Selective enrichment of comammox Nitrospira as the nearly sole nitrifier using formate as the carbon source within an extremely short period

Nitrification has long been regarded as a two-step biological process catalyzed by two distinct groups of microorganisms, but the discovery of complete ammonia oxidizers (comammox) has challenged this traditional view. Comammox Nitrospira has been detected in a wide range of environmental samples, but due to the long enrichment time and unclear key factors for its enrichment, there is still a lack of enriched samples for physiological studies. In this study, the enrichment of comammox Nitrospira was achieved in less than 100 days under mainstream wastewater conditions using formate as the sole carbon source. The successful enrichment of comammox Nitrospira was confirmed through 16S rRNA gene amplicon sequencing and quantitative PCR targeting the functional marker gene amoA. Comammox Nitrospira became the nearly exclusive nitrifier in the system, with the comammox amoA gene copy number increased 337-fold relative to its abundance in the inoculated sludge and was 113 times higher than that of AOB on Day 193. Furthermore, only a single Nitrospira related metagenome-assembled genome (MAG) was obtained by metagenomic sequencing and binning. Phylogenetic analysis and average nucleotide identity comparisons showed a high degree of relatedness to Ca. Nitrospira nitrosa, which was tentatively named Nitrospira sp. FTF1. In addition to the nitrification process, the MAG of Nitrospira sp. FTF1 also encodes various energy metabolism pathways, including genes related to formate dehydrogenase, filling a gap in our understanding of formate metabolism in Clade A comammox Nitrospira. The subsequent kinetic and N2O emission factor studies revealed the differences in substrate affinity and the high N2O emission potential of the comammox Nitrospira enriched with formate. These findings provide new insights into the ecological adaptability of comammox Nitrospira and the selective pressures driving its enrichment in engineered environments.