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Removing nitrogen and phosphorus from low ratio of chemical oxygen demand to total nitrogen and temperature municipal wastewater stays a challenge. In this study, a pilot-scale anaerobic/aerobic/anoxic sequencing batch reactor (A/O/A-SBR) system first treated 15 m 3 /d actual municipal wastewater at 8.1-26.4 °C for 224 days. At the temperature of 15.7 °C, total nitrogen in influent and effluent were 45.5 and 10.9 mg/L, and phosphorus in influent and effluent were 3.9 and 0.1 mg/L. 16 s RNA sequencing results showed the relative abundance of Competibacter and Tetrasphaera raised to 1.25 % and 1.52 %. The strategy of excessive, no and normal sludge discharge enriched and balanced the functional bacteria, achieving an endogenous denitrification ratio more than 43.3 %. Sludge reduction and short aerobic time were beneficial to energy saving contrast with a Beijing municipal wastewater treatment. This study has significant implications for the practical application of the AOA-SBR process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In anaerobic/aerobic/anoxic (A/O/A) process, endogenous denitrification (ED) is critically important, and achieving steady endogenous partial denitrification (EdPD) is crucial to carbon saving and anammox application. In this study, EdPD was rapidly realized from conventional activated sludge by expelling phosphorus accumulating organisms (PAOs) in anaerobic/anoxic (A/A) mode during 40 days, with nitrite transformation rate (NTR) surging to 82.8 % from 29.4 %. Competibacter was the prime EdPD-fulfilling bacterium, soaring to 28.9 % from 0.5 % in phase II. Afterwards, balance of high NTR and phosphorus removal efficiency (PRE) were attained by well regulating competition and cooperation between PAOs and glycogen accumulating organisms (GAOs) in A/O/A mode, when the Competibacter (21.7 %) and Accumulibacter (7.3 %, mainly Acc_IIC and Acc_IIF) were in dominant position with balance. The PRE recovered to 88.6 % and NTR remained 67.7 %. Great balance of GAOs and PAOs contributed to advanced nitrogen removal by anammox., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Advanced nitrogen removal cannot be achieved through the conventional biological nitrogen removal process, which requires higher carbon sources and aeration energy. The proposal of intermittent aeration in the aerobic chambers offered an innovative approach to enhance nitrogen removal in low carbon-to-nitrogen ratio (C/N) municipal sewage, using a plug-flow reactor with anaerobic/aerobic/anoxic (AOA) process. Due to the effective utilization of internal carbon sources through the intermittent aeration, the total inorganic nitrogen removal efficiency (NRE) increased to 77.9 ± 3.2 % with the mean aerobic hydraulic retention time of only 3.2 h and a low C/N of 3.3 during the operation of 210 days. Polyhydroxyalkanoates dominated the nitrogen removal in this AOA system, accounting for 48.0 %, primarily occurring in the alternant aerobic/anoxic chambers. Moreover, the microbial community structure remained unchanged while the NRE increased to 77.9 %. This study provided an efficient and economic strategy for the continuous plug-flow AOA process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In suspended sludge system, efficient enrichment and retention of anammox bacteria are crucial obstacles in mainstream wastewater treatment by anammox process. In this study, anammox bacteria was self-enriched in a pilot-scale suspended sludge system of two-stage nitrification-denitrification process serving municipal wastewater treatment. With the low ammonia (NH 4 + -N) of 9.3 mg/L, nitrate (NO 3 - -N) of 15.6 mg/L and COD/NO 3 - -N of 2.2 under extremely low nitrogen loading rate of 0.012 kg N/m 3 /d, anammox activity bloomed after its abundance increasing from 5.9 × 10 7 to 4.6 × 10 9 copies/g dry sludge. Significant NH 4 + -N removal was occurred and maintained stably in the denitrification reactor with anammox bacteria accounting for 1.13%, even under temperature decreasing to 20.0℃. The adequately anoxic environment, efficient retention with the static settlement, and NO 2 - production via NO 3 - reduction provided favorable environment for anammox bacteria. This study demonstrated the feasibility and great potential in mainstream anammox application without seeding specific sludge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Despite the advantages of the combined anammox and fermentation-driven denitrification process in nitrogen removal and energy consumption, stable performance at decreased temperatures remains a challenge. In this study, a robust and high-efficient nitrogen removal efficiency (95.0-93.1 ∼ 86.8-93.4%) with desirable effluent quality (3.0-4.1 ∼ 7.9-4.9 mg/L) under long-term decreased temperatures (30 °C→25 °C→20 °C) was achieved in a zero-external carbon Partial Nitritation/Anammox combined with in-situ sludge Fermentation-Denitrification process treating sewage. Excellent sludge reduction averaged at 14.9% assuming no microbial growth. Increased hzsB mRNA (2.2-fold) and reduced E a (80.9 kJ/mol) proved resilient anammox to lower temperature. RT-qPCR tests revealed increased NarG/NirK (5.1) and NarG/NirS (4.9) mRNA at 20 °C, suggesting higher NO 3 - →NO 2 - over NO 2 - →N 2 pathway. Metagenomics unraveled dominant anammox bacteria (Candidatus_Brocadia, 2.27%), increased denitritation bacteria containing more NarG (Hyphomicrobium, 0.8%), fatty acid biosynthesis and CAZymes genes. Enhanced denitritation with recovered organics from sludge reserved nitrite for anammox and facilitated higher anammox contribution to N removal at 20 °C (42.4%) than 30 °C (39.5%). This study proposed an innovative low-temperature strategy for in-situ sludge fermentation, and demonstrated stability of advanced municipal wastewater treatment and sludge disposal through energy savings and carbon recovery under decreased temperatures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The application of mainstream anammox is highly desirable for municipal wastewater treatment. However, enrichment of anammox bacteria (AnAOB) is challenging, particularly given the vicious competition from denitrifying bacteria (DB). Here, suspended sludge biomass management, a novel operational strategy for hybrid process (suspended sludge/biofilm), was investigated for 570 days based on a modified anaerobic-anoxic-oxic system treating municipal wastewater. By successively decreasing the suspended sludge concentration, the traditional hybrid process was successfully upgraded to a pure biofilm anammox process. During this process, both the nitrogen removal efficiency (NRE) and rate (NRR) were significantly improved (P < 0.001), from 62.1 ± 4.5% to 79.2 ± 3.9% and from 48.7 ± 9.7 to 62.3 ± 9.0 g N/(m 3 ·d), respectively. Mainstream anammox was improved in the following: Candidatus Brocadia was enriched from 0.70% to 5.99% in anoxic biofilms [from (9.94 ± 0.99) × 10 8 to (1.16 ± 0.01) × 10 10 copies/g VSS, P < 0.001]; the in situ anammox reaction rate increased from 8.8 ± 1.9 to 45.5 ± 3.2 g N/(m 3 ·d) (P < 0.001); the anammox contribution to nitrogen removal rose from 9.2 ± 2.8% to 67.1 ± 8.3% (P < 0.001). Core bacterial microbiome analysis, functional gene quantification, and a series of ex situ batch experiments demonstrated that the stepwise decreases in suspended sludge concentration effectively mitigated the vicious competition of DB against AnAOB, enabling high-level AnAOB enrichment. This study presents a straightforward and effective strategy for enriching AnAOB in municipal wastewater, shedding fresh light on the application and upgradation of mainstream anammox., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Since unsustainable partial nitrification prone to unstable nitrogen removal rates, cultivation and enrichment of AnAOB, further improve autotrophic nitrogen removal contribution have been challenges in the mainstream anammox process. This study proposed a new strategy to enrich AnAOB motivated by endogenous partial denitrification (EPD) in total floc sludge system through the AOA process with sustainable nitrification. The results showed that in the presence of NH 4 + and NO 3 - at the anoxic stage of N-EPDA, Ca. Brocadia was enriched (0.005%→0.92%) in floc sludge via internal carbon source metabolism of EPD. The C/N and temperature of N-EPDA were also optimized to achieve higher activities of EPD and anammox. The N-EPDA was operated at low C/N ratio (3.1) with anammox nitrogen removal contribution of 78% during the anoxic stage, Eff.TIN of 8.3 mg/L and NRE of 83.5% during phase III, achieved efficient autotrophic nitrogen removal and AnAOB enrichment in the absence of partial nitrification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Efficient retention and enrichment of anammox bacteria (AnAOB) are essential for the application of municipal wastewater anammox. Herein, an innovative process for highly enriching AnAOB within suspended carrier was developed in a single-stage anaerobic/oxic/anoxic reactor with 5.5 % carrier filling ratio for real sewage. Addition of sludge fermentation products promoted stable maintenance of partial nitrification (nitrite accumulation rate > 90.0 %) and achieved efficient external sludge reduction (27.6-37.9 %). Continuous nitrite supply and carrier addition promoted AnAOB enrichment (2.4 × 10 11 gene copies/g dry sludge). Candidatus Brocadia was the predominant bacteria in carriers (18.6 %). The average effluents of total inorganic nitrogen (TIN) and NH 4 -N were 1.9 and 0.8 mg/L with removal rates of 97.0 % and 98.7 %. In the anoxic stage, TIN removal rate reached 71.5 %, and the proportion of anammox to nitrogen removal accounted for 82.7 %. This study broadens the application of mainstream sewage anammox and the resource utilization of waste activated sludge.+ -N were 1.9 and 0.8 mg/L with removal rates of 97.0 % and 98.7 %. In the anoxic stage, TIN removal rate reached 71.5 %, and the proportion of anammox to nitrogen removal accounted for 82.7 %. This study broadens the application of mainstream sewage anammox and the resource utilization of waste activated sludge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

It is of great significance to start up the anammox process in the most commonly used anaerobic-anoxic-oxic (A 2 /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 A 2 /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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

The stable nitrite (NO 2 - -N) generation and rapid startup of anammox-based process are the main bottlenecks hindering its application in mainstream municipal wastewater treatment. In this study, a Partial-Denitrification (PD) system reducing nitrate (NO 3 - -N) to NO 2 - -N was rapidly developed within 40 days, using the nitrification/denitrification sludge from wastewater treatment plant. The NO 3 - -N to NO 2 - -N transformation ratios achieved 80.6 %. Significantly, a fast self-enrichment of anammox bacteria in this system was subsequently obtained, resulting in the successful transformation to an efficient PD/Anammox (PD/A) process after 79-day operation. The total nitrogen removal efficiency increased from 12.4 % to 90.0 % with influent ammonia and nitrate of 45.9 mg N/L and 62.2 mg N/L, corresponding to the anammox activity significantly increasing to 6.0 mgNH 4 + -N/g VSS/h without seeding anammox sludge. Abundance of anammox increased from 6.7 × 10 8 to 2.0 × 10 11 copies/g dry sludge. High-throughput sequencing results showed that Candidatus Brocadia was the only known anammox genus and accounted for 1.08 % during the PD/A stage. Functional bacteria for PD, assumed to be the Thauera, was enriched from 1.99 % to 60.06 % but decreased to 32.49 % during the improvement of anammox activity. It demonstrated that the PD system with stable NO 2 - -N accumulation enabled a rapid self-enrichment of anammox bacteria and sufficient nitrogen removal with ordinary nitrification/denitrification sludge. This provides new insights into the scaling application of anammox by integrating PD with shortened startup periods and improved TN removal efficiency., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Enhancement of anaerobic ammonium oxidation (anammox) process and enrichment of anammox bacteria in the oxic zone of mainstream sewage treatments are complex. Also, quantification of the anammox contribution for nitrogen removal in the oxic zone is hindered owing to the simultaneous occurrence of anammox and nitrification. An alternating anaerobic/oxic/anoxic/oxic bioreactor whose oxic zone boosted partial nitrification coupling anammox (PN/A) and anoxic zone boosted partial denitrification coupling anammox (PD/A), respectively, was operated to treat real sewage for >380 days. Desirable nitrogen removal (effluent total inorganic nitrogen (TIN) of 4.7 ± 1.9 mg N/L) was obtained from low carbon/nitrogen (3.6 ± 0.5) sewage with ammonium concentration of 52.5 ± 2.2 mg N/L in the influent. Under the condition of dissolved oxygen (DO) of 1.5-3 mg/L, anammox bacteria was still enriched within the aerobic biofilms, with the relative abundance increasing to 8.2 % (day 345) from 0 % (no biomass on day 1), which was higher than the value in the flocculent sludge (0.03 %) (P < 0.001). PN driven by flocculent sludge with high activity of ammonium oxidized bacteria (AOB) ensured sufficient nitrite supply for the anammox process with the existence of continuous DO. During the steady operation period, the maximum anammox contribution in the oxic zone was quantified to be 10.6 % by withdrawing aerobic biofilms from the system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The application of partial nitrification-anammox (PNA) in continuous flow processes for treating low COD/N (C/N) sewage remains a critical challenge. Here, a traditional continuous anoxic/oxic (A/O) process was operated to investigate nitrogen removal from municipal wastewater by the bio-augmentation of partial nitrification sludge combined with the inoculation of biocarriers under decreasing temperatures. Stable enhanced nitrogen removal via PNA was achieved. The average total inorganic nitrogen in influent and effluent was 44.3 and 7.1 mg N/L under a low C/N ratio (3.4) and a short hydraulic retention time (8.2 h). The bio-augmentation of partial nitrification sludge enhanced the PNA process under low temperatures (16.9 ± 0.6 °C). The nitrogen removal efficiency remained stable at 83.3 ± 5.7 % as the temperature decreased from 29.1 to 16.3 °C, and the relative abundance of Ca. Brocadia in carrier biofilms increased from 2.22 % to 4.31 % and 3.27 % in two aerobic chambers after 70 days of operation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A novel two-stage partial nitrification-anammox (PN-A) process was developed, achieving nitrogen removal from low carbon/nitrogen ratio municipal sewage under two typical operational modes and seasonal ambient temperatures. When complete nitritation-anammox was performed at temperatures greater than 19.4 °C, the effluent concentration of total inorganic nitrogen (TIN) was 4.1 mg/L, corresponding to a nitrogen removal efficiency (NRE) of 94.3 %. In contrast, when partial nitritation-anammox was performed at temperatures below 19.4 °C, the effluent TIN was 12.3 mg/L, corresponding to a NRE of 83.6 %. The relative abundance of Nitrosomonas and Nitrosomonadaceae increased from 0.02 % to 0.28 %, while Ca. Brocadia decreased from 1.85 % to 1.30 %, with the contribution of anammox to nitrogen removal being highest under low temperatures (19.4℃ to 13.8℃), at 59.0 %. This novel two-stage PN-A process provides a new approach for the stable operation of wastewater treatment plants (WWTPs) under low ambient temperatures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Directly integrating anammox into sewage treatment is attractive, but anammox bacteria (AnAOB) enrichment is complex due to vicious competition from heterotrophic bacteria (HB). A novel strategy of optimal organics management using a preanaerobic stage and subsequent limited-oxygen conditions (0.32 ± 0.15 mg-O 2 /L) is applied, and a hybrid autotrophic-heterotrophic denitrification process is developed to treat sewage-like wastewater with a COD/N ratio of 3.1 for 420 days. The stable process was achieved, and a high total nitrogen removal rate of 0.53 kg-N/(m 3 ·d) was obtained compared to conventional nitrification/denitrification. The 16S rRNA high-throughput sequencing analysis suggested that the relative abundance of the nonendogenous HB ( Denitratisoma and Thauera ) was drastically reduced ( P ≤ 0.001), whereas the endogenous denitrifying HB ( Candidatus ( Ca. ) Competibacter) was significantly enriched in the anammox granules (9.98%, P ≤ 0.001). Moreover, Ca. Competibacter as an inner core and Nitrospira and Ca. Brocadia as an outside coating of the anammox granules indicated the cooperation of AnAOB with HB as revealed by laser-scanning confocal microscopy and qPCR. In situ tests further confirmed nitrite from two pathways (partial nitritation and endogenous partial denitritation) that favored AnAOB enrichment. Optimal organics management can mitigate the competition of HB with AnAOB by redirecting the metabolic pathways and microbial community, which is critical to directly integrating anammox into sewage treatment.

The collapse of simultaneous partial nitrification, denitrification and anammox (SPNDA) system, caused by the destruction of partial nitrification (PN), is the most likely phenomenon to occur. Therefore, recovering the process quickly and maintaining efficient nitrogen removal is a valuable topic for research. In the anaerobic/aerobic/anoxic operation mode, SPNDA process was used to treat domestic sewage in a sequencing batch biofilm reactor. After the deterioration of PN effect, with the addition of hydroxylamine, the activity of ammonia-oxidizing bacteria in the nitrobacteria increased (61.0-91.3 %), whereas the accumulation of nitrite quickly recovered to 90.4 % within 5 days. Meanwhile, the nitrogen removal efficiency improved (61.8-95.6 %) and the effluent TN was 2.1 mg/L. Furthermore, Candidatus Brocadia was enriched (0.50-1.82 %) in the system. The results indicated that the addition of hydroxylamine was an effective strategy to recover and economically maintain the SPNDA process for advanced nitrogen removal from domestic sewage., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In low-ammonia sewage anammox process, cultivation and enrichment of anammox bacteria (AnAOB) is a challenge especially from traditional activated sludge. To this end, a novel strategy solely using activated sludge as inoculum and biocarrier in a dynamic fixed-bed reactor was proposed in this study. During 115-day operation, excellent performance was achieved with influent total inorganic nitrogen (TIN) and effluent TIN of 55.3 mg·L -1 and 4.1 mg·L -1 , respectively. Rapid enrichment of AnAOB (doubling time: 8.5 days) was demonstrated by augmented specific anammox activity (trace value to 1.85 mg N·g VSS -1 ·h -1 ) and increased hzsB gene number (10 6 to 10 9 copies·g -1 dry sludge), with predominance of Candidatus&#95;Brocadia. Large-flocs aggregate was the primary habitat for AnAOB with highest abundance and capacity. The distinctive sludge properties, symbiotic microbial interactions and dynamic operation scheme facilitated AnAOB growth and retention. This study provides a simple, economic and workable approach for the start-up of mainstream anammox process., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 × 10 9 to 4.5 × 10 10 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A continuous-flow anaerobic/aerobic/anoxic reactor with complete suspended activated sludge using sludge alkaline fermentation products as carbon source was utilized to strengthen nitrogen removal performance for low C/N ratio (<4) wastewater. Long-time performance indicated that the nitrite accumulation rate reached 60.40%, which strengthened the contribution of anammox. The average total inorganic nitrogen removal efficiency improved 19.40%. The abundance of ammonia oxidizing bacteria has not changed, but the abundance of nitrite oxidizing bacteria reduced from 5.79% to 0.69%. Quantitative PCR results demonstrated that the abundance of anammox bacteria has raised by 80.5 times. These results indicated that side-stream sludge alkaline fermentation promoted the mainstream partial nitrification, consequently accelerating the in-situ enrichment of anammox bacteria. No external carbon source dosing and short oxic hydraulic retention time (5.3 h) save energy and reduce consumption significantly in this system., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The pH control to promote short-chain fatty acids (SCFAs) production during anaerobic alkaline fermentation basically focused on constant pH control. In this study, a simple and consumption-reducing intermittent pH control strategy at moderate temperature (23 ± 2 °C) was investigated with adjusting pH to 10 when naturally reduced to 8. The intermittent pH control strategy could alleviate the inhibition of acid-producing bacteria by strong alkaline and high FA concentration. Meanwhile, microbial diversity promoted by 6% and 69% while the relative abundance of acid-producing bacteria increased by 36% and 61% compared to blank and constant pH fermenters. The relative genes abundance related to amino acid metabolism and fatty acid production were mostly promoted and led to enhanced SCFAs production. In the long-term fermenter, the intermittent pH control strategy could result in a 68% reduction in alkali consumption and a 37% increase in SCFAs production compared to that of the constant pH at 10., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The start-up, efficient, and secure operation of Anammox treating low ammonia sewage, is an important research focus. In this study, a partial nitrification-denitrification coupled with simultaneous Anammox and denitrification (PND-SAD) process was achieved in sequencing batch reactor/up-flow anaerobic sludge bed (SBR-UASB). The key measures to maintain high efficiency PND were: (i) controlling dissolved oxygen in the SBR below 0.5 mg/L, which is not only conducive to PN, but also promotes the contribution of simultaneous nitrification and denitrification to nitrogen removal; (ii) monitoring the nitrate (NO 3 - -N exceeds 1.0 mg/L. The nitrite accumulation rate reached 97.6%. SBR effluent and domestic sewage entered the UASB. Although Candidatus Brocadia only accounted for 0.8%, its contribution to nitrogen removal reached 76.8%. In PND-SAD system, the aerobic HRT was only 3.8 h, nitrogen removal efficiency up to 97.3%.3 - -N exceeds 1.0 mg/L. The nitrite accumulation rate reached 97.6%. SBR effluent and domestic sewage entered the UASB. Although Candidatus Brocadia only accounted for 0.8%, its contribution to nitrogen removal reached 76.8%. In PND-SAD system, the aerobic HRT was only 3.8 h, nitrogen removal efficiency up to 97.3%., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 × 10 10 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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aggregation of anammox bacteria is essential to maintain high biomass concentrations and prevent the loss of biomass in anammox processes. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used in this study to predict the metagenomic potentials and characterize the microbial community structure and functional features in anammox aggregates (e.g., sludge flocs, biofilms, and granules). The results showed that Candidatus Brocadia was the most dominant anammox genera in all aggregates (38.0% in flocs, 69.4% in biofilm, and 52.0% in granules) and the functional gene involved in the anammox process was detected in the highest amount in biofilms, followed by granules and flocs. Furthermore, the anammox microbial aggregation pathway was explored that anammox bacteria have strong motility and high capability for early attachment. Anammox bacteria could produce large amounts of EPS (extracellular polymeric substances) regulated by quinolone and transport to extracellular environment through type II secretion system. The strong ability of c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate) synthesis enabled a stable architectural structure of aggregation. This study elucidated the aggregation mechanism of anammox microorganisms at the genetic level to enhance the stability of anammox processes. PRACTITIONER POINTS: Candidatus Brocadia was the most dominant anammox genera in aggregates. Anammox bacteria have strong motility and high attachment capability. Anammox bacteria possess strong EPS synthesis regulated by quinolone. c-di-GMP synthesis enables a stable structure of aggregation., (© 2021 Water Environment Federation.)

Denitrifying phosphorus removal (DPR) technology is one of the most effective approach to simultaneously realize nitrogen (N) and phosphorus (P) removal from low COD/N ratio wastewater. Identifying the interaction of denitrifying phosphate-accumulating organisms (DPAOs), denitrifying glycogen organisms (DGAOs) and denitrifying ordinary heterotrophic organisms (DOHOs) is critical for optimizing denitrification and anoxic P uptake efficiency in DPR processes. In this study, a novel DPR system of anaerobic anoxic oxic - biological contact oxidation (AAO-BCO) was employed to dispose actual sewage with various influent COD/N ratios (3.5-6.7). High efficiency of TIN (76.5%) and PO 4 3- -P (94.4%) removal was observed when COD/N ratio was between 4.4 and 5.9. At the COD/N ratio of 5.7 ± 0.2, prominent DPR performance was verified by the superior DPR efficiency (88.7%) and anoxic phosphorus uptake capacity (PUA DPAOs /ΔTIN = 1.84 mg/mg), which was further proved by the preponderance of DPAOs in C, N and P removal pathways. GAOs have a competitive advantage over PAOs for COD utilization at low COD/N ratio of 3.7 ± 0.2, which further limited the N removal efficiency. High proportion of N removal via DOHOs (21.2%) at the COD/N ratio of 6.5 ± 0.2 restrained the DPR performance, which should be attributed to the outcompete of DOHOs for NO 3 - . The nutrient removal mechanisms were explicated by stoichiometric calculation methodology to quantify the contribution of diverse functional microorganisms, contributing to improving the robustness of AAO-BCO system when facing the fluctuation of influent carbon source concentration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The two-stage partial nitrification (PN)-Anammox process, during long term treatment of high-ammonia nitrogen leachate, faces challenges such as the adaptation of nitrite oxidation bacteria (NOB) and failure of real-time control of pH. Resultant instabilities including NH 4 + -N and NO 3 - -N accumulation were overcome by culturing sludge fermentation liquid (SFL)-driven partial denitrification (PD) in situ in the Anammox process. Biodegradation of slowly biodegradable organics (SBO) in SFL created organics restriction condition, which limited the activity of denitrification bacteria and achieved its balance with Anammox bacteria. Produced NO 3 - -N is reduced to NO 2 - -N through PD, which further improved the removal of NH 4 + -N through Anammox. NO 2 - -N was utilized timely by Anammox bacteria, which avoid further reduction of NO 2 - -N to N 2 , and result in a high nitrate to nitrite transformation ratio (NTR) of 93.3%. Satisfactory nitrogen removal efficiency (NRE) and nitrogen removal rate (NRR) of 99.6% and 822.0 ± 9.0 g N/(m 3 ∙d) were obtained, respectively. Key genera related to degradation of SBO, PD and Anammox were enriched. The value of narG/(nirK+nirS) increased from 0.05 on day 1-0.15 on day 250. Combining SFL-driven PD with two-stage Anammox process provided a novel insight for applying this process to realize advanced nitrogen removal in practical engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Rapid achievement of nitritation of mainstream municipal wastewater in a continuous-flow process is attractive since it favors the involvement of the anammox process and reduces the operational costs. In this study, a feasible and economical strategy is proposed to rapidly achieve the nitritation of municipal wastewater. By aggressively discharging excess sludge during the seasonal warming period (temperature increasing from 18°C to 22°C), nitritation was established in 15 days with a nitrite accumulation ratio of 85.09% in a continuous-flow anaerobic/oxic (An/O) reactor. Meanwhile, qPCR results revealed that amoA abundance increased from (1.78±0.10) × 10 8 copies/(g VSS) to (1.05±0.11) × 10 10 copies/(g VSS) while the abundance of nitrite-oxidizing bacteria decreased from (1.1±0.02) × 10 10 copies/(g VSS) to (5.01±0.02) × 10 8 copies/(g VSS). The temperature gradually stabilized at 26°C during the following operational period and stable nitritation was maintained with a nitrite accumulation ratio above 90%, which was mainly attributed to a short sludge retention time (SRT) of 4.3 days and a low dissolved oxygen of 0.86 ± 0.5 mg/L. Falling temperature negatively impacted the stability of nitritation, but nitritation could be restarted by aggressively discharging excess sludge during another temperature increase period. Overall, this study provides a feasible strategy to start-up nitritation that has great potential applications for municipal wastewater treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The realization of stable partial nitrification and advanced nitrogen removal are not acquired effectively in conventional pre-denitrification biological nitrogen removal processes treating domestic sewage. Herein, a novel anaerobic/aerobic/anoxic/aerobic (AOAO) continuous plug-flow reactor, characterized with double sludge reflux and a bypass of anaerobic mixed liquor conveyed to anoxic zone, was first constructed to realize stable partial nitrification in treating domestic sewage. The alternating anoxic/aerobic conditions and longer anoxic sludge retention time might be responsible for the partial nitrification. Nitrite accumulation ratio reached 89.3 ± 3.3% with the maximum activity ratio of AOB to NOB increasing from 0.72 to 8.17. A content total inorganic nitrogen (TIN) removal efficiency (93.7 ± 2.2%) and effluent TIN concentration (2.9 ± 0.9 mg N/L) were obtained after 238 days' operation. Specifically, nitrogen balance of the typical cycle showed that about 30.1% of TIN was removed through simultaneous partial nitrification and denitrification (SND) in aerobic zone and 48.2% by endogenous denitrification in anoxic zone. The AOAO process is an economic treatment for domestic sewage with aerobic hydraulic retention time (HRT) of 4 h., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Partial-denitrification (PD; NO 3 - →NO 2 - ) has recently been proposed to be a feasible choice of NO 2 - -N supply for anammox bacteria. In this study, an aerobic/anoxic process for treating domestic wastewater was operated for 176 days to evaluate the feasibility of using sludge fermentation liquid for partial denitrification of the wastewater. Results show that, with the ratio of C/N (COD/ NO 3 - -N) increased at anoxic stage, the average NO 2 - -N concentration in the effluent and nitrate-to-nitrite transformation ratio (NTR) at anoxic stage showed relative growth. High-throughput sequencing analysis demonstrated that the enhancement of PD can be explained by the increases of Thauera, Paracoccus and Enterobacteriaceae. Moreover, Candidatus&#95;Brocadia (0.13%) was detected as the predominant anammox bacteria. Ex-situ isotopic tracing technique analysis assessed that the ratio of anammox role (ra%) was 7.29%. This study has a great potential for being coupled with the anammox bacteria for advanced nitrogen removal., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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 (A 2 /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 A 2 /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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Denitrifying phosphorus removal sludge are usually faced with various famine environments in wastewater treatment plants (WWTPs). Endogenous metabolisms under aerobic, anoxic, and anaerobic starved conditions were characterized to investigate their impact on survival and activities of denitrifying polyphosphate accumulating organisms (DPAOs). DPAOs utilized intracellular polymers to survive and presented diverse consumed priorities of PHA types under various starvations. The biomass decay rate was approximately 2.7 and 1.7 times lower for aerobic condition than for anoxic and anaerobic conditions owing to the maximum maintenance energy requirement for aerobic condition (68.6 mmol/C-molVSS ATP). During short-term starvations, significant activity decay for anaerobic starved sludge was attributed to its distinctive endogenous metabolisms. For long-term starvations, the higher amounts and preponderant type of PHA (PHB) reserve favored to the greater DPAO activities for anoxic starved sludge. The results show that anoxic condition may be an implementable strategy for maintaining denitrifying phosphorus removal performance in WWTPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study used salinity (0.5 wt%, 0.75 wt%) to accelerate the formation of ammonia oxidizing bacteria (AOB)-enriched aerobic granular sludge in a lab-scale anaerobic/micro-aerobic simultaneous partial nitrification, denitrification and phosphorus removal (SPNDPR) reactor. Results confirmed that the average granule diameter increased from 298.7 to 425.4 µm after 45 days of salinity stress even with low dissolved oxygen. Extracellular polymeric substances increased from 149.5 to 387.7 mg/g VSS after salinity (0.75 wt%) treatment, in turn accelerating granulation. Partial nitrification was maintained under the salinity condition due to the relative high activity and abundance of AOB, and the observed nitrite accumulation ratio averaged 98.9%. Salinity favored glycogen-accumulating organisms over polyphosphate-accumulating organisms (PAOs)/denitrifying-PAOs, with the abundance of Candidatus&#95;Competibacter increasing from 4.86% to 15.34% and the simultaneous partial nitrification-denitrification efficiency increasing from 74.4% to 91.1%, promoting N-removal potential. The P-removal performance was good under 0.5 wt% salinity but was inhibited under 0.75 wt% salinity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Partial nitrification is crucial for application of autotrophic nitrogen removal which is beneficial for treating carbon-limited wastewater. This study presents an alternative strategy for achieving partial nitrification of domestic wastewater treatment, by treating activated sludge of a nitrifying reactor on long-term nitrite exposure. Initially, the nitrifying reactor obtained complete nitrification. After the sludge of the reactor was treated with nitrite at concentration of 5-30 mg·L -1 without feeding for 32 days, the nitrite accumulation ratio (NAR) above 90% was achieved in 30 days, and then the NAR stabled at 97.9% under low temperature of 16.6 °C. Further analysis showed that the activity decay rate of ammonium-oxidizing bacteria (AOB) (0.020 d -1 ) was lower than that of nitrite-oxidizing bacteria (NOB) (0.035 d -1 ) under nitrite stress. Meanwhile, the NOB were effectively suppressed while AOB were dominant. These observations supported the feasibility of achieving stable partial nitrification by treating sludge on long-term exposure to nitrite., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Side-stream sludge treatment using free nitrous acid (FNA) is a novel strategy to achieve nitritation in mainstream of wastewater treatment plants (WWTPs). To optimize nitritation, the effect of starvation on this strategy was investigated in this study. The results showed that pre-starvation, which is the starvation before FNA treatment, enhanced the resistance of sludge to FNA. This led to a decrease in the nitrite accumulation rate (NAR), which dropped from 70% to 27% after aerobic pre-starvation. This was further confirmed in the FNA treatment using the sludge collected from the secondary settling tank (anoxic pre-starvation) and the aerobic zone (without starvation) of an Anaerobic-Anoxic-Oxic system. The post-starvation, which was the starvation after FNA treatment, decreased NAR from 63% to 14%. To obtain a higher NAR, the sludge used for FNA treatment should be collected from aerobic zone, and be returned to aerobic zone after treatment to avoid pre-starvation and post-starvation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

High inorganic salts inevitably impose a toxic impact on biological treatment processes. In this study, the effect of salinity on the performance and microbial community structures of partial-denitrification (PD) was firstly investigated. Results showed the denitrifying activities of non-domesticated PD sludge were completely inhibited under a temporary high salinity (≥1.5 wt%). However, after domestication, denitrifying activities maintained above 50% of the maximum with salinity build-up step-by-step from 0.0 wt% to 3.0 wt%. High nitrite production was stably achieved during 120 days with nitrate-to-nitrite transformation ratio around 90%. Further investigation showed extracellular polymeric substances content of PD sludge increased from 184.59 mg gVSS -1 to 560.64 mg gVSS -1 , accompanied by the elevation of average particle size. This occurred against high salinity as a protective response of PD bacteria. Moreover, Thauera, the functional bacteria of PD system, was still dominant with the relative abundance increasing to 83.36% (3.0 wt%) from 51.33% (0.0 wt%)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The knowledge of the effect of aerobic starvation on the functional bacterial activities and community structures is important for the recovery of nutrient removal in activated sludge system. Four aerobic starvation processes (3, 7, 14 and 30days) for nitrifiers and polyphosphate accumulating organisms (PAOs) were studied. The results showed that nitrifiers could utilize the released ammonium for growth during the first 7days of the aerobic starvation, and then gradually decayed. In the recovery period, the slower recovery ability of nitrite-oxidizing bacteria (NOB) than ammonium-oxidizing bacteria (AOB) contributed to the nitrite accumulation. Besides, the sequential consumption of polyhydroxyalkanoates (PHA), glycogen and the utilization of polyphosphate (poly-P) in PAOs provided the energy to maintain bacterial metabolic activity. High-throughput sequencing analysis revealed that aerobic starvation had substantial impacts on the succession of microbial community, and the majority of original dominant species within the phylum Proteobacteria and Bacteroidetes declined while Firmicutes increased., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An anaerobic-aerobic-anoxic sequencing batch reactor (AOA-SBR) using sludge fermentation products as carbon source was developed to enhance nitrogen and phosphorus removal in municipal wastewater with low C/N ratio (<4) and reduce sludge production. The AOA-SBR achieved simultaneous partial nitrification and denitrification (SND), aerobic phosphorus uptake and anoxic denitrification through the real-time control and the addition of sludge fermentation products. The average removal efficiencies of total nitrogen (TN), phosphorus (PO 4 3- -P) and chemical oxygen demand (COD) after 145-day operation were 88.8%, 99.3% and 81.2%, respectively. Nitrite accumulation ratio (NAR) reached 99.1% and sludge reduction rate reached 44.1-52.1%. Specifically, 34.4% of the TN removal was carried out by SND and 57.5% by denitrification. Illumina MiSeq sequencing indicated that ammonium-oxidizing bacteria (Nitrosomonas) were enriched and nitrite-oxidizing bacteria (Nitrospira) did not exist in AOA-SBR. The system demonstrated potential to solve the dual problem of insufficient carbon source and sludge reduction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Obtaining desirable partial nitritation (PN) is crucial for successful application of the combined PN and anammox process. In this study, the partial nitritation and simultaneously phosphorus removal (PNSPR) 1 granular sludge reactor treating low-strength domestic sewage was rapidly started up in 67days through seeding denitrifying phosphorus removal (DPR) 2 sludge. The nitrite/ammonium ratio in effluent was approximately 1 and the nitrite accumulation rate (NAR) was more than 95%, about 93% of orthophosphate was removed. The DPR sludge rich in phosphate accumulating organisms (PAOs) with few nitrifying bacteria could promote the achievement of PNSPR. Quantitative microbial analysis showed that the ammonium oxidizing bacteria (AOB) gene ratio in sludge increased from 0.21% to 3.43%, while nitrite oxidizing bacteria (NOB) gradually decreased to 0.07%. The average particle size of sludge increased from 114 to 421μm, indicating the formation of PNSPR granules. The high phosphorus content in sludge and phosphorus removal facilitated rapid granulation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The free ammonia (FA) inhibition on ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) under anaerobic condition was investigated in this study. The results indicated that NOB was more sensitive to the FA anaerobic treatment than AOB. The FA anaerobic inhibition on nitrifier gradually heightened with the increase of FA concentration. Accompanied with FA concentration increase from 0 to 16.82mgNH 3 -N·L -1 (the highest concentration adopted in this study), the activity of AOB reduced by 15.9%, while NOB decreased by 29.2%. After FA anaerobic treatment, nitrite was accumulated during nitrification. However, the nitrite accumulation disappeared on the sixth cycle of activity recovery tests with excessive aeration. Based on this result, a novel strategy for achieving nitritation is proposed, which involves recirculating a portion of the activated sludge through a side-line sludge treatment unit, where the sludge is subjected to treatment with FA under anaerobic condition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Sludge aggregation and biofilm formation are the most effective approaches to solve the washout of anammox microorganisms. In this study, the structure and composition of EPS (extracellular polymeric substances) were investigated to elucidate the factors for the anammox aggregation property. Anammox sludge taken from 18 lab-scale and pilot-scale reactors treating different types of wastewater was analyzed using EEM-PARAFAC (excitation-emission matrix and parallel factor analysis), FTIR (Fourier transform infrared spectroscopy) and real-time PCR combined with multivariate statistical analysis. The results showed that slime and TB-EPS (tightly bound EPS) were closely related with water quality and sludge morphology, and could be used as the indicators for anammox microbial survival ability and microbial aggregate morphology. Furthermore, slime secreted from anammox bacterial cells may be exhibited higher viscosity to the sludge surface and easily formed the gel network to aggregate. Large amounts of hydrophobic groups of protein in TB-EPS promoted the microbial aggregation. The mechanisms of anammox aggregation explored in this study enhanced the understanding of anammox stability in wastewater treatment processes.

Anaerobic treatment is the most widely used method of waste activated sludge (WAS) stabilization. Using a semi-continuous stirring tank with condensed WAS, we investigated effects of decreasing the solid retention time (SRT) from 32days to 6.4days on sludge reduction, soluble chemical oxygen demand (SCOD) release and dehydration capability, along with anaerobic digestion operated at medium temperature (MT-AD) or anaerobic digestion operated at room temperature (RT-AD). Results showed that effects of temperature on SCOD release were greater at SRT of 32d and 6.4d. When SRT was less than 8d, total solids (TS), volatile solids (VS) and capillary suction time (CST) did not change significantly. CST was lowest at SRT of 10.7days, indicating best condition for sludge dehydration. Principal component analysis (PCA) showed that the most optimum SRT was higher than 10.7d both in MT-AD or RT-AD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tightly bound extracellular polymeric substances (TB-EPS) are important components of sludge, playing a crucial role in the behavior of activated sludge. They are located in the innermost layer of EPS which closely combine with the cell surface and are difficult to be extracted. To fully understand the role of TB-EPS, it is extremely important to find an appropriate TB-EPS extraction protocol, which provides maximum yields of TB-EPS under the premise of minimal contribution of cell lysis. Ultrasonic method has been widely applied for TB-EPS extraction due to its unique advantages, but no one has developed a systematic and scientifically optimized method for this protocol. In this study, a novel method based on response surface methodology (RSM) was successfully developed to optimize the conditions of TB-EPS extraction. The optimal conditions were determined at an ultrasound time of 1.00 min, ultrasonic density of 5.59 W/mL and a mixed liquor suspended solid (MLSS) of around 1700-1800 mg/L. Furthermore, combined analysis of microscopy, particle size, and excitation-emission matrix (EEM) was successfully applied to evaluate the optimal conditions. The result indicates that the optimal conditions are efficient, reliable, and reproducible which effectively solves the bottleneck problem of the conflict between cell viability and TB-EPS yield, and little effect on its chemical structure.

For low carbon-to-nitrogen ratio municipal wastewater, full utilization of carbon sources is positive in reducing carbon emissions and carbon source addition. This study evaluated and proposed appropriate anaerobic duration control to improve nitrogen and phosphorus removal performance in Denitrifying Phosphorus Removal and Partial Denitrification coupled with Anammox (DPR-PDA) system. Excessive anaerobic duration resulted in wasted carbon source, anaerobic stage should be stopped after carbon source had been utilized. Hydraulic Retention Time-Anaerobic (HRT-An) of R1 was shortened from 5h to 0.56h, while R2 was maintained at 5h. The removal efficiency of nitrate (NO 3 −) were 91.7% in R1 and 71.5% in R2. Peak of NO 2 − accumulation (Peak-NO 2 --N) during denitrification in R1 was 4.5 times than in R2. Candidatus_Brocadia as anammox bacteria was self-enriched, the abundance in R1 was 4.67 times than in R2. This provided a practical guide for optimization of wastewater treatment processes and application of anammox technology. [Display omitted] • The effect of HRT-An on N and P removal was examined in two DPR-PDA systems. • Re-NO 3 --N of R1 (HRT-An: 5. → 0.56h) and R2 (HRT-An: 5h) were 91.7% and 71.5%. • Peak-NO 2 --N during denitrification process in R1 was 4.5 times higher than in R2. • Candidatus_Brocadia was self-enriched, abundance in R1 was 4.67 times than in R2. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel starch-driven HF-EPDA system was established. • Advanced NRE of 95.84% was achieved with domestic wastewater treatment. • Positive feedback established in system promotes the robustness and recoverability. • Candidatus Brocadia effectively contributed 69.53% to TIN removal in system. Anammox-based processes are pivotal for elevating nitrogen removal efficiency in municipal wastewater treatment. This study established a novel HF-EPDA system combined in-situ hydrolytic fermentation (HF) with endogenous partial denitrification (EPD) and anammox. Slowly-biodegradable organic matter (SBOM) was degraded and transformed into endogenous polymers for driving production of sufficient nitrite by EPD, further promoted the nitrogen removal via anammox process. Processes above formed positive feedback, guaranteeing the robustness and recoverability of system. After a 92-day suspension during operation, advanced nitrogen removal was still achieved with excellent nitrogen removal efficiency of 95.84 ± 1.73 %, treating with actual domestic wastewater and synthetic nitrate wastewater. Candidatus Brocadia and Candidatus Competibacter were dominant bacteria on biofilms responsible for the anammox and EPD process respectively, while the main hydrolytic fermentation organisms norank_o SBR1031 was enriched in floc sludge. This study highlights the reliable potential for expanding anammox application with simultaneous improvement of SBOM utilization. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ultrafast mainstream PN rate of more than 1 kg N m−3 d−1 was first obtained. • Ultrarapid PN start-up was achieved in 8 days at 10-day SRT under saturated DO. • Ultrastable PN with a NAR of 98.5 % maintained over 250 days under fluctuations. • NOB groups and activity decreased by 99.9 % and high-rate AOB species was enriched. • PN reproducibility and robustness were validated under real-world conditions. Achieving stable and high-rate partial nitrification (PN) remains a worldwide technical conundrum in low-strength mainstream conditions. This study successfully achieved ultrarapid mainstream PN within 8 days under a saturated dissolved oxygen (DO) supply strategy, reaching a record-breaking PN rate of over 1.0 kg N m−3 d−1 treating municipal wastewater. Stable PN was maintained for over 200 days with an ultrahigh nitrite accumulation ratio of 98.5 ± 0.9 %, resilient to seasonal fluctuations in temperature (16.0–25.6 °C) and load (NH 4 +-N, 40–80 mg N/L). Kinetics revealed a remarkable 159.1-fold increase in the maximum activity ratio of ammonia-oxidizing bacteria (AOB) to nitrite-oxidizing bacteria (NOB). The faster response of AOB to saturated DO stimulated its highest activity difference with NOB, contributing to the AOB (Nitrosomonas oligotropha) boom and the elimination of NOB groups (−99.9 %). Our results highlight the importance of promoting AOB rather than solely focusing on NOB suppression for initiating and stabilizing high-rate mainstream PN. [ABSTRACT FROM AUTHOR]

• A pilot-scale plant of A2O-BCO was established and operated continuously for 385 days. • PDPRA was proposed and applied by leveraging DPAOs as NO 2 - suppliers for anammox. • PDPRA enabled a mainstream anammox activity as high as 6.14 μmol-N/(L·h). • PDPRA facilitated a high-level enrichment of AnAOB (Ca. Brocadia, 2.46 ± 0.52 %). • CRE, ARE, NRE, and PRE achieved up to 83.5 %, 99.8 %, 77.1 %, and 99.3 %, respectively. Applying anaerobic ammonium oxidation (anammox) in municipal wastewater treatment plants (MWWTPs) can unlock significant energy and resource savings. However, its practical implementation encounters significant challenges, particularly due to its limited compatibility with carbon and phosphorus removal processes. This study established a pilot-scale plant featuring a modified anaerobic-anoxic-oxic (A2O) process and operated continuously for 385 days, treating municipal wastewater of 50 m3/d. For the first time, we propose a novel concept of partial denitrifying phosphorus removal coupling with anammox (PDPRA), leveraging denitrifying phosphorus-accumulating organisms (DPAOs) as NO 2 − suppliers for anammox. 15N stable isotope tracing revealed that the PDPRA enabled an anammox reaction rate of 6.14 ± 0.18 μmol-N/(L·h), contributing 57.4 % to total inorganic nitrogen (TIN) removal. Metagenomic sequencing and 16S rRNA amplicon sequencing unveiled the co-existence and co-prosperity of anammox bacteria and DPAOs, with Candidatus Brocadia being highly enriched in the anoxic biofilms at a relative abundance of 2.46 ± 0.52 %. Finally, the PDPRA facilitated the synergistic conversion and removal of carbon, nitrogen, and phosphorus nutrients, achieving remarkable removal efficiencies of chemical oxygen demand (COD, 83.5 ± 5.3 %), NH 4 + (99.8 ± 0.7 %), TIN (77.1 ± 3.6 %), and PO 4 3− (99.3 ± 1.6 %), even under challenging operational conditions such as low temperature of 11.7 °C. The PDPRA offers a promising solution for reconciling the mainstream anammox and the carbon and phosphorus removal, shedding fresh light on the paradigm shift of MWWTPs in the near future. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Denitrifying phosphorus removal was validly established using AOA operation mode. • Appropriate C/P is beneficial for inhibiting the excessive growth of GAOs. • The abundance of Dechloromonas significantly increased as typical DPAOs. • PAOs-GAOs balance was obtained under low DO concentration with aeration for 10 min. Realizing the quick enrichment and development of denitrifying phosphorus accumulating organisms (DPAOs) in actual household wastewater and industrial nitrate wastewater has significant research significance. In this study, a novel operation mode of anaerobic-oxic-anoxic (AOA) was adopted to successfully realize the enrichment and cultivation of DPAOs in urban domestic wastewater. Adjusting influent COD to PO 4 3−-P ratio, shortening the aerobic time and decreasing the aeration volume were conducive to select DPAOs in microbial populations. The system was operated for 180 days and the DPAOs were well enriched during the stable operation with the percentage of Dechloromonas increased to 5.1 %. Accordingly, the effluent PO 4 3−-P was < 0.3 mg P/L, the removal efficiency of phosphorus was 96.9 % and the removal efficiency of nitrate was 92.5 %. Above all, DPR can be successfully applied to AOA systems with good phosphorus removal performance. [ABSTRACT FROM AUTHOR]

• SBR achieved up to 93 % above partial nitrification of municipal wastewater. • SBR effluent and mature landfill leachate in UASB had an 89.4 % anammox contribution. • UASB achieved simultaneous anammox and partial denitrification with an HRT of 3.8 h. • The PN-SAPD process achieved an Eff.TIN of 3.4 mg/L and a removal efficiency of 95 %. • Ca. Brocadia had the highest abundance and was the only AnAOB for nitrogen removal. Anaerobic ammonia oxidation (anammox) of municipal wastewater is a research focus, especially the combined treatment with mature landfill leachate is a current research hotspot. In this study, municipal wastewater was treated by partial nitrification via sequencing batch reactor (SBR), and its effluent and mature landfill leachate were then mixed into an up-flow anaerobic sludge blanket (UASB) for simultaneous anammox and partial denitrification reaction. Through partial nitrification, a high nitrite accumulation rate (93.0 ± 3.8 %) was achieved by low dissolved oxygen (0.5–1.6 mg/L) and controlled aerobic time (3.5 h) in SBR. The UASB system was responsible for 78.8 ± 2.1 % nitrogen removal of the entire system with a hydraulic reaction time (HRT) of 3.8 h, accompanied by the anammox contribution up to 89.4 ± 6.0 %. The overall partial nitrification-simultaneous anammox and partial denitrification (PN-SAPD) system was controlled at a total COD/TIN of 2.8 ± 0.3 and a total HRT of only 10.2 h, achieving the nitrogen removal efficiency and effluent TIN were 95.2 ± 2.2 % and 3.4 ± 1.5 mg/L, respectively. The qPCR results showed functional genes (hzsA(B), hdh) associated with anaerobic ammonia-oxidizing bacteria (AnAOB), whose high gene copy abundance and transcription expression ensured the removal of major nitrogen from municipal wastewater and mature landfill leachate. 16S amplicon sequencing showed that the Ca. Brocadia (9.72–12.6 %) was further enrichment after sodium acetate was added, and the transcription expression of Thauera (0.5–7.0 %) caused nitrate to nitrite. The high abundance of related enzymes (hao, hzs, hdh, narGHI) involved in anammox and partial denitrification processes were found in the macrogenomic sequencing, and only Ca. Brocadia was involved in multi-pathway nitrogen metabolism in AnAOB. Based on the efficient nitrogen removal by AnAOB and denitrifying bacteria, this modified PN-SAPD process provides a new option for the co-treatment of mature landfill leachate in municipal wastewater treatment plants. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • High NO 2 – accumulation was achieved and NTR reached 87.0 % within 18 days. • Sludge size increased from 131.5 μm to 400.9 μm in 49 days to achieve granulation. • PD bacteria can quickly adapt to the treatment of electroplating tail wastewater. • Thauera was significantly enriched with abundance increasing from 12.5% to 76.4%. The nitrite (NO 2 –) accumulation in partial denitrification (PD) offers the possibility of widespread application of anammox process. In this study, the rapid establishment of PD granular system was achieved by increasing nitrogen loading rates (NLR) from 0.9 to 4.8 kg N/(m3·d), with the nitrate-to-nitrite transforming ratio (NTR) increasing rapidly to 87.0 % within 18 days. Growth evidence indicated that the functional genus Thauera was significantly enriched (12.5 %→76.4 %), with nitrate (NO 3 –) reduction rates (S NO3) improving by 5.4 times from 13.0 to 70.7 mg N/(g VSS·h). Importantly, the rapid aggregation of PD biomass as granules ensured robustness and resistance of PD feeding with the electroplating tail wastewater (NO 3 –-N of 103.0 ± 5.0 mg/L), obtaining stable NTR above 91.5 %. This study demonstrated the achievability of the fast development of PD granules and the adaptability and robustness of treating nitrate-containing industrial wastewater, which provided a promising method for efficient nitrogen transformation in industrial applications. [ABSTRACT FROM AUTHOR]

[Display omitted] • The TN in the influent and effluent were 50.9 mg/L and 8.1 mg/L at the C/N of 2.8. • The contribution rate by anammox after strengthening PN/A and PD/A more than 45%. • Anammox bacteria on the biocarrier in oxic chambers increased from 0.61% to 1.02%. • It is easy to realize advanced nitrogen removal by PN-PD /A in step-feed process. Combined partial nitrification-denitrification/anammox (PN-PD/A) processes have attracted great attention from researchers in recent years to achieve high nitrogen removal from low carbon /nitrogen (C/N) municipal wastewater. In this context, a step-feed anoxic/oxic (A/O) process was conducted in this study through the combination of the partial nitrification-anammox (PN/A) and partial denitrification-anammox (PD/A) to remove N from municipal wastewater with low C/N. The enhancement of the PN-PD/A process resulted in N removal efficiency of 85.6% at C/N of 2.8. The contributions of the anammox reached 36.4 and 8.8% in the anoxic and oxic chambers, respectively. The biocarriers added to the anoxic and oxic chambers increased the relative abundance of the anammox bacteria in biofilms from 0.61% to 1.51% and 1.02%, respectively. This study demonstrated that employing the step-feed A/O process can create optimal conditions for the anammox bacteria growth, thereby ensuring advanced N removal from low C/N municipal wastewater. [ABSTRACT FROM AUTHOR]

[Display omitted] • SPDAF system showed adaptability to gradual cooling at 19.6–16.5 ℃. • SPDAF system was robust to sudden cooling and drastic temperature fluctuations. • Hydrolysis and acidogenesis bacteria promoted the use of complex organics. • In-situ fermentation supplemented electron donors for NO 3 –-N reduction. A synergistic partial denitrification, anaerobic ammonium oxidation (Anammox), and in-situ fermentation (SPDAF) system was established to solve problems of wastewater treatment plants (WWTPs) in combined treatment of domestic sewage, and nitrate wastewater discharged from industrial areas. The SPDAF system was started up at decreasing temperatures (26.8–18.9 ℃), and remained robust at abrupt temperature drop and drastic temperature fluctuations (20.7–14.1 ℃). The influent and effluent total inorganic nitrogen (TIN) were 97.0 ± 3.7 mg/L and 10.3 ± 4.0 mg/L, respectively. In-situ fermentation supplemented electron donors for NO 3 –-N reduction. A high TIN removal efficiency, of 89.5 ± 3.9% was obtained. Specifically, Anammox contributed 90.9 ± 5.2% to TIN removal. Furthermore, the abundances of hydrolysis and acidogenesis bacteria were 14.02% and 29.47% in the low and high zones, respectively, which promoted fermentation and the use of complex organics. This study provided novel insights for actual operation of WWTPs. [ABSTRACT FROM AUTHOR]