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[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]

• Improved NRE from 72.2% to 77.2% was firstly achieved as temperature decreases. • More tolerance of PD at low temperature increased nitrite substrates for anammox. • Ca. Brocadia increased by 167% and focused on biofilm (0.27%) at low temperature. • Anammox contribution improved from 41.8% to 60.2% and responsible for better NRE. Low temperature is one of the obstacles in mainstream anammox. This study aimed to evaluate the stability of a step-feed bioreactor dominated by denitratation/anammox (PD/A) for treating municipal wastewater with decrease in temperature (21.5 °C→12.6 °C). During long-term operation, nitrogen removal efficiency (NRE) was improved from 72.2% (21.5 °C→14.9 °C) to 77.2% (stable at 13.9 °C) with influent and effluent total inorganic nitrogen of 44.6 and 10.1 mg N/L. Better low temperature tolerance of nitrate reduction than nitrite reduction enhanced denitratation and increased nitrite substrates for anammox. Microbial structure analysis revealed that Ca. Brocadia increased by 167% and reached 0.27% on biofilm at low temperature. Finally, anammox contribution improved from 41.8% to 60.2%, saving organic carbon for denitrification, which was responsible for better NRE. Moreover, Ferribacterium, Dechloromonas , and Thauera were the major denitrifiers distributed in suspended sludge. Overall, this study provides an optimistic prospect for promoting anammox in mainstream applications at low temperature. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • PN/A-PD/A was efficiently realized in typical UCT process to treat municipal sewage. • TIN in influent and effluent were 51.3 and 11.0 mg/L even with COD/TIN ratio of 2.9. • Anammox activity improved from 6.52 to 9.68 mg NH 4 +-N/gMLSS/d with abundance raised. • Anammox contributed 47% for NRE with two coupled ways for continuous supply of NO 2 –. Improving contribution and nitrogen removal efficiency (NRE) of partial anammox in municipal wastewater is a researching hotspot. This study developed an innovative PN/A-PD/A process with fixed biocarriers in anaerobic/anoxic chambers for actual sewage treatment in a typical step-feed reactor over 390 days. Two coupled pathways providing continuous NO 2 – (partial nitrification in oxic chambers and partial denitrification in anaerobic/anoxic chambers) for anammox were introduced to the process, achieving 47% nitrogen loss by anammox in stable phase. The influent and effluent total inorganic nitrogen (TIN) were 51.3 and 11.0 mg/L, respectively, even with chemical oxygen demand (COD)/TIN ratio of 2.9. Anammox activity improved from 6.52 to 9.68 mg NH 4 +-N/gMLSS/d and abundance on the biocarriers raised to 3.16 × 1010 gene copies/g dry sludge. Overall, this study confirmed partial anammox, spatially coupled with partial nitrification and partial denitrification via oxic/anoxic distribution with step feed mode, as an alternative for application of mainstream anammox. [ABSTRACT FROM AUTHOR]

• The NRE reached 77.8 ± 4.3% even COD/TIN ratio at 2.9 during 548 days of study. • The effect of PD/A will be enhanced after the added anammox biocarriers adapted. • Anammox bacteria abundance on biocarriers remained stable and was higher than WWTPs. • PD/A contributed about 32–47% nitrogen loss in hypoxic zones in step-feed AAO reactor. The application of partial denitrification coupling with anammox (PD/A) in municipal sewage treatment is one of the most promising research field, and it has just begun. In 548 days of experiment treating real domestic sewage, the continuous flow reactor was started up by adding biocarriers containing anammox bacteria and operated stably after acclimatization. The PD/A was successfully achieved in the step-feed AO system. Furthermore, mass balance of ammonium revealed that anammox could contribute 32–47% to nitrogen loss. Compared with no biocarriers addition phase, the nitrogen removal efficiency (NRE) increased to 77.8 ± 4.3% and the effluent total inorganic nitrogen (TIN) decreased to 11.0 ± 2.1 mg/L at the COD/TIN ratio of 2.9 from day 428 to day 548 under the condition of anammox biocarriers addition and without external carbon source. Quantitative polymerase chain reaction (qPCR) and high-throughput sequencing analysis showed that anammox bacteria on the biological carrier accounted for 3.06% of the total bacteria in the stable phase, which was much higher than the reported <0.003% in wastewater treatment plants (WWTPs). After 247 days of operation with anammox biocarriers addition, the abundance of anammox bacteria on the carriers tended to be stable at 3.36 × 1010 gene copies g−1 dry sludge. The activity of anammox decreased and maintained at 13.14 g NH 4 +-N m−3 d−1 under the biocarriers-only condition, while increased from 11.71 g NH 4 +-N m−3 d−1 to 25.65 g NH 4 +-N m−3 d−1 in the hybrid condition of biocarriers and flocs. This study indicated that PD/A is effective to strengthen nitrogen removal with low COD/TIN ratio. [ABSTRACT FROM AUTHOR]

• Multiple coupling PN, PD and EPD with anammox was realized in a suspended sludge SBR. • Superior N-removal efficiency of 94% was achieved without external carbon source. • Anammox contributed 78% to TIN removal with 5 h aeration times (DO=0.5–0.8 mg/L). • Anammox activity and abundance increased during the PNA-(E)PDA process. • PNA-(E)PDA provides a novel option on mainstream municipal wastewater treatment. Achieving advanced nitrogen removal based on anammox for treating mainstream municipal wastewater in a single suspended sludge bioreactor is a challenging research topic. In this study, multiple coupling nitritation, denitritation and endogenous denitritation with anammox (PNA-(E)PDA) was simultaneously achieved in a 10 L step-feed bioreactor, which enhanced stable nitrogen removal. After 223 days of operation, the total nitrogen concentrations of the influent and effluent were 70.7 ± 6.1 and 4.3 ± 1.8 mg/L, respectively, when treating municipal wastewater even at C/N ratio of 2.24 with only 5 h of aerobic time (DO: 0.5–0.8 mg/L). After the evolution of nitritation/anammox to PNA-(E)PDA, the contribution of anammox to nitrogen removal increased to 78.6% and the anammox activity increased from 4.3 ± 0.2 to 15.2 ± 0.7 mg NH 4 +-N/gVSS/d. qPCR results showed that the abundance of anammox bacteria increased from 4.1 × 109 to 4.5 × 1010 copies/ (g VSS). High-throughput sequencing further revealed that the relative abundance of Candidatus Brocadia , the dominant anammox genus, increased from 0.09 to 0.46%. Based on the strong competitiveness of anammox on nitrite, this novel PNA-(E)PDA process provides a potential strategy for enriching anammox bacteria in municipal wastewater treatment plants. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Anammox contributed over 70% to N-removal with salinity of 0, 0.4, 0.7, and 1.0%. • Highest NRE of 81.2% and anammox contribution of 76.5% was achieved at 1.0%. • Salinity intensifies nitritation and denitratation processes further enhancing anammox. • Ca. Brocadia enriched on biofilms while Nitrosomonas enriched in suspended sludge. Saline wastewater present in municipal pipe networks poses challenges to biological nitrogen removal due to its inhibition on microorganisms. This study focuses on the effects of low salinity (0.0%, 0.4%, 0.7% and 1.0%) on a system featuring a combination of nitritation/anammox in oxic stage and denitratation/anammox in anoxic stage (double-anammox) in a step-feed SBR for municipal wastewater over a period of 130 days. The results showed that a maximum nitrogen removal efficiency of 81.2% was achieved at a salinity of 1.0% with anammox contribution of 76.5%. Analysis of anammox contribution and sludge activities discovered that low salinity promoted both nitritation and denitratation, further enhancing the coupling with anammox. Further, microbial analysis confirmed that Ca. Brocadia was enriched on biofilms from 0.21% to 0.51% and Nitrosomonas was enriched in flocs from 0.50% to 1.04%. Overall, the double-anammox process appears to be a promising method for the treatment of saline wastewater. [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]

In this study, enhanced nitrogen removal through in situ enrichment of anammox bacteria was successfully obtained in a full-scale municipal wastewater treatment plant (WWTP). The WWTP was an anaerobic-anoxic-oxic (AAO) process and upgraded by adding moving carriers into the anoxic zone. Enhanced nitrogen removal was obtained during almost two years of operation. The significant nitrogen removal might be associated with the in situ enrichment of anammox bacteria on the adding carriers, as revealed by the comprehensive results of molecular analysis and 15N-stable isotope tracing tests. Quantitative PCR results indicated that anammox bacteria in the anoxic-carrier biofilms presented a higher abundance than flocculent sludge (16S rRNA: P < 0.005; HzsB : P < 0.042). The 16S rRNA amplicon sequencing showed significant differences in the phylum Planctomycetes (P < 0.001) between anoxic-carrier biofilms and flocculent sludge. And metagenomic sequencing analysis further revealed the anammox relative abundance in the anoxic-carrier biofilms was significantly higher than the reported level in the flocculent sludge of conventional WWTPs. In addition, 15N-stable isotope tracing tests showed that anammox could be combined with nitrate reduction by the anoxic-carrier biofilms. Thus, enriched anammox bacteria might contribute to nitrogen loss and lead to improvements in the nitrogen removal, which was also supported by the mass balance analysis of organic carbon, nitrogen, and phosphorus of the WWTP. Overall, this study suggests that anoxic-carrier biofilms might be a candidate to enhance nitrogen removal through partial anammox in municipal WWTPs. Image 1 • The carriers added in the anoxic zone of the municipal WWTP could enhance N-removal. • The anoxic-carrier biofilms enriched with both denitrifies and anammox bacteria. • Anoxic biofilms show comparable AMX activity as denitrification by 15N isotope tracer. • Coupling nitrate reduction with anammox might enhance N-loss via anoxic biofilms. [ABSTRACT FROM AUTHOR]

[Display omitted] • Anammox-based process achieved highest NRE of 81.2% at salinity of 2.0%. • With real municipal sewage, influent and effluent TIN are 44.5 and 8.2 mg N/L. • Increased salinity inhibited NOB, nitrite reduction bacteria, and AOB by order. • Enhanced nitrite production and biofilm formation ensure high anammox contribution. • Anammox bacteria occupied 1.07% maximally and has strong adaptability to salinity. Nitrogen removal from saline municipal wastewater has recently attracted attention; thus, developing energy-efficient technology such as anaerobic ammonium oxidation (anammox) for advanced nitrogen removal of hypersaline wastewater is significant. In this study, the long-term salt adaptation and evolution mechanisms of an anammox-based process driven by both nitritation (NH 4 +→NO 2 —, PN) and denitratation (NO 3 —→NO 2 —, PD) were investigated under salinities ranging from 1.0% to 3.0% (10–30 g NaCl/L). At 1.0% salinity, PN evolved into the major nitrite production pathway with thorough suppression of nitrite oxidation. At 1.5% salinity, nitrite reduction was inhibited along with suspended sludge loss due to poor sludge settleability. Under 2.0% salinity with only-biofilm, the highest nitrogen removal efficiency (81.2%) was observed with influent and effluent total inorganic nitrogen concentrations of 44.5 and 8.2 mg N/L, respectively. Notably, results showed that anammox bacteria occupied up to 1.07% of the microbial community (qPCR: 1.14×1011 copies/(g dry sludge)) and has strong adaptability to salinity. With salinity increases, anammox contribution kept over 85% and Ca. Kuenenia (0.57%→0.22%) was more tolerant than Ca. Brocadia (0.46%→0.02%). Metagenomic sequencing revealed that the multiple nitrite substrates production by Nitrosomonas (amoA -enriched), Denitratisoma and Denitromonas (narG -enriched) guaranteed the high anammox contribution. This study provides a multifaceted understanding of the anammox-based process, which can enable improved application in real saline wastewater treatment. [ABSTRACT FROM AUTHOR]

[Display omitted] • Nitritation was stable with NAR always higher than 82% when temperature fluctuated. • Ca. Brocadia was enriched from 0.03% (20.4℃) to 0.24% (12.9℃) in suspended sludge. • Granulation was achieved in domestic wastewater system as temperature dropped. • Average particle size of sludge increased from 128.5 μm to 245.6 μm. • Relative abundance of Ca. Brocadia in granule was as high as 16.09% Low temperature is an important factor affecting the municipal wastewater treatment systems. The aim of this study was tracking the variations in the abundance of anammox bacteria (AnAOB) and the sludge form as the temperature decreased. Mutual boost of granulation and enrichment of AnAOB was achieved even though the temperature dropped from 20.4 °C to 12.9 °C. The average particle size of the sludge increased from 128.5 μm to 245.6 μm. With low dissolved oxygen (DO) aeration (0.2–0.5 mg/L) and short oxic hydraulic retention time (HRT) (5 h), nitritation in the anaerobic/oxic/anoxic (AOA) system was stable enough to provide NO 2 – for AnAOB. Ca. Brocadia , a type of typical AnAOB, was enriched from 0.03% to 0.24% in the suspended sludge and reached 16.09% in the granular sludge. Overall, this study presents the prospects of anammox and granule technologies when treating municipal wastewater at a low temperature. [ABSTRACT FROM AUTHOR]

[Display omitted] • Double-anammox driven by nitritation and denitratation was successfully achieved. • Anammox contributed over 70 % of nitrogen removal with real municipal wastewater. • Gradient reduction of C/N ratio was key for enhancing denitratation (NO 3 –→NO 2 –) process. • Ca. Brocadia maintained and concentrated on biofilms using one-stage step-feed SBR. • Denitratation gene (NarG) and nitritation gene (AmoA) were enriched in flocculent sludge. A novel double-anammox process for advanced mainstream nitrogen removal was established using step-feed sequencing batch reactor (SBR) system with integration of suspend sludge and biofilms. Following optimization of influent distribution ratio, the effluent total inorganic nitrogen (TIN) was < 10.2 mg N/L, with influent TIN of 43.4 mg N/L, and anammox contributed 71.4% to TIN removal. Biological processes and batch tests revealed that gradient C/N reduction promoted denitratation/anammox in anoxic stage, and simultaneous nitritation and anammox were achieved in oxic stage. Specially, anammox maintained on biofilms with abundance over 109 copies/ (g dry sludge). High-throughput sequencing revealed that Thauera and Nitrosomonas were enriched in flocs. Furthermore, metagenomic sequencing confirmed that Thauera owns narG and napA (NO 3 –→NO 2 –) and Nitrosomonas owns amoA (NH 4 +→NO 2 –), support stable NO 2 – supply for double-anammox. This mainstream anammox-dominant process could potentially be used for stable nitrogen removal in municipal wastewater treatment plants. [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]

Anaerobic ammonium oxidation (anammox) has been extensively investigated for cost-efficient nitrogen removal from wastewater. However, the major issues of nitrate (NO 3 −-N) residue and instability in the current combination of nitritation and anammox process necessitates being addressed efficiently. The recently proposed partial-denitrification (PD), terminating NO 3 −-N reduction to nitrite (NO 2 −-N), has been regarded as a promising alternative of NO 2 −-N supplying for anammox bacteria. Given the engineering practices, the steadily high NO 2 −-N production, alleviating organic inhibition, and reducing greenhouse gas of PD process offers a viable and efficient approach for anammox implementation. Moreover, it allows for the extending applications of anammox process due to the NO 3 −-N removal availability. Here we comprehensively review the important new outcomes and discuss the emerging applications of PD-based anammox including the process development, mechanism understanding, and future trends. Significant greater stability and enhanced nitrogen removal efficiency have been demonstrated in the novel integrations of PD and anammox process, indicating a broad perspective in dealing with the mainstream municipal sewage, ammonia-rich streams, and industrial NO 3 −-N contained wastewater. Furthermore, researches are still needed for the predictable and controllable strategies, along with the detailed microbiological information in future study. Overall, the achievement of PD process provides unique opportunity catalyzing the engineering applications of energy-efficient and environmental-friendly wastewater treatment via anammox technology. • Partial-Denitrification provides a stable and efficient alternative for nitrite supply. • PD process offers extending application of anammox treating nitrate wastewater. • PD/A holds potential in improved efficiency for mainstreams and sidestreams treatment. • Emerging opportunities are given for wastewater treatment engineering by PD/A. • Predictable controlling strategies and penetrating microbial mechanism are needed. [ABSTRACT FROM AUTHOR]