Metabolic inhibitor-free assessment of the heterotrophic ammonia-oxidizing activity in activated sludge

When the contributions of three ammonia-oxidizing pathways (heterotrophic or autotrophic aerobic ammonia oxidization, and anammox) to wastewater biological nitrogen removal systems was compared by determining their ammonia-oxidizing activities, the key question is how to accurately determine the potential heterotrophic aerobic ammonia-oxidizing (PHAe) activity when the potential autotrophic aerobic ammonia-oxidizing (PAAe) activity (by ammonia-oxidizing bacteria (AOB) or archaea, or complete ammonia oxidization bacteria) also contributes to ammonia oxidization in PHAe activity assay medium. Using a AOB species and three heterotrophic AOB species as inocula, we demonstrated the feasibility of PHAe activity evaluation in the absence of a metabolic inhibitor, i.e., by subtracting the PAAe activity determined in PAAe activity assay medium from a combination of PAAe and PHAe activity determined in PHAe activity assay medium. Binary organic carbon sources (i.e., glucose and acetate) were included in the PHAe activity assay medium to fulfill the carbon requirements of most heterotrophic AOB genera. Higher ammonia-oxidizing activity in AOB biomass than heterotrophic AOB biomass (35.6 vs. 2.6–10.0 mg NH4+–N g−1 MLSS h−1) provides the remarkable advantages of autotrophic aerobic ammonia oxidization in biological nitrogen removal systems. Ammonia removal in three full-scale biological nitrogen removal systems for sewage treatment was predominantly mediated by PAAe activity (1.9–3.3 vs. 0.0–0.3 mg NH4+–N g1 MLSS h−1). [Display omitted] •It determines heterotrophic nitrifying activity in metabolic inhibitor-free assay medium.•Autotrophic aerobic ammonia-oxidizing activity is far higher than heterotrophic one.•Binary carbon sources in assay medium fulfill the requirements of most heterotrophic AOB genera.•Autotrophic aerobic microorganisms dominate ammonia oxidization in full-scale BNR systems.