Thermophilic Sulfide-Driven autotrophic Denitrification: High-Rate nitrogen removal and metagenomic insights into...

Sulfide-driven autotrophic denitrification (SDAD) plays a pivotal role in linking nitrogen and sulfur cycles, particularly in thermophilic environments. However, the performance and metabolic mechanisms of thermophilic SDAD systems remain underexplored. This study successfully enriched thermophilic sulfide-oxidizing denitrifiers from hot spring sediments (60 °C) in an expanded granular sludge bed (EGSB) reactor, achieving a stable nitrate removal rate of 250 mg N L^-1 d^-1. This thermal system demonstrated a higher tolerance threshold for sulfides (250 mg TDS L^-1) than mesophilic processes. The influent sulfide-to-nitrate (S/N) molar ratio critically influenced nitrogen removal efficiency with nitrite accumulation at low S/N ratios (1:1), whereas higher ratios (2:1) restored complete nitrate reduction. Microbial community analysis demonstrated Thermus (52.89 %) as the predominant genus in the SDAD system, marking the first report of its dominance in thermophilic nitrogen-sulfur coupling, alongside uc_Aquificales (21.78 %). Metagenomic insights further revealed two high-quality genomes: Thermus scotoductus exclusively executed complete sulfide-to-sulfate oxidation via the Sox system and partially catalyzed denitrification (narGHI); while Aquificaeae_UBA11096 adopted Sox-independent enzyme system and complete denitrification. Strikingly, Thermus scotoductus encoded nrfH, and it might be the overlooked " nitrogen retainer" in geothermal ecosystems. This work elucidates the cooperative and competitive interactions between thermophilic denitrifiers, and establishes a multi-level thermal adaptability analysis. These findings advance understanding of their biogeochemical roles in geothermal ecosystems, and provide a sustainable strategy for nitrogen removal in high-temperature wastewater treatment.