Enhanced denitrification performance via biochar-mediated electron shuttling in Pseudomonas guariconensis: mechanistic insights from enzymatic and electrochemical analyses

Nitrogen pollution constitutes a critical environmental challenge for aquatic ecosystems, where biological denitrification serves as a critical mechanism for nitrogen removal in wastewater treatment plants (WWTPs). Pseudomonas guariconensis , a key denitrifying bacterium in WWTPs, demonstrates stable symbiotic relationships with organic carbon substrates under operational conditions. This study investigated the biochar-mediated enhancement mechanism of denitrification performance using Pseudomonas guariconensis strain XYH-2. Experimental results revealed that 0.5 % (w/w) biochar addition significantly improved denitrification efficiency, elevating NO 3 − -N removal from 39 % to 94 % within 16 h while reducing N 2 O accumulation by 81.7 % (from 837 to 153 ppm). Electrochemical characterization and extracellular polymeric substances (EPS) analysis demonstrated biochar's role as an electron shuttle, evidenced by a 48.5 % increase in electron transfer system activity (ETSA) and enhanced redox currents. Notably, biochar stimulation elevated the activities of four key denitrifying enzymes: nitrate reductase (NAR, +75.6 %), nitrite reductase (NIR, +25.4 %), nitric oxide reductase (NOR, +33.3 %), and nitrous oxide reductase (N 2 OR, +159.4 %). Structural characterization revealed biochar-induced conformational modifications in N 2 OR that enhanced substrate binding affinity, particularly explaining the dramatic N 2 O mitigation. These findings provided mechanistic insights into biochar-bacteria synergism, proposing an effective strategy for optimizing denitrification processes in nitrogen-contaminated wastewater treatment systems.