Suspended sludge-biofilm niche cooperation enables stable mainstream nitrogen removal via mixotrophic partial...

The sustainable application of partial denitrification/anammox (PD/A) under mainstream wastewater conditions depends critically on relieving the strong competition between heterotrophic and autotrophic microorganisms for substrates and ecological space. In this study, ferrous sulfide (FeS) was employed as a biofilm carrier to establish a mixotrophic PD/A (MPD/A), which maintained a total nitrogen removal efficiency of 90.25% under fluctuating organic carbon conditions in mainstream wastewater. To elucidate how niche differentiation between suspended sludge and biofilms supports synergistic nitrogen removal, community composition and predicted functional genes were analyzed to resolve the coexistence and division of labor of key microorganisms. The results showed that FeS, protein-like EPS, and multiple functional microbial populations jointly supported biofilm establishment and stability, enabling the preferential retention and enrichment of anammox bacteria (AnAOB) (14.09%), autotrophic denitrifiers, and other microorganisms involved in synergistic nitrogen removal. Together with suspended sludge, biofilms formed a cooperative nitrogen removal network integrating autotrophic and heterotrophic partial denitrification, complete denitrification, and anammox. Functional gene responses further revealed that autotrophic and heterotrophic partial denitrification within the biofilm sustained NO₂^--N supply, while FeS strengthened electron transfer and growth metabolism in AnAOB, thereby enhancing nitrogen removal kinetics and process stability. These findings provide a new strategy for expanding the ecological niche of anammox in mainstream denitrification tanks and a practical basis for the stable retention, enrichment, and sustained functional expression of AnAOB.