Isotopic and genomic interrogation unravels the sustaining mechanism of nitrate-dependent Fe(II) oxidation via organic carbon-driven internal iron cycle.

Nitrate-dependent Fe(II) oxidation (NDFO) offers a promising approach for wastewater denitrification, but its sustainability is constrained by substantial Fe(II) demand and the resultant iron encrustation. By harnessing organic carbon in low C/N wastewater to drive dissimilatory Fe(III) reduction, this study establishes a self-sustaining internal iron cycle that overcomes this limitation. The organic carbon initiates the N/Fe co-metabolic cycle by reducing nitrate and Fe(III) to produce nitrite and Fe(II). The regenerated Fe(II) subsequently supports microbially mediated nitrite reduction to N2, completing the cycle. Without external Fe(II) supplementation, the system achieved 96% total nitrogen removal at a C/N ratio of 2.0, sustained by dynamic Fe(II)/Fe(III) transformations. Integrated DNA-SIP and metagenomic analyses revealed that organic carbon reshaped the microbiome, shifting functional dominance from autotrophic Thiobacillus to mixotrophic Thauera, which possesses genetic potential for both denitrification and Fe(III) reduction. This shift established a narG-nirS-dominated denitrification pathway, a genomic feature associated with high system performance. Furthermore, organic carbon alleviated iron encrustation and enhanced iron availability, with siderophore-related genes showing increased abundance, suggesting a potential role in reducing crust formation on cell surfaces. Overall, this study demonstrates that the inherent organic carbon in low C/N wastewater serves as a functional resource to drive a self-sustaining iron cycle, mitigating encrustation and eliminating external Fe(II) dependence for sustainable NDFO.