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In biofilm-based engineered ecosystems, the reactor performance was closely linked to interspecies interactions within a biofilm ecosystem, whereas the ecological processes underpinning such linkage were still unenlightened. Herein, the principles of community succession and assembly were integrated to capture the ecological laws of biofilm development by molecular ecological networks and assembly model analysis based on the 16S rRNA sequencing analysis and metagenomics in a well-controlled moving bed biofilm reactor. At the initial colonization phase (days 0−2, driven by initial colonizers), interspecific cooperation (74.18%) facilitated initial biofilm formation, whereas some pioneers, and keystone species disappeared at later phases. At the accumulation phase (days 3−30, rapid biofilm development), interspecific cooperation (81.41 ± 5.07%) contributed to rapid biofilm development and keystone species were mainly involved in quorum sensing or positively correlated with extracellular polymeric substance production. At the maturation phase (days 31−106, a well-adapted quasi-equilibrium state), increased interspecific competition (32.74 ± 4.77%) and higher small-world property facilitated the rapid information transportation and pollutant treatment, and keystone species were positively correlated with the removal of COD and NH 4 + -N. Homogenizing dispersal diminished the contemporary community dissimilarities, while turnover but rather nestedness governed the temporal variations in the biofilm succession period. This study highlighted the specificity of ecological processes at distinct biofilm development phases, which would advance our understanding on the development-to-function linkages in biofilm-based treatment processes.

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