The Role of Ammonia Oxidising Bacteria in Mixed-Species Biofilm Formation and Chloramine Decay

Abstract

Ammonia oxidizing bacteria (AOBs) are chemoautotrophic organisms that produce complex organic compounds through the metabolism of ammonia and carbon dioxide, providing nutrients that can be used by heterotrophic bacteria (HTBs) for growth. Therefore, the role of AOBs in the formation of mixed-species biofilms within the context of drinking water distribution systems was investigated, including the effects of such biofilms on monochloramine, a commonly used drinking water disinfectant. The effect of cell-to-cell signalling on these systems, particularly N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS), was also determined. The formation of mixed-species bacterial biofilms by AOBs was shown, and this effect was due to the provision of organic nutrients. When grown under autotrophic conditions, bacteria of the Nitrosomonadaceae family, which are known AOBs, were the dominant organisms. Pseudomonas spp. and Vibrio spp. were also strongly represented, as determined by Length Heterogeneity-PCR and amplicon sequencing. Supplementation with glucose during inoculation resulted in the dominance of these HTBs, yet AOBs were nonetheless retained as a minority in the biofilm and produced nitrite. When exposed to glucose, pre-formed mixed-species biofilms were restricted in further growth, and this restriction was removed when the biofilms were also treated with nitrification inhibitors (NIs). This suggests a novel role for AOBs in regulating the total biomass of biofilms, although the mechanism for this interaction is unknown. The AOBs enhanced the recovery of biofilms after exposure to chloramine. Biofilms treated with NIs were more susceptible to disinfection by chloramine, although AOBs and HTBs within biofilms were equally affected by chloramine. Together the data suggest that the localised production of nitrite in these biofilms provides a biochemical barrier that protects all species in the biofilm from disinfection. Finally, QS was observed to differentially affect biofilm formation and ammonia oxidation, depending on nutrient availability. However, QS did not directly regulate the expression of amoA, which encodes an ammonia monooxygenase enzyme. As the most common organisms in the mixed species biofilm are capable of producing and detecting AHLs, these results suggest cross-species signalling within nitrifying biofilms that controls biofilm development of the mixed species community.