Multi-Species Communities Containing Ammonia-Oxidising Bacteria 16S metabarcoding raw sequence reads

Despite significant research, the biofilm-forming capabilities of Nitrosomonas europaea are poorly understood; studies indicating biofilm-forming traits of N. europaea typically involve the use of either an immobilizing support medium, or multi-species communities containing N. europaea in addition to other organisms. This study combined biofilm assays and molecular techniques to demonstrate that N. europaea is a poor biofilm-forming organism, relying almost exclusively on the activity of associated heterotrophic bacteria in mixed-species communities. However, N. europaea has a vital role in the proliferation of mixed-species communities under carbon-limited conditions, such as in drinking water distribution systems, through the provision of organic carbon via ammonia oxidation. The results showed that the addition of nitrification inhibitors to mixed-species nitrifying cultures under carbon-limited conditions disrupted biofilm formation and caused the dispersal of pre-formed biofilms. This dispersal effect was not observed when an organic carbon source, glucose, was included in the medium. Additionally, the biomass of mixed-species nitrifying biofilms was further enhanced when pre-established biofilms were supplemented with glucose and the nitrification inhibitor, 2-ethynylpyridine, in contrast to when supplemented with glucose alone. These enhanced biofilms also exhibited increased diversity and evenness on the Shannon-Weiner diversity index and Pielou's evenness index (H'=2.4, J'=0.92) compared to the average of the rest of the biofilms (H'=2.1, J'=0.79) This suggests that active AOB partially suppress or limit the overall growth of the heterotrophic bacteria through an unknown mechanism, such as the production of growth-limiting compounds or coincidental metabolic interference. The model developed here suggests that ammonia-oxidising bacteria (AOB) are involved in both the formation and maintenance of multi-species biofilm communities, not only supporting biofilm formation by providing organic carbon, but restricting and controlling total biomass through an as yet unknown mechanism.