Distinct growth stages shaped by an interplay of deterministic and neutral processes are indispensable for functional anammox biofilms

Complex microbial biofilms orchestrating mainstream anaerobic ammonium oxida-tion (anammox) represent one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from anthropogenic waste waters. However, little is known about the ecological processes that are driving microbial community assem-bly leading to functional anammox biofilms in engineered ecosystems. Here, we use fluorescence in situ hybridization and 16S rRNA sequencing combined with network modelling to elucidate the contribution of stochastic and deterministic processes dur-ing anammox biofilm development from first colonization to maturation in a carrier-based anammox reactor. We find that distinct stages of biofilm development emerge naturally in terms of structure and community composition. These stages are charac-terized by dynamic succession and an interplay of stochastic and deterministic pro-cesses. The staged process of biofilm establishment appears to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure with the desired metabolic capacities. We discuss the relevance of this im-proved understanding of anammox community ecology and biofilm development concerning its practical application in the start-up and configuration of anammox biofilm reactors.