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• Fast start-up of mainstream PD/A without inoculation in A2/O process in 60 days. • Rapid in situ enrichment of AnAOB (2.49%) on the virgin carriers in anoxic zone. • NRE improved 16.9% with a stable effluent TIN of 11.6 mg/L under low COD/N ratio. • Anammox contributed to nitrogen loss of 37.9 ± 6.2% treating actual municipal sewage. • Anoxic biofilm performed a better coordination between denitrifiers and AnAOB. It is of great significance to start up the anammox process in the most commonly used anaerobic-anoxic-oxic (A2/O) process in treating mainstream municipal wastewater. Recently, partial-denitrification/anammox (PD/A) has attracted increasing interest as a new avenue in mainstream. This study investigated the in situ start-up of PD/A process in a traditional A2/O process. The PD/A system was rapidly started up within 60 days by adding virgin carriers into the anoxic zone and then run stably for the next 90 days. The in situ anammox activity reached 1.0 ± 0.1 mg NH 4 +-N/L/h contributing 37.9 ± 6.2% of total nitrogen removal. As a result, the nitrogen removal efficiency of the system increased by 16.9%. The anammox bacteria (AnAOB) on the anoxic biofilms were enriched with a doubling time of 14.53d, and the relative abundance reached 2.49% on day 150. Phylogenetic analysis showed the dominant AnAOB was related to Ca. Brocadia sp. 40, which was the only detected anammox genus in the anoxic biofilm from start-up to stable operation. Batch tests and qPCR results revealed that compared with the floc sludge, the anoxic biofilms exhibited NO 2 − accumulation driven by PD and performed a better coordination between denitrifiers and AnAOB. Overall, this study provides great confidence for the in situ fast start-up of mainstream anammox using conventional activated sludge. [Display omitted] [ABSTRACT FROM AUTHOR]