Anammox bacteria have attracted attention due to their apparent importance in saving energy and reducing organic chemical demands. Here, we report the detection and quantification of anammox bacteria with an improved primer set in a validation wastewater treatment plant. The improved primer set was shown to detect a broad range of anammox bacteria (47.3%) facilitating more accurate analyses of nitrogen removal mechanisms. Nitrogen removal efficiency and dominant nitrogen removal mechanisms were compared in the modification-Johannesburg (Mod-JHB), modified Ludzack-Ettinger (MLE) single-feed, and anoxic-oxic-anoxic-oxic (AOAO) step-feed modes. In the Mod-JHB configuration, simultaneous nitrification and denitrification (SND) and anammox were found to be responsible for more than 80% of total inorganic nitrogen (TIN) removal (98.5 +/- 0.8% of TIN removal). Decrease of anoxic SRT from 5 to 2.5days did not have any obvious effect on nitrogen removal or the abundance of functional microorganisms. Microbial batch tests demonstrated that both partial nitrification and dissimilatory nitrate reduction to ammonium (DNRA) were responsible for maintaining the anammox process. Short SRT (2days) in the aerobic zone may explain the presence of partial nitrification. This study provides insights to the analysis of nitrogen removal mechanisms in validation wastewater treatment plants (WWTPs) aiming for high nitrogen removal efficiency.