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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]

[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]

• Anammox was stably enriched on treating municipal wastewater over 500 days' tests • Partial-denitrification/anammox contributed 29.2±6.7% of nitrogen removal • The Ca. Brocadia (4.34%) dominated in the anoxic-carrier biofilms rather than flocs • The Ca. Brocadia was key narG contributor to NO 3 − → NO 2 − in anoxic-carrier biofilms This study proposes a novel strategy of stably enriching anammox in mainstream, based on the competitive difference to NO 2 − between anoxic biofilms and suspended sludge. A modified anaerobic-anoxic-oxic (A2/O) process run for 500 days with actual municipal wastewater. Microbial analysis revealed that anoxic-carrier biofilms had a significantly higher abundance of anammox (qPCR: 0.74% – 4.34%) than suspended sludge (P< 0.001). Batch tests showed that anammox within anoxic-carrier biofilms contributed to significant nitrogen removal, coupled with partial-denitrification (NO 3 − → NO 2 −). The anammox genus, Ca. Brocadia, was highly enriched when suspended sludge was accidentally lost. Further batch tests found that reducing suspended biomass helped anammox enrichment in anoxic-carrier biofilms, because the suspended sludge had strong NO 2 − competition (NO 2 − → N 2) with anammox (increased nirK). Metagenomic sequencing revealed that Ca. Brocadia dominates in the anoxic-carrier biofilms, and is the most important narG contributor to NO 3 − → NO 2 −, which could have promoted the competition of NO 2 − with heterotrophic bacteria. For this A2/O process, the low effluent total nitrogen (8.9 mg ± 1.0 mg N/L) was attributed to partial-denitrification coupling with anammox, demonstrating that this process is applicable to the general influent N-concentration range (30 mg – 50 mg NH 4 +-N/L) of municipal wastewater treatment plants (WWTPs). Based on the special competitive preference of anammox for NO 2 −, this study provides a promising and practical alternative for enriching anammox bacteria in municipal WWTPs. [Display omitted] [ABSTRACT FROM AUTHOR]