© 2019 Society of Chemical IndustryBACKGROUND: The investigation of the anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) pathways has improved our insight into nitrogen removal from wastewater. The detection of dominant pathways is beneficial for gaining knowledge on the fate of nitrogen during wastewater treatment. The aim of this study is to explore the nitrogen removal pathways in case of fluctuation in nitrogen loading rate (NLR). For this purpose, the effect of fluctuation in NLR under different feeding strategies (short-term and long-term feeding) on the relative abundance of functional genes related to nitrogen removal pathways were investigated. With the analysis of genes encoding enzymes instead of the 16S rRNA genes, it was aimed to focus on processes rather than the microbial species. RESULTS: The inoculum consisted of aerobic and anaerobic sludge was determined as optimum inoculum for nitrogen removal via anammox when the long-term feeding was applied. The fluctuation in NLR resulted with dominance of different pathways in reactors operated with different feeding strategies. The statistical analyses indicated the importance of DNRA in achieving low nitrogen concentrations. Also, a positive significant correlation between nitrogen removal efficiency with aerobic ammonium oxidation revealed the importance of aerobic ammonium oxidation in nitrogen removal even under anoxic conditions. CONCLUSION: The main conclusions of this study may be summarized as (i) feeding strategy determines dominant nitrogen removal process, (ii) selecting appropriate inoculum is important to shorten the acclimation period, (iii) functional genes are useful as early warning system for track changes in process. © 2019 Society of Chemical Industry.