Prokaryotic community interchange between distinct microhabitats causes community pressure on anammox biofilm development.

Biofilms are an efficient way to underpin the biological process of wastewater treatment. However, little is known about the driving forces of biofilm formation and development in industrial settings. Long-term observation of anammox biofilms indicated the interplay between different microhabitats (biofilm, aggregate, plankton) was important in sustaining biofilm formation. SourceTracker analysis showed that 88.77 ± 2.26% of initial biofilm originated from the aggregate, however, independent evolution was led by anammox species in the later stage (182d and 245d). Noticeably, the source proportion of aggregate and plankton increased when temperature varied, suggesting an interchange of species between different microhabitats could be helpful to biofilm recovery. The microbial interaction pattern and community variation displayed similar trends, but the unknown source proportion of interaction was very high during the entire incubation (7-245d), thereby the same species may develop different relationships within the distinct microhabitats. The core phyla, Proteobacteria and Bacteroidota, accounted for ∼80% of interactions in all lifestyles, which is consistent with the fact that Bacteroidota played important role in the early stage of biofilm assembly. Although anammox species evolved few links with other OTUs, Candidatus Brocadiaceae still outcompeted the NS9 marine group to dominate the homogeneous selection process in the later stage (56-245d) of biofilm assembly, implying that the functional species may be decoupled from the core species in the microbial network. The conclusions will shed a light on the understanding of biofilm development in large-scale biosystems of wastewater treatment.