Moderately acid-tolerant Nitrospira strains obtained by single-colony isolation on plates

Nitrospira are difficult to cultivate, and pure cultures are mainly obtained by dilution-to-extinction combined with other methods, a process that usually takes years to over a decade. Here, we isolated three Nitrospira strains from activated sludge and sediment within one year using conventional single-colony isolation. Pure cultures of two strains could grow and reform single colonies within 25-60 days on all three types of plates prepared with agar, agarose, and gellan gum. Phylogenetic and phylogenomic analyses placed them within lineage I and indicated that they represent potential novel species, with the provisional names Nitrospira monocolonialis for strain Z02 and Nitrospira limosa for strains Z47 and Z70. All three strains were moderately acid-tolerant, with a minimum tolerable pH of 5.0; strain Z02 showed the highest acid tolerance, retaining 74% of its activity at pH 5.5. Strain Z02 was also highly adaptable to oxygen concentrations of 1-21%, retaining 78% and 85% of its maximum growth rate at 1% and 21% O2, respectively. Compared with the representative lineage I strain Nitrospira defluvii, all three strains contain superoxide dismutase encoding genes and some unique genes. Comparative transcriptomic analysis suggested that strain Z02 upregulated genes involved in stress responses, cell envelope modification, and putative type IVa pilus systems under acidic condition. Overall, this study provides the first demonstration that Nitrospira can grow on plates prepared with common gelling agents and form single colonies within a short period, and that Nitrospira can also tolerate moderately acid conditions.