Fe(II)-driven abiotic-biotic relay alleviates denitrification bottleneck via chemical nitrite reduction and...

The coexistence of iron and nitrate (NO₃^-) in natural and engineered environments invites complex abiotic and biotic interactions, yet how such abiotic-biotic synergies operate under fluctuating carbon availability and how light modulates them remain poorly resolved. Using a nitrate-reducing, nitrite-accumulating enrichment culture derived from lake sediment, we uncovered a synergistic abiotic-biotic relay that overcame the kinetic bottleneck of denitrification. During initial heterotrophic denitrification of 2 mM NO₃^-, 85.10-89.72% of the substrate was accumulated as NO₂^-. In contrast, ferrous iron (Fe(II)) amendment triggered subsequent iron-dependent nitrate reduction (IDNR) and significantly reduced NO₂^- accumulation. Abiotic controls confirmed that Fe(II) chemically reduced the accumulated NO₂⁻ to the downstream products. In parallel, metagenomic and metatranscriptomic analyses of the bioactive samples demonstrated that these gaseous intermediates (e.g., NO, N₂O) were enzymatically reduced to N₂ based on upregulated denitrification-associated genes. More importantly, when exogenous acetate was depleted, the community sustained IDNR not through strict autotrophy but via heterotrophic metabolism using intracellular poly-3-hydroxybutyrate (PHB) and microbial necromass as the carbon/energy sources. This metabolic plasticity drove a functional succession from organotrophic denitrifiers (e.g., Pseudomonas) toward PHB- and necromass-utilizing microbial consortia mainly composed of Pseudomonas, Alicycliphilus and some phototrophic populations. Supporting evidence showed that illumination further accelerated the relay via light-driven reactive oxygen species, and secondary iron minerals (e.g., bernalite, lepidocrocite, and goethite) formed as fingerprints of the Fe(II) oxidation. Collectively, this work deciphers a dual-mechanism model, abiotic nitrite reduction followed with endogenous carbon-fueled denitrification, that governed efficient nitrate reduction under carbon-limited conditions. Leveraging such abiotic-biotic relays offers promising strategies for sustainable nitrogen removal in both natural and engineered systems.