Deciphering the synergy between autotrophic and heterotrophic electron donors in pyrite driven mixotrophic denitrification for advanced nitrogen removal.

Pyrite-driven autotrophic denitrification (PAD) is restricted by the low solubility of pyrite (FeS), leading to slow reaction rate and suboptimal nitrogen removal. To address this problem, we developed a pyrite-driven mixotrophic denitrification (PMD) system, which combined sodium acetate (as bioavailable carbon) with sustained electron release of pyrite. Under the chemical oxygen demand (COD) to total nitrogen (TN) (C/N) ratio of 3.0, the TN removal efficiencies of 87.99 % and 86.03 % in batch reactor and continuous-flow biofilter were achieved, with the total phosphorus (TP) removal of 88.90 % and 96.32 %, respectively. The PMD system showed a TN removal rate constant (k = 0.116 h), which exceeded those of both autotrophic (k = 0.033 h) and heterotrophic denitrification (k = 0.085 h) systems. In batch reactor, the pyrite-carbon synergy enriched heterotrophic (e.g., Thauera), autotrophic (e.g., Thiobacillus) and mixotrophic (e.g., Azonexus) denitrifiers. Moreover, the genes encoding periplasmic nitrate reductase (NAP), nitrite reductase (NIR), sulfur dehydrogenase (Fcc), sulfite reductase (CysJI), nicotinamide adenine dinucleotide (reduced form) (NADH) dehydrogenase and cytochrome c (Cyt c), etc., were upregulated. In continuous-flow biofilter, the increased C/N ratios expanded PMD-active zones with elevated Thiobacillus abundance. The carbon source also promoted microbial protein secretion to accelerate pyrite corrosion and alleviated passivation. Furthermore, the pyrite and carbon source provided ample external electrons, weakening the contribution of endogenous denitrification and nitrate assimilation reduction processes in nitrogen removal, which was conducive to sustaining sludge activity and reducing nitrogen accumulation. This study elucidated the mechanism of synergistic electron donors in PMD systems, offering practical strategies for enhanced nitrogen removal.