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[Display omitted] • The 54-day start-up period of PD mainly attributed to shortening anoxic duration. • Both nitrate-reducing and nitrite-generating slowed down by acetate-using PD biofilm. • The critical role of Simplicispira in PD was proved via metagenomics. • Side stream incubating PD biofilm may serve the upgrading of mainstream Anammox. Recently, adding carriers into anoxic zone is proposed for mainstream Anammox upgrade, which relied on the denitrifiers responsible for partial denitrification (PD) to generate essential nitrite for Anammox bacteria. Still, their low abundance in the naturally formed biofilm leads to insufficient nitrite supply. This study investigated the sequential culturing of PD biofilm. By inoculating ordinary activated sludge, the PD process was quickly established within 54-day. During that, decreasing carbon to nitrogen ratio and anoxic duration in order might be effective strategies. Adding carriers shifted the microbial community, especially the proliferation of Flavobacterium. When solely using the mature PD biofilm, high nitrate to nitrite transformation ratio (>70%) was obtained. Meanwhile, both nitrate-reducing and nitrite-generating processes slowed down and lasted ∼90 min. In addition, abundant Simplicispira candidate for PD was detected in biofilm. This study also suggests that regularly harvesting PD-related functional bacteria from a side-stream incubator promotes mainstream Anammox upgrade. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mainstream anammox process was started up in situ via simple operation strategy. • Desirable NRE of about 86% was obtained despite the fluctuation in sewage quality. • Validity of the start-up strategy was verified with three kinds of seed sludge. • Biofilm and anaerobic/aerobic/anoxic mode favored enrichment of anammox bacteria. • Seed sludge with high AOB activity promoted the in situ start-up of anammox process. The feasibility of in situ start-up of mainstream anammox process was investigated in three parallel sequencing batch biofilm reactors (SBBRs) inoculated with nitrification sludge, partial nitrification sludge, and denitrifying phosphorus removal sludge, respectively. The SBBRs were operated under alternate anaerobic/aerobic/anoxic pattern at ambient temperature (16.5–26.8 °C). The influent organic and nitrogen loading rates were increased stepwise. Anammox bacteria grew exponentially with relative abundance and overall bacterial activity increasing from 0 to 0.004% to 0.29–0.40% and 'not detected' to 6–7 mg N/L/h, respectively. Desirable nitrogen removal efficiency of about 86% was obtained in 3–4 months for the influent nitrogen of 40.5–73.6 mg N/L. Anammox was the primary nitrogen transformation pathway. For the anammox bacterial enrichment, biofilm, alternate anaerobic/aerobic/anoxic pattern, and limited aeration played important roles. Seed sludge with high ammonium oxidizing bacterial activity further promoted the start-up of anammox process. The in situ start-up strategy could promote the full-scale application of mainstream anammox. [ABSTRACT FROM AUTHOR]

• Stable sewage nitrogen removal was obtained in a PNA-SBBR at low temperatures. • Microbial community structure in the PNA biofilm system remained reasonably stable. • Psychrotolerant microbes were enriched at low temperature, improving system stability. • Increased secretion of TB-EPS favored the stable nitrogen removal at low temperatures. Stable sewage nitrogen removal with nitrogen removal efficiency of 87.5 ± 2.2% was achieved in a partial nitrification-anammox (PNA) biofilm system at low temperatures (12.8–16.3 °C). High-throughput sequencing analysis indicated that the microbial community structure in the sequencing batch biofilm reactor (SBBR) remained reasonably stable. Candidatus Brocadia was the only detected anammox genus and remained stable at 0.3–0.5%. Some psychrotolerant microorganisms that could secrete cryoprotective extracellular polymeric substances (EPS), including Flavobacterium and Thermomonas , were enriched at low temperatures. This could be conducive to the stable operation of the PNA-SBBR. Moreover, according to the EPS composition and characteristics analysis, the secretion of tightly-bound EPS that bound to the cell surface containing plentiful protein was stimulated at low temperatures, further improving the system stability. Overall, the reasonably stable microbial community structure, enrichment of psychrotolerant microorganisms, and increased secretion of EPS could play important roles for stable sewage nitrogen removal at low temperatures. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Partial nitrification-anammox process was started up without adding anammox sludge. • Anammox reaction could be the primary nitrogen removal pathway in the reactor. • Reactor could efficiently treat low-strength and low C/N ratio domestic sewage. • Sequencing batch biofilm reactor was stable even at low temperatures (<15 °C). Abstract In this study, enhanced removal of nitrogen from real domestic wastewater with low organic carbon to nitrogen ratio of 2.1–3.6 was achieved in a sequencing batch biofilm reactor (SBBR) with enriching anammox bacteria in situ. The SBBR was inoculated with ordinary nitrification sludge and operated under alternative anaerobic, aerobic and anoxic patterns. During the start-up period, influent nitrogen and carbon loading rate gradually increased. According to the quantitative microbial analysis, the growth of ammonium oxidizing bacteria and anammox bacteria were promoted, resulting in an increase from 6.56 × 104 to 1.51 × 107 and from 1.11 × 104 to 1.69 × 106 gene copies·mL−1, respectively. During the steady period (Day 102–205), nitrogen removal efficiency of 88.2% was achieved with an effluent total nitrogen of 6.3 mg N·L−1 for average influent ammonium of 53.9 ± 4.0 mg·L−1. Nitrogen removal was steady when temperature decreased from 27.0 to 12.8 °C. Variations in nitrogen compounds suggested that anammox reaction could be the main pathway for nitrogen removal, according to the results of activity batch and continuous tests. For the high nitrogen removal performance, biofilm and post-anoxic unit could play important roles. Overall, this study provided an alternative method to improve the removal of nitrogen from sewage by directly enriching anammox bacteria. [ABSTRACT FROM AUTHOR]

[Display omitted] • Mainstream SNAD-IFAS reactor was started up by seeding more accessible inoculum. • Advanced nitrogen removal efficiency of 91.2 ± 3.6 % was achieved. • New anammox biofilm was formed and could be used as inoculum for other anammox system. • Microbial correlation networks suggested cooperation between DNB, FB and AnAOB. To solve the shortage of inoculum, the feasibility of establishing simultaneous partial nitrification, anammox, and denitrification (SNAD) reactor through inoculating nitrification–denitrification sludge, anammox biofilm and blank carriers was investigated. Advanced nitrogen removal efficiency of 91.2 ± 3.6 % was achieved. Bacteria related to nitrogen removal and fermentation were enriched in anammox biofilm, blank carriers and flocs, and the abundance of dominant anaerobic ammonia oxidizing bacteria (AnAOB), Candidatus Brocadia , reached 3.4 %, 0.5 % and 0.3 %, respectively. Candidatus Competibacter and Calorithrix became the dominant denitrifying bacteria (DNB) and fermentative bacteria (FB), respectively. The SNAD system was successfully established, and new mature biofilms formed in blank carriers, which could provide inoculum for other anammox processes. Partial nitrification, partial denitrification and aerobic_chemoheterotrophy were existed and facilitated AnAOB enrichment. Microbial correlation networks revealed the cooperation between DNB, FB and AnAOB that promoted nitrogen removal. Overall, the SNAD process was started up through inoculating more accessible inoculum. [ABSTRACT FROM AUTHOR]

Biological nitrogen removal from sewage via anammox is a promising and feasible technology to make sewage treatment energy-neutral or energy-positive. Good retention of anammox bacteria is the premise of achieving sewage treatment via anammox. Therefore the anammox metabolism and its factors were critically reviewed so as to form biofilm/granules for retaining anammox bacteria. A stable supply of nitrite for anammox bacteria is a real bottleneck for applying anammox in sewage treatment. Nitritation and partial-denitrification are two promising methods of offering nitrite. As such, the strategies for achieving nitritation in sewage treatment were summarized by reviewing the factors affecting nitrite oxidation bacteria growth. Meanwhile, the methods of achieving partial-denitrification have been developed through understanding the microorganisms related with nitrite accumulation and their factors. Furthermore, two cases of applying anammox in the mainstream sewage treatment plants were documented. [ABSTRACT FROM AUTHOR]

[Display omitted] • SPNA-IFAS reactor was started up by seeding nitrification sludge and anammox biofilm. • NOB migrated from flocs to biofilm while AnAOB enriched in flocs when SRT decreased. • Short SRT of flocs may play a limited role in inhibiting NOB. • Short SRT of flocs may decrease AnAOB abundance and deteriorate nitrogen removal. • Simultaneous fermentation, partial denitrification-anammox was speculated in biofilm. The feasibility of starting up mainstream single-stage partial nitrification-anammox (SPNA) system by inoculating nitrification sludge and anammox biofilm was investigated. The SPNA system treating low-strength synthetic wastewater was rapidly started up with TN removal efficiency of 88.5 ± 1.8% and effluent nitrate concentration of 7.2 ± 1.2 mg/L. Both the abundance and maximum activity of nitrite oxidizing bacteria (NOB) in flocs decreased obviously. Interestingly, the abundance of anaerobic ammonium oxidizing bacteria (AnAOB) in flocs increased from 0.213% to 0.346% despite the sludge retention time (SRT) of flocs decreased to 60 days, the AnAOB in biofilm was 0.434%. That meant AnAOB gradually enriched in flocs and accounted for a fairly high proportion. The inhibition of NOB, partial denitrification and increased aerobic_chemoheterotrophy function in flocs might be the main reasons for AnAOB enrichment. The possibility of simultaneous fermentation, partial denitrification and anammox reaction was predicted in biofilm, further improving the stability of the system. [ABSTRACT FROM AUTHOR]