Bacterial assembly during the initial adhesion phase in wastewater treatment biofilms

Biofilm start-up is a critical and time-consuming process in moving bed biofilm reactors (MBBRs), with the procedure beginning with bacteria being statically bound on surfaces. Studies addressing this critical process have mainly focused on constructing models based on single strains, although consideration of the unstable adhesion process of structured bacterial communities remains underexplored. In this study, impedance based real-time cell analysis (RTCA) was employed to quantitatively characterize the unstable adhesion process of structured bacterial communities collected from the aerobic tanks of eight full-scale wastewater treatment plants (WWTPs). The unstable adhesion time ranged from 8.85 ± 1.53 h to 17.06 ± 0.64 h, indicating significant differences in bacterial colonization properties. Using principal components analysis (PCA), Na⁺, K⁺ and proteins were found to significantly influence the biofilm unstable adhesion process. Furthermore, the differences in unstable adhesion times were closely related to the abundance of the most abundant operational taxonomic units (OTUs). The dominant OTUs mainly belonged to Aeromonadaceae and Enterobacteriaceae, with 73% found to be negatively corelated with unstable adhesion time. Furthermore, bacterial assembly during the initial adhesion phase was driven by bacterial interactions and key OTUs (exhibiting maximum connectivity in phylogenetic molecular ecological networks (pMENs)). Analysis of pMENs indicated that bacterial cooperation was a dominant factor in the initial adhesion, which may involve bacterial co-colonization, co-aggregation and communication. Considering keystone taxa were not identified, OTUs with max connectivity in pMENs were considered as key species. Although these key species play important roles in the connection of networks, their relative abundances were low and no significant relationships were observed with the unstable adhesion time. Overall, unstable adhesion in MBBRs is regulated by the dominant bacterial species and the alleviation of environmental variables by repulsive forces, providing potential strategies of dosing quorum sensing signals and key cations at the initial adhesion phase in reactors, to facilitate initial biofilm formation.