PRJDB5488 Nitrospira sp. KM1 Genome sequencing Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitrospira has been recognized as the numerically dominant nitrite-oxidizing bacterial genus, and is primarily responsible for the second step of aerobic nitrification. Nevertheless, the physiological ad genomic properties of Nitrospira remain poorly understood because the organisms are difficult to isolate and culture. Here we report a novel cultivation strategy for obtaining members of the Nitrospira lineage II in pure culture. The method combines 1) selective enrichment of Nitrospira using a continuous feeding reactor, and 2) purification followed by sub-cultivation via a cell sorting system by focusing on the unique characteristics of Nitrospira forming spherical microcolonies. New insight into previously uncultured Nitrospira would accelerate the genomic and biochemical understandings of this important group of organisms. Nitrospira sp. KM1 Genome sequencing bioproject PRJDB5488 PRJDB5488 true Nitrification is an important process in the biogeochemical nitrogen cycle and is widely exploited in biological wastewater treatment. Recently, Nitrospira has been recognized as the numerically dominant nitrite-oxidizing bacterial genus, and is primarily responsible for the second step of aerobic nitrification. Nevertheless, the physiological ad genomic properties of Nitrospira remain poorly understood because the organisms are difficult to isolate and culture. Here we report a novel cultivation strategy for obtaining members of the Nitrospira lineage II in pure culture. The method combines 1) selective enrichment of Nitrospira using a continuous feeding reactor, and 2) purification followed by sub-cultivation via a cell sorting system by focusing on the unique characteristics of Nitrospira forming spherical microcolonies. New insight into previously uncultured Nitrospira would accelerate the genomic and biochemical understandings of this important group of organisms.