Defining culture conditions for Nitrolancea-a hidden nitrite-oxidizing bacterium
Nitrification is a key process for N-removal in engineered and natural environments, but recent findings of novel nitrifying microorganisms with surprising features revealed that our knowledge of this functional guild is still incomplete. Especially nitrite oxidation – the second step of nitrification – is catalyzed by a phylogenetically diverse bacterial group and only recently, bacteria of the phylum Chloroflexi have been identified as thermophilic nitrite-oxidizing bacteria (NOB). Among these, Nitrolancea hollandica was isolated from a laboratory-scale nitrifying bioreactor operated at 35°C with a high load of ammonium bicarbonate. However, very few closely related environmental 16S rRNA sequences have been retrieved so far and its distribution remains cryptic. In this study, we demonstrate how such thermophilic NOB can be enriched using modified mineral media inoculated with samples from a full-scale wastewater side-stream reactor operated at 39°C. Distinct cultivation conditions resulted in quick and reproducible high enrichment of two different genotypes of Nitrolancea, closely related to N. hollandica. The same cultivation approach was applied to a complex nitrite-oxidizing pre-enrichment at 42°C, which had been inoculated with biomass from the geothermal spring 'Las Máquinas' in the Copahue volcano area in Neuquen, Argentina. Here, an additional, distinct representative of the genus Nitrolancea was obtained. This novel species had 16S rRNA and nitrite oxidoreductase alpha subunit (nxrA) gene sequence identities to N. hollandica of 98.5% and 97.2%, respectively. A genomic average nucleotide identity between the Argentinian strain and N. hollandica of 91.9% indicates that it indeed is a distinct species.  All Nitrolancea cultures formed lancet-shaped cells identical to Nl. hollandica and revealed similar physiological features, including the capability to grow at high nitrite concentrations. Growth was optimal at temperatures of 35-37°C and was strongly enhanced by ammonium supplementation. Genomic comparisons revealed that the four Nitrolancea share a core set of 2114 genes and encode similar key functions. Our results define general growth preferences for Nitrolancea that enable their selective enrichment from artificial and natural environments. While these NOB might be of low abundance in nature, their proliferation is dependent on the balanced presence of nitrite and ammonium and an incubation temperature around 37°C.