Quantification and Phylogenetic Analysis of Ammonia Oxidizers on Biofilm Carriers in a Full-Scale Wastewater Treatment Plant

Abstract

Biofilm carriers have been used to remove ammonia in several wastewater treatment plants (WWTPs) in Japan. However, the abundance and species of ammonia oxidizers in the biofilms formed on the surface of carriers in full-scale operational WWTP tanks remain unclear. In the present study, we conducted quantitative PCR and PCR cloning of the amoA genes of ammonia-oxidizing bacteria and archaea (AOB and AOA) and a complete ammonia oxidizer (comammox) in the biofilm formed on the carriers in a full-scale WWTP. The quantification of amoA genes showed that the abundance of AOB and comammox was markedly greater in the biofilm than in the activated sludge suspended in a tank solution of the WWTP, while AOA was not detected in the biofilm or the activated sludge. A phylogenetic analysis of amoA genes revealed that as-yet-uncultivated comammox Nitrospira and uncultured AOB Nitrosomonas were predominant in the biofilm. The present results suggest that the biofilm formed on the surface of carriers enable comammox Nitrospira and AOB Nitrosomonas to co-exist and remain in the full-scale WWTP tank surveyed in this study.

Keywords: WWTP; ammonia-oxidizing bacteria; biofilm; comammox; real-time PCR.

Fig. 1.

Schematic illustration of the full-scale WWTP in the present study. Solution containing activated sludge was collected from an anoxic (AO) tank and microaerophilic (MA) tank. A solution containing activated sludge and biofilm carriers (BC) was collected from an aerobic (A) tank.

Fig. 2.

Average concentrations of NH₄⁺, NO₂^–, and NO₃^– in anoxic (AO), microaerophilic (MA), and aerobic (A) tanks. Different letters above the columns indicate significant differences (P<0.05). SE represents the standard error.

Fig. 3.

Average abundance of AOB (A), comammox (B), and total bacteria (C) in the unit wet weight of the activated sludge in AO-, MA-, and A-tanks and the biofilm on BC. Different letters above the columns indicate significant differences (P<0.05). The comammox in the activated sludge of the tanks was below the detection limit (shown as ND). SE represents the standard error.

Fig. 4.

Percentages of AOB and comammox numbers in total bacterial numbers in the activated sludge of AO-, MA-, and A-tanks and the biofilm on BC. Different letters above the columns indicate significant differences (P<0.05). SE represents the standard error.

Fig. 5.

Average abundance of AOB (A), comammox (B), and total bacteria (C) in the unit volume (L) of solutions in AO-, MA-, and A-tanks, calculated based on numbers shown in Fig. 3. There were no significant differences among the samples (P>0.05). SE shows the standard error.

Fig. 6.

Neighbor-joining phylogenetic tree of betaproteobacterial AmoA amino acid sequences of clones retrieved from BC samples. AmoA of Nitrosomonas species and environmental clones detected as the best BLASTx hits were used as reference sequences. Nitrosospira multiformis ATCC 25196 was used for the outgroup. The numbers at nodes represent bootstrap values (%, 1,000 resampling); only values greater than 50% are indicated.

Fig. 7.

Neighbor-joining phylogenetic tree of comammox AmoA amino acid sequences of clones retrieved from BC samples. AmoA of Comammox Nitrospira species and environmental clones detected as the best BLASTx hits were used as reference sequences. Nitrosospira multiformis ATCC 25196 was used for the outgroup. The numbers at nodes represent bootstrap values (%, 1,000 resampling); only values greater than 50% are indicated.