Fungi-Scrap Iron Filings Mixotrophic Denitrification Changed Mechanisms of Regulating Intracellular Metabolic Ecosystems at the Transcriptome Molecular Level: A New Insight into Enhanced Nitrogen Pollution Remediation in Oligotrophic Surface Water

Iron-coupled fungal denitrification provides promising strategies for oligotrophic water remediation, although its molecular drivers remain unclear. This study applied scrap iron filings (SIFs) to enhance aerobic denitrification in three fungi (Penicillium sp. N8, Leptobacillium leptobactrum N9, Aspergillus sp. DH4) at C/N = 2, achieving 38.44–62.95% higher nitrate degradation with optimized nitrogen flux. Kinetics model suggested that culture conditions changes influenced competition between biotic and abiotic reduction in the SIFs-enhanced group. Transcriptomic analysis revealed that SIFs triggered fungal metabolic reprogramming to sustain survival and denitrification. Sugar transport channels were upregulated to enhance organic substrate transport (4–6 upregulated subunits) and activate the Embden–Meyerhof–Parnas pathway and tricarboxylic acid cycle to produce sufficient energy. Concurrently, glutamine synthetases facilitate ammonia assimilation into biomass, supporting long-term fungal viability. SIFs addition also stimulated the divalent iron transporters (0.10–0.97-fold) and facilitated iron transport to synthesize more iron-containing protein active sites in reinforcing electron transport chain and nitrogen metabolism. Crucially, SIFs optimized the allocation of electrons to denitrifying enzymes, minimizing nitrite accumulation. These adaptive mechanisms, including enhanced substrate utilization and metabolic coordination, address survival and denitrification challenges in oligotrophic conditions. This study elucidated fungal denitrification strategies under iron additions, providing a mechanistic foundation for optimizing sustainable nitrogen removal technologies.