Impacts of Ammonia Stress and Iron-based Conductive Materials on the Stability of Anaerobic Granular sludges: Compositions, Exoprotein Structures, and Microbial Origins of Extracellular Polymeric Substances

Excessive ammonia in industrial wastewater can lead to the disintegration of anaerobic granular sludge (AnGS) and biogas reduction. While supplementations with nano magnetite (nMag) and nano zero-valent iron (nZVI) have emerged as promising mitigation strategies, the mechanisms underlying AnGS disintegration and recovery remain unclear. This study comprehensively investigates the long-term impacts of ammonia stress (1.5–5.0 g/L) and nMag/nZVI-supplementation on AnGS, and compositions, structures, and microbial origins of extracellular polymeric substances (EPS). Results showed that ammonia stress reduced sludge hydrophobicity and elevated electronegativity, causing loose granular structures. Despite exopolysaccharides (PS) concentrations increased by 13.5–187.8% under ammonia stress, key monosaccharides (fucose, rhamnose, galactose) participating in metal ion-bridging interactions decreased by 32.9–51.5%, thereby weakening microbial aggregation. Concurrent reductions in hydrophobic amino acids content and down-regulation of exoproteins (PN) with abundant β-sheet-associated structures (e.g., (β/α)-barrel, β-helix, and β-propeller) weakened hydrophobic interactions, particularly metal ion-binding proteins and carbohydrate active enzymes (CAZymes). Exoproteomics also reveals that ammonia stress down-regulated extracellular electrons transfer-associated PN, compromising microbial survivability. In contrast, nMag-supplementation outperformed nZVI in alleviating these inhibitory effects, likely through selective enrichment of extracellular CAZymes, particularly GH43 family proteins that may improve AnGS strength via β-propeller-PS-microbes cross-linking network. PN- and PS-producers occupied distinct ecological niches, with filamentous populations exhibiting previously overlooked roles in PN production beyond structural scaffolding. These findings provide mechanistic insights into ammonia-induced AnGS disintegration and establish a theoretical foundation for optimizing mitigation strategies by nMag/nZVI-supplementation in AnGS systems.