Functional degeneracy in translationally active Nitrospira populations confers resilience to out-selection in partial nitritation activated sludge

Energy-efficient nitrogen removal in activated sludge wastewater treatment systems could be achieved by oxidizing a part of the influent ammonium to nitrite (partial nitritation; PN) to provide growth substrates for anammox. However, the metabolic and physiological diversity of nitrite oxidizing bacteria (NOB) makes their out-selection a challenge in mainstream activated sludge, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the long-term (~100 day) stability of PN in mainstream activated sludge obtained by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/L. The nitrite accumulation ratio peaked at 42% by day 40 in the reactor with sidestream FA-treatment, while it did not increase in the control reactor without FA added to the sidestream. A subsequent decrease in nitrite accumulation within the FA-treated reactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout sidestream exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV out-selected in the FA-treated reactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, functionally degenerate Nitrospira populations likely provided resilience to the nitrite-oxidizing community during FA-treatment by shifting activity to physiologically resistant members. These results highlight how in situ physiology approaches like BONCAT can resolve ecological niches in the activated sludge microbiome, and how functional degeneracy observed within Nitrospira populations likely necessitates a combination of out-selection strategies to achieve stable mainstream PN.