Contribution of ammonia oxidizing microorganisms and effect of paranitrophenol onammonia oxidation of nitrifying sludge 

The oxidation of ammonia to nitrite is the initial and rate-limiting step for most biological nitrogen removal approaches in wastewater treatment. However, the contribution of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia oxidation in wastewater treatment plants (WWTPs) has yet been clearly clarified. In this study, two laboratory nitrifying reactors (NRI and NRII) were seeded and operated under different conditions; therefore, different proportions of AOA and AOB arose in both reactors. AOA amoA genes outnumbered AOB amoA genes in reactor NRI, while only AOB amoA genes were the only detectable ammonia oxidizer in reactor NRII. The AOA amoA gene sequences from reactor NRI belonged to the Nitrososhaera sister cluster within the Group 1.1b Thaumacheota. Allythiourea (ATU) and 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), which were shown in previous studies to specifically inhibit AOB and AOA, respectively, were applied individually and as a mixture to observe the ammonia-oxidizing activity of AOA and AOB in NRI and NRII sludge. The results demonstrated that AOA and AOB jointly oxidized ammonia in NRI sludge, while AOB played the main role in ammonia oxidation in NRII sludge. DNA stable isotope probing (DNA-SIP) with 13C-HCO3- was performed on NRI sludge.  The 13C was incorporated into AOA and AOB amoA genes implying that both microorganisms may perform autotrophy during ammonia oxidation. DNA-SIP also showed that AOA can incorporate the 13C into the amoA genes while AOB cannot grow when 80 µM ATU was added. The results confirmed that ATU of 80 µM can be applied to clarify the ammonia-oxidizing activity of AOA in NRI sludge. ATU was applied to sludge from 5 full-scale WWTPs where the numbers of AOA and AOB amoA genes in the sludge varied. The results demonstrated that AOB played the main role in ammonia oxidation in all sludge. In the sludge that AOA outnumbered AOB, AOA involved around 20% of ammonia oxidation under presence of ATU at 80 µM. Inhibitory effect of paranitrophenol (PNP) was studied with sludge from reactor NRII. PNP at concentrations of ≥ 50 mgL-1 showed complete inhibition of ammonia oxidation. Analyses of bacterial cell viability and active nitrifying microorganisms using fluorescence in situ hybridization (FISH) technique indicated that PNP at concentrations of 10 and 200 mgL-1 tended to reduce bacterial cells and active AOB. The findings of this study can further lead to an improvement of wastewater treatment design and operation.