Disentangling community succession in full-scale sidestream partial nitritation-anammox biofilms

Context. To retain slow growing bacteria, biofilms are commonly used in biological nitrogen removal of wastewater treatment. One of these processes is partial nitritation-anammox (PNA), where ammonia-oxidizing bacteria (AOB) and anammox bacteria coexist in the biofilm.

Gap. Studies on biofilm bioreactors are often limited to describe the community structure and dynamics of mature biofilms. A mature PNA biofilm has a community with AOB and anammox bacteria converting ammonia to nitrogen gas, but there is little knowledge about the path towards stable bioreactor operation. Moreover, full-scale PNA biofilm reactors are commonly seeded with biomass from other reactors for a rapid start-up, which may not be a necessary habit.

Aim. Our aim was to study the development and community succession of biofilms on virgin substratum until a fully functional and stable full-scale PNA process is attained.

Methods. We used shotgun metagenomics, resulting in 218 prokaryotic and five eukaryotic metagenome-assembled genomes.

Findings. Bacteria and eukaryotes rapidly colonized the substratum. Species turnover, species loss/gain occurred trough the study. Some populations were rapidly replaced by others, and only 30 MAGs were observed across the entire study. Among nitrogen converting bacteria, Nitrosomonas cluster 6A, Nitrospira comammox and nitrite-oxidizing bacteria (NOB) co-occurred and were among the early colonizers. At day 28, NO₂^- and NO₃^- peaks were observed in the reactor, which coincided with the highest relative abundance of Nitrosomonas, NOB and comammox. These nitrifier populations were replaced by Nitrosomonas cluster 7, and later on anammox bacteria emerged (Figure 1).

Utilization. We show that the path towards a fully functional full-scale PNA reactor, which took around 180 days, consists of stages with rapid turnover of nitrifiers as well as other microbial populations, with emergence of anammox bacteria after around three months. This contribute to our knowledge on biofilm community assembly and general microbial ecology.