Concentration-dependent roles of hydrazine in immobilized denitrifying biofilm for industrial wastewater treatment

Hydrazine-bearing industrial wastewater is challenging to treat biologically because hydrazine can simultaneously act as a reducing substrate and a microbial inhibitor. In this study, an immobilized denitrifying biofilm system was used to evaluate the concentration-dependent effects of hydrazine on denitrification performance, electron contribution, and microbial response under anoxic conditions. Under sufficient co-substrate conditions, 5-10 mg/L hydrazine was effectively removed, with a maximum removal efficiency of approximately 94%, while stable denitrification was maintained. Nitrogen-15 isotope tracing showed that approximately 31% of the electrons released from hydrazine oxidation were transferred to denitrification-coupled nitrate reduction, indicating that hydrazine can partially contribute reducing equivalents in the denitrifying biofilm. However, elevated hydrazine concentrations impaired hydrazine oxidation and denitrification, induced nitrite and ammonium accumulation, and reduced carbon utilization. Mechanistic analyses showed that this deterioration was associated with oxidative stress, membrane damage, and inhibition of key enzymes, particularly nitrite reductase and hydroxylamine oxidoreductase. Metagenomic analysis further revealed a stress-induced shift in the microbial community from central carbon metabolism toward compensatory pathways. Overall, this study provides mechanistic and process-level insights into the feasibility and operational limitations of using immobilized denitrifying biofilms for treating hydrazine-bearing industrial wastewater.