Ferric iron enhances granule densification and pollutant removal in microalgal-bacterial granular sludge via iron-homeostatic microbial rewiring

Microalgal-bacterial granular sludge (MBGS) is a promising low-energy wastewater treatment technology, but the role of Fe³⁺ concentration in regulating its structure and function remains unclear. MBGS was cultivated under four Fe³⁺ concentrations (0.1, 1, 3, and 5 mg/L). At 5 mg/L Fe³⁺, granules became more compact, with SVI₅ decreasing from 80.0 to 60.1 mL/g and average size increasing from 1.72 to 1.90 mm. These changes were accompanied by enhanced extracellular polymeric substances (EPS) hydrophobicity despite reduced EPS production. The 5 mg/L treatment also increased dark-cycle COD and PO₄^3--P removal, while NH₄⁺-N removal remained unchanged. At 5 mg/L Fe³⁺, Thauera and iron-associated Rubrivivax formed a synergistic consortium, consistent with the superior dark-cycle pollutant removal. Metagenomic analysis revealed limited variation in C/N/P metabolic genes but strong iron-related regulation, with fur decreasing by 40.4% at 5 mg/L relative to the initial inoculum, suggesting a community shift toward iron-homeostatic configuration rather than a stress response. Four metagenome-assembled genomes (MAGs (Thauera mechernichensis MAG43, Rubrivivax sp. MAG118 and MAG36, and Microcoleus sp. MAG30) showed distinct C/N/P/Fe gene distributions, reflecting functional differentiation among community members. These findings provide a mechanistic basis for optimizing iron supplementation strategies to enhance the stability and performance of MBGS in practical applications.