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• Anammox bacteria were in situ highly enriched to 2.22% under the mainstream. • Anammox loss was decreased when SS exist, while denitrification was increased. • Metagenomic sequencing revealed SS weakened NO→N 2 but enhanced NO 3 −→NO. • Migration mechanism of anammox in biofilm alleviated the adverse effect of SS. • Novel real-time pH strategy on controlling PD/A process was firstly proposed. The basic premise of anammox-technical application reliability in municipal wastewater treatment is substantially enriched anammox bacteria. To enrich the anammox, the special interaction mechanism between the suspended sludge (SS) and anoxic biofilm was investigated over three months in a partial denitrification/anammox biosystem subjected to dynamic changes in SS (absence→ presence→ absence). Results show that the introduction of SS significantly decreased the anammox nitrogen removal efficiency (83.8 ± 6.5%→ 48.7 ± 17.0%). With the presence or absence of SS, the spatial distribution of anammox bacteria within the anoxic biofilm gradually changed between the inner and outer layers, as detected by CLSM-FISH. qPCR and metagenomic sequencing show that changes in the presence and absence status of SS significantly reduced the abundance of the NO reducing functional gene, while the NO supply capacity (NO 3 −→NO) was improved, further favoring the anammox process. Batch tests and typical cycles further demonstrated that the anammox bacteria can stably acquire NO 2 −, and anammox bacteria in the anoxic biofilm competed far more NO 2 − than denitrifying bacteria according to the typical pH curve. Accordingly, the abundance of Candidatus Brocadia, as detected by high throughput sequencing, decreased in the anoxic biofilms with the introduction of SS, but greatly increased (0.82%→2.22%) after SS discharge. This study sheds new light on the high in-situ enrichment of anammox in mainstream. [Display omitted] [ABSTRACT FROM AUTHOR]