Anaerobic ammonia oxidation (Anammox) is an economical and sustainable wastewater nitrogen removal technology, and its application in the mainstream process is the inevitable trend of the development of Anammox. However, how to effectively enriching Anammox bacteria from the activated sludge remains challenging and restricts its extensive applications. In this study, the rapid and efficient enrichment of Anammox bacteria was achieved by raising the reflux ratio and nitrogen loading rate (NLR) using conventional activated sludge as the inoculant. In the screening phase (days 1–90), the reflux ratio was increased to discharge partial floc sludge, resulting in the relative abundance of Candidatus Brocadiaceae increased from 0.04% to 22.54%, which effectively reduced the matrix and spatial competition between other microorganisms and Anammox bacteria. On day 90, the stoichiometric ratio of the Anammox process closely approached the theoretical value of 1:1.32:0.26, indicating that the Anammox reaction was the primary nitrogen removal process in the system. In the enrichment phase (days 91–238), the NLR increased from 0.43 to 1.20 kgN/(m3·d) and removal efficiency was 71.89%, resulting in the relative abundance of Candidatus Brocadiaceae increased to 61.27% on day 180. The reactor operated steadily from days 444 to 498, maintaining the nitrogen removal rate (NRR) of 3.00 kgN/(m3·d) and achieving successful sludge granulation with the particle size of 392.4 µm. In short, this study provided a simple and efficient approach for enriching Anammox bacteria from the activated sludge, supporting to start an Anammox process efficiently.
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