A rapid collection of yet unknown ammonia oxidizers in pure culture from activated sludge

Nitrification is an important reaction in the biological nitrogen removal process in wastewater treatment plants (WWTPs). As ammonia-oxidizing microbes are slow-growing and sensitive to environmental factors such as free ammonia, pure strains are hard to obtain, preventing our understanding of their physiological characteristics. To conquer this hurdle, we report a high-throughput isolation technique based on scattering signatures, which exploits the tendency of many ammonia-oxidizing bacteria (AOB) to form microcolonies in activated sludge. The AOB microcolonies were directly sorted from the activated sludge without long incubation and enrichment bias, and were sequentially inoculated into 96-well microtiter plates containing growth medium. Phylogenetic analysis of the pure strains isolated in this study revealed a deeply branching and unrecognized lineage and diversity within the genus Nitrosomonas, beyond our expectation.