Thiocyanate-driven denitrification with mixotrophic flexibility for real coking wastewater treatment: Novel insights into nitrogen cycling

Industrial coking wastewater, characterized by high thiocyanate (SCN⁻), nitrate, and complex toxic organics, challenges conventional biological nitrogen removal and impedes resource recovery. To shift the treatment objective from mere detoxification to predictable nitrogen partitioning, a SCN⁻-driven biological nitrogen removal (SCN⁻-BNR) bioreactor was operated for 200 days, comprising a 160-day synthetic stoichiometric optimization phase and a 40-day validation phase with undiluted real coking wastewater. We identified the influent SCN⁻-S/NO₃⁻-N mass ratio (S/N) as the primary operational lever governing nitrogen fate. Increasing this ratio to ∼4.0 drove >99% nitrate removal, with DNRA contributing 49.1% of the total nitrate reduction. Crucially, ¹⁵N stable isotope tracing and metagenomics elucidated a synergistic cross-feeding mechanism: Chlorobium sp. likely initiates SCN⁻ cleavage, followed by cyanate hydrolysis (cynS) and dissimilatory nitrate reduction to ammonium (DNRA, nrfA) driven by distinct populations (SpSt-501 sp. And JADFDR01 sp.). DNRA was highly activated under electron-donor-surplus conditions, directly contributing up to 22.8% of the generated effluent ammonium. This metabolic division of labor proved exceptionally resilient; the mixotrophic consortium maintained stable >95% SCN⁻ and >90% NO₃⁻ removal during real wastewater validation, demonstrating strong tolerance to phenol, quinoline, and salinity. This study provides a verifiable operational-mechanistic framework for engineering next-generation SCN⁻-driven bioreactors, integrating robust complex wastewater detoxification with circular nitrogen management.