Deciphering stratified structure and microbiota assembly of biofilms from a pyrite-based biofilter driven by mixotrophic denitrification

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) (λ = 0.32), dissolved organic matter (DOC) (λ = 0.29), and NH₄⁺-N (λ = 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.