Obtaining complete and canonical ammonia-oxidizing bacteria through specific labeling and cell sorting.

Mitigation of the negative environmental consequences of excess anthropogenic nitrogen input requires a thorough understanding of the processes driving the biogeochemical nitrogen cycle. Nitrification is one of the key nitrogen-cycling processes and is performed by ammonia-oxidizing bacteria and archaea, nitrite-oxidizing bacteria, and complete nitrifiers. However, the fastidious growth of nitrifiers largely hampered their isolation using classical cultivation techniques, as most nitrifiers do not grow on solid media. Here, we present a workflow for the targeted enrichment and isolation of complete and canonical ammonia-oxidizing bacteria by combining function-specific in vivo fluorescent labeling with cell sorting. Optimized floc disruption and labeling techniques enlarged the fraction of planktonic cells and the fluorescent signal intensity, respectively, while maintaining cell viability. Sorted fractions were incubated in ammonium-containing mineral media and were screened for nitrite and nitrate production. Nitrifying cultures were upscaled and characterized with 16S ribosomal ribonucleic acid and gene-targeted polymerase chain reactions and fluorescence hybridization. Overall, we obtained one axenic and one enriched , and seven comammox enrichment cultures from five bioreactors, a recirculating aquaculture system biofilter, and agricultural soil. In conclusion, the presented workflow enables the fast and targeted retrieval of ammonia oxidizers from complex samples, allowing for in-depth physiological characterization.