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[Display omitted] • Anammox contribution to nitrogen removal was robustly maintained at 91.3 ± 6.6 %. • Compared with stable NO 3 – → NO 2 –, NO 2 – → N 2 was sensitive to temperature reduction. • Anammox abundance was increased (0.56% to 1.22%) despite of decreased temperature. • NO 2 – accumulation at lower temperature was unexpectedly helpful to enrich anammox. Effluent quality deterioration caused by seasonal temperature reductions in wastewater treatment systems using partial anammox technology is a challenge that cannot be ignored. Here, relationships of denitrification and anammox under decreasing temperature were investigated in an anoxic moving bed biofilm reactor (MBBR). Compared with stable partial-denitrification (NO 3 – → NO 2 –), the NO 2 – reduction to N 2 was considerably inhibited when the temperature decreased, conversely helping to the competition of NO 2 – for anammox. Namely, this transformation provided sufficient substrates for anammox bacteria. Although the TIN removal decreased slightly, anammox contribution was robustly maintained at 91.3 ± 6.6 %, even increased. High-throughput sequencing results revealed that anammox bacteria were enriched (0.56 % to 1.22 %). Moreover, qPCR results showed that increased ratio of hzsB /(nirK + nirS) further supported anammox gained an enhancement. This study demonstrated partial-denitrification/anammox process using anoxic MBBR could maintain stable autotrophic nitrogen removal contribution when encountering temperature decrease, providing a new perspective on the application of mainstream anammox. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anammox coupled with partial denitrification enhanced to nitrogen removal above 55%. • Ultra-high abundance of Ca. Brocadia (5.5, 6.5%, 5.8%) in connected anoxic biofilms. • Ca. Brocadia sapporoensis is acetate-resistant, explaining its high enrichment in biofilms. • Anammox activity increased with decreasing denitrifying activity along anoxic zones. For municipal wastewater treatment via partial-denitrification/anammox (PD/A) in the anoxic zone, the primary challenge is effective enrichment of anaerobic ammonium oxidation (anammox) bacteria with a stable autotrophic nitrogen removal performance. This long-term study investigated a pure-biofilm anoxic/oxic reactor using real municipal wastewater with an ammonia concentration of 49.1 ± 3.6 mg N/L, achieving a total inorganic nitrogen concentration of 11.2 ± 3.5 mg N/L in the effluent, resulting in a removal efficiency of 77.4 ± 7.2%. Driven by partial denitrification, the anammox contribution to total nitrogen removal represented an ultra-high level over 55%, and was considerable in the first anoxic zone. Candidatus Brocadia, the only anammox genus detected by 16S rRNA gene sequencing, reached robust abundances of 5.46, 6.49 and 5.79% in the three sequentially connected anoxic zones. Metagenomic sequencing further identified that the main species was Ca. Brocadia sapporoensis, which is inhibited by nitrite but not by acetate, supporting its high-level enrichment under acetate conditions in the anoxic zone. Batch tests indicate that heterotrophic denitrification activity decreased while anammox activity increased along the anoxic zones, and that excessive heterotrophic denitrifying activity was not conducive to increasing the anammox contribution. This study achieved robust anammox bacterial abundance using a pure-biofilm system, shedding fresh light on the application of mainstream anammox in municipal wastewater treatment. [ABSTRACT FROM AUTHOR]

• 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]