Elucidating the role of extracellular polymeric substances (EPS) in modulating autotrophic-heterotrophic interactions in a pyrite-assisted autotrophic denitrification biofilm

Autotrophic biofilm systems have been applied for nitrate removal from organic matter-poor waters. Nevertheless, heterotrophs can survive long-term fully autotrophic conditions, with stable or even increasing populations. Extracellular polymeric substances (EPS) are reported to sustain heterotrophic growth in such systems, but their role in regulating biofilm structure, population networks, and gene abundance remains unclear. In this study, we investigated microbial, functional gene, and EPS dynamics in a pyrite-assisted autotrophic denitrification (PAD) biofilter operated for 180 days. Despite fully autotrophic operating conditions, sulfur-oxidizing denitrifiers and their genes decreased over time, while EPS sustained heterotrophic anaerobes throughout the experimental periods. Biofilm maturation led to EPS conversion into filaments with potential conductive properties that facilitated electron transfer between microbes, pyrite, and nitrate. Polysaccharides in EPS were preferentially consumed over proteins, increasing the protein/polysaccharide ratio. Based on these results, a three-phase biofilm development model (initial attachment, maturation, and disassembly/restructuring) was proposed, linking temporal changes in EPS composition and microbial community structure to biofilm architecture and sustained denitrification.