The precise structure and assembly process of pyrite-based biofilms remain poorly understood. The polysaccharides (PN), proteins (PS), and extracellular DNA were enriched in the soluble extracellular polymeric substance (EPS), loosely bound EPS, and tightly bound EPS, respectively, indicating a significant stratified structure of biofilms. The tryptophan facilitated mixotrophic metabolic processes. Both dominant (>1%) and rare species (<0.01 %) harbored core bacteria, including sulfur autotrophic bacteria, sulfate-reducing bacteria, and heterotrophic bacteria. Furthermore, partial least-squares path modeling quantified the contributions of total phosphorus (TP) (2 = 0.32), dissolved organic matter (DOC) (2 = 0.29), and NH4+-N (2 = 0.26) to variations in the microbial community. Nonmetric multidimensional scaling analysis revealed three distinct stages in biofilm development: colonization (0-36 d), succession (36-149 d), and maturation/old (149-215 d). Furthermore, neutral community model indicated that stochastic processes drove the colonization and maturation/old stages, while deterministic processes dominated the succession stage. This study offered valuable insights into the regulation of pyrite-based engineered ecosystems.

Further reducing total nitrogen (TN) and total phosphorus (TP) in the secondary effluent needs to be realized effectively and in an eco-friendly manner. Herein, four pyrite/sawdust composite-based biofilters were established to treat simulated secondary effluent for 304 days. The results demonstrated that effluent TN and TP concentrations from biofilters under the optimal hydraulic retention time (HRT) of 3.5 h were stable at <2.0 and 0.1 mg/L, respectively, and no significant differences were observed between inoculated sludge sources. The pyrite/sawdust composite-based biofilters had low N2O, CH4, and CO2 emissions, and the effluent's DOM was mainly composed of five fluorescence components. Moreover, mixotrophic denitrifiers (Thiothrix) and sulfate-reducing bacteria (Desulfosporosinus) contributing to microbial nitrogen and sulfur cycles were enriched in the biofilm. Co-occurrence network analysis deciphered that Chlorobaculum and Desulfobacterales were key genera, which formed an obvious sulfur cycle process that strengthened the denitrification capacity. The higher abundances of genes encoding extracellular electron transport (EET) chains/mediators revealed that pyrite not only functioned as an electron conduit to stimulate direct interspecies electron transfer by flagella but also facilitated EET-associated enzymes for denitrification. This study comprehensively evaluates the water-gas-biofilm phases of pyrite/sawdust composite-based biofilters during a long-term study, providing an in-depth understanding of boosted electron transfer in pyrite-based mixotrophic denitrification systems.