Ecological reorganization of fungal communities driven by floc-size stratification and process upgrading in a full-scale landfill leachate treatment plant

Abstract

Floc size in activated sludge systems plays a critical role in shaping bacterial communities by regulating microenvironmental heterogeneity, yet its ecological influence on fungal assemblages during process upgrading remains largely unexplored. This study aimed to systematically investigate the effects of floc-size stratification on fungal community composition, assembly mechanisms, source contribution patterns, and molecular ecological co-occurrence network structures during the transition from conventional nitrification–denitrification (CND) to partial nitrification–denitrification (PND) in a full-scale landfill leachate treatment plant. Fungal communities from large (L, >100 μm), medium (M, 25–100 μm), and small (S, <25 μm) flocs were characterized across three operational stages (CND, Trans, and PND) using high-throughput ITS sequencing. Analytical approaches including neutral community model, normalized stochasticity ratio, fast expectation–maximization for microbial source tracking, and molecular ecological network modeling were applied. Results showed that S-flocs consistently contributed the largest proportion to the supernatant fungal community across all stages. Community assembly transitioned from predominantly stochastic in the CND stage to increasingly deterministic in the PND stage, especially within L and M flocs. Co-occurrence network analysis revealed a shift from small-world to scale-free modular topologies, indicating substantial remodeling of fungal co-occurrence correlations. Dissolved oxygen, representing an operational factor, and sludge physicochemical indicators jointly influenced fungal community differentiation across floc-size gradients. This study highlights the regulatory role of floc-size stratification in shaping fungal ecology and network architecture during engineered nitrogen removal process upgrading. These findings provide new insights into spatial microbial dynamics and inform floc-scale strategies for optimizing biological wastewater treatment performance.