Context. Realizing energy-efficient biological nitrogen removal requires strategies that maintain consistent modulation of nitrifying populations within the activated sludge microbiome to cease the oxidation of ammonium at nitrite.
Gap. Engineering strategies designed to out-select nitrite oxidizing bacteria (NOB) are challenged by their metabolic versatility, warranting measurements of their in situ physiologies under these selective pressures.
Aim. This study illuminated the physiologies and community dynamics of NOB populations in activated sludge responding to a selective pressure induced by treating 20% of return activated sludge with free ammonia (FA).
Methods.
Parallel experimental and control activated-sludge sequencing batch reactors (SBRs) were operated for ~100 days with and without side-stream FA-treatment at 200 mg NH3-N/L, respectively. We applied time-series 16S rRNA gene amplicon sequencing along with biorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to elucidate changes in NOB community structure and resolve their in situ physiological responses at the cellular translational level.
Findings.
The nitrite accumulation ratio peaked at 42% by day 40 in the experimental bioreactor, while no nitrite accumulation was observed in the control. Time-series community monitoring revealed that a subsequent decrease in nitrite accumulation within the experimental bioreactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). BONCAT-FACS confirmed that the single Nitrospira ASV washed-out of the experimental bioreactor had reduced translational activity following FA-treatment, whereas the activities of the two Nitrospira ASVs that emerged during process acclimation were not impacted (Fig.1). Thus, the coexistence of functionally degenerate Nitrospira populations with distinct physiological tolerances to FA conferred community-level reliance against the applied out-selection pressure.
Utilization. This study highlights how BONCAT-FACS can resolve in situ ecophysiologies within shared niches in the activated sludge microbiome, and thus inform strategies for effective community management.
