Comammox Nitrospira was the dominant ammonia oxidizer in an acidic biofilm reactor at pH 5.5 and pH 5

Nitrification is a vital process in the biological removal of inorganic nitrogen compounds. In order to ensure the stability and effectiveness of this process, buffer solutions should be added to the system to maintain neutral to slightly alkaline conditions. With a focus on the newly discovered comammox Nitrospira, this research investigates the transition of the nitrifying community within a biofilm reactor under different acidic levels (initiated at pH 6 and gradually decreased to pH 5). During the 305-day continuous operation experiment, it was observed that responsible ammonia oxidizers transitioned from ammonia-oxidizing bacteria (AOB) during the initial stages (setup stage and early stage of pH 6) to comammox Nitrospira under pH 5.5 and pH 5. Further analysis using next-generation sequencing targeting both the 16S rRNA region and amoA region revealed a shift in the dominant cluster of both Nitrospirae and comammox Nitrospira under varying pH conditions. Our study identified a distinct cluster of comammox Nitrospira that is phylogenetically closed to sequences found in acidic environments, but exhibits dissimilarity from known comammox Nitrospira isolates and the majority of environmental sequences. This cluster was found to be prevalent in the acidic biofilm reactor studied and thrived particularly well at pH 5. These findings underscore the potential significance of this distinct, uncultivated group of comammox Nitrospira in performing ammonia oxidation under acidic conditions. KEY POINTS: • Ammonia was effectively removed under pH 5.5 and 5 in the biofilm reactor • The dominant ammonia oxidizer was comammox Nitrospira when pH was 5.5 and 5 • A potential acidophilic cluster of comammox Nitrospira was identified in this acidic biofilm reactor.