation analyses indicated that nitrate reduction partitioning in the coculture was governed by the nitrite reductase (Nrf) activity of G. metallireducens, which was Fe(III)-dependent. Further validation in urban river water confirmed that Fe(III) supplementation significantly enhances DNRA activity. Our findings establish Fe(III) as a previously unrecognized regulator of microbial nitrogen retention, showing insights into strategies for managing nitrogen fluxes in agricultural and aquatic systems.IMPORTANCENitrogen is essential for life, but its loss from ecosystems through microbial processes like denitrification harms agricultural productivity and contributes to greenhouse gas emissions. Retaining nitrogen as ammonium via microbial dissimilatory nitrate reduction to ammonium (DNRA) could mitigate these issues, but the factors governing microbial prioritization of DNRA over denitrification remain unclear. Our study reveals that Fe(III) plays a critical, previously unrecognized role in steering this process. We show that Fe(III) availability determines whether the nitrate-reducing community conserves nitrogen as ammonium or releases it as gas, with implications for managing nitrogen in soils and waterways. By demonstrating Fe(III)'s ability to enhance nitrogen retention in environmental systems like urban rivers, our findings offer a new lever for sustainable agriculture and pollution control. This work bridges microbial ecology and environmental management, highlighting how trace metals shape nutrient cycles in ways that can be harnessed to protect ecosystem health.