Successful enrichment of low-abundant comammox Nitrospira from nitrifying granules under ammonia-limited conditions

ABSTRACT In artificial engineered systems, nitrification is a key reaction that accounts for the removal of biological nitrogen. Recently, a single microbe capable of oxidizing ammonia to nitrate, known as a complete ammonia oxidizer (comammox), has been discovered. Although the abundance and diversity of comammox Nitrospira in engineered systems have been identified through molecular-based approaches, the enrichment and isolation of comammox Nitrospira remains a challenge. Therefore, the aim of this study was to enrich comammox Nitrospira from nitrifying granules, which were used to increase the efficiency of biological nitrogen removal in wastewater treatment plants. We sought to accomplish this through the use of a fixed-bed continuous feeding bioreactor. Fluorescence in situ hybridization, 16S rRNA gene amplicon sequencing and qPCR of functional genes were utilized to monitor the growth of nitrifiers including comammox Nitrospira. Cloning of comammox amoA genes identified amoA phylogeny of enriched comammox Nitrospira. This work is an example demonstrating that continuous supply of low ammonium concentrations alongside biomass carriers is effective in cultivating comammox Nitrospira from engineered systems.