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A novel biological nitrogen removal process can be developed based on the newly discovered nitrite-dependent anaerobic methane oxidation (n-damo) process. In this work, the short- and long-term effects of mineral nutrients on the n-damo process were investigated by single-factor and orthogonal experiments, respectively. The pH buffering capacities of different media were determined by acid–base titration. KHCO₃ and KH₂PO₄ greatly influenced the activity of n-damo bacteria and the pH changes of the media. An improved n-damo medium was proposed, containing 0.3 g L⁻¹ CaCl₂, 0.2 g L⁻¹ MgSO₄, 0.3 g L⁻¹ KH₂PO₄ and 0.2 g L⁻¹ KHCO₃. Moreover, the presence of 1% of CO₂ in gas could maintain the pH near neutral in the improved medium, which was first estimated in theory and then verified in practice. Finally, these results were applied in an n-damo reactor. The nitrogen removal rate was slightly …