Connecting biodiversity and potential functional to promote motivated labor in iron sulfide-amended partial denitrification/anammox systems

Mitigating the intense competition for substrates and ecological niches between heterotrophic and autotrophic microorganisms is essential for sustaining nitrogen removal in partial denitrification/anammox (PD/A) systems. Herein, an innovative high-intensity PD/A process was designed, utilizing iron sulfide and minimal organic carbon input. The nitrogen loading rate (NLR) reached 0.96 ± 0.06 kg N/(m 3 ·d) at a C/N ratio of 0.5, considerably higher than all results reported to date. Elucidating microbial ecology in this lab-scale mixotrophic PD/A system is vital for large-scale application. Results showed that sulfur-oxidizing denitrifiers ( Thiobacillus and Pseudorhodoplanes ; 0 % ∼ 1.11 %), functioning as network hubs, redistributed nitrate burden from heterotrophic denitrifiers ( Denitratisoma ; 8.44 %) via inorganic electron uptake, while freeing niches for the anammox bacteria ( Ca. Brocadia; 143.67-fold enrichment). A synergistic microbial community integrating multiple metabolic pathways achieved 96.24 ± 0.11 % nitrogen removal. Furthermore, genetic evidence indicated that membrane-bound narG accelerated nitrate reduction, while hydroxylamine-dependent anammox ( via hao instead of nirS ) eliminated toxic NO intermediates. This enhanced bioreactor stability and reduced hydraulic retention time from 12 h to 2 h, maximizing NLR. Our findings propose a novel paradigm to advance PD/A systems toward low-carbon, high-rate operation, ensuring desirable effluent quality and microbial ecological balance.