Enhancing Long-Term Denitrification Stability through Operational Strategies in Iron- and Calcite-Rich Industrial Waste-Derived Sulfur Media: Performance and Biogeochemical Insights

Abstract This study evaluated how operational strategies affect the long-term stability of sulfur-based denitrification using iron- and calcite-rich industrial waste-derived media, testing four packed-bed reactors, namely, desulfurized waste (DSW), acid mine drainage sludge (AMDS), goethite–gypsum mixture (GGM), and gypsum–calcite mixture (GCM), which were operated continuously for 564 days under intermittent alkalinity (NaHCO3) and organic carbon (methanol) supplementation to assess how media mineralogy and operational control jointly govern denitrification performance, pH stability, sulfate production, and microbial community structure. Reactor stability was governed primarily by buffering capacity and operational strategy: AMDS and DSW, enriched in calcite and iron phases, sustained higher pH and nitrate removal than GGM. Periodic alkalinity supplementation mitigated acidification and controlled methanol dosing improved resilience and denitrification efficiency under stress; these benefits persisted after reactors returned to autotrophic operation. Stable systems were dominated by Thiobacillus and Hyphomicrobium, reflecting synergistic mixotrophic interactions, whereas unbuffered GGM shifted toward Rhodanobacter-dominated communities under acidic stress. Mineralogical analysis confirmed progressive calcite dissolution, gypsum depletion, and formation of phosphate-associated precipitates. These findings show that pairing iron- and calcite-rich industrial waste-derived sulfur media with targeted operational strategies can substantially improve the durability and performance of denitrification systems.