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Partial-denitrification coupled with anammox (PD/A) process is regarded as an emerging technology to improve nitrogen removal efficiency during municipal wastewater treatment. In this work, the shift from low to high nitrite accumulation in one-stage anoxic biofilm reactor using PD/A process was studied. When dinirogen gas (N 2) was pumped into the bioreactor to remove CO 2 , effluent nitrite concentration sharply increased from 0.3 mg N/L on day 10 without N 2 to 14.6 mg N/L on day 21 with N 2 , but the coupled anammox reaction almost stopped. Batch tests indicate that the metabolic process of PD/A underwent a notable shift, transitioning from initially low nitrite (<0.5 mg N/L) to high nitrite accumulation (13.5 mg N/L). Metagenomic sequencing demonstrated that Thauera was associated with the genes napA, napB and narG , supporting the improved nitrate-to-nitrite pathway. The shift in coupling mechanisms demonstrated that the one-stage PD/A process has significant practical potential. [Display omitted] [ABSTRACT FROM AUTHOR]

• 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 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]