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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 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]

• 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]

• Anammox (Ca.Brocadia , 0.69%) were enriched in biofilms, markedly higher than flocs. • 15N-isotope tracer tests showed partial denitrification is a prerequisite for anammox. • Anammox contributed to 68% of the nitrogen loss via anoxic biofilms in batch tests. • Nitrogen removal efficiency improved 16.9% by combining denitrification and anammox. • Mainstream partial-anammox might has great potential of engineering application. Introducing anammox technology into low-ammonia sewage treatment offers sustainable nitrogen removal, but there remain challenges to its future applications. Here, anammox bacteria were enriched by integrating into partial denitrification (NO 3 −-N to NO 2 −-N) in the anaerobic/anoxic/oxic (A2/O) reactor with low COD/N ratios (2.7 ± 0.4) sewage. This significantly enhanced nitrogen removal through partial anammox process over stable operation of 300 days. Microbial community analysis showed that the anammox bacteria were enriched in anoxic-carrier biofilms (Ca. Brocadia , 0.69%), and their abundance was significantly higher than the flocculent sludge (16 s rRNA sequencing: P < 0.001; qPCR: P < 0.001). Further activity testing via 15N-isotope tracing revealed that partial denitrification is a prerequisite for in situ occurrence of anammox reaction. Anammox contributed to 68% of the total nitrogen loss via anoxic-carrier biofilms in batch tests. Combining denitrification and anammox, the nitrogen removal efficiency of the A2/O process improved by approximately 16.9% for low COD/N ratios real sewage. [ABSTRACT FROM AUTHOR]