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•NOB adapted to free-ammonia (FA) sludge treatment via community shift•The NOB population adapted to FA has higher growth rate and survival chances•NOB adapted to low dissolved oxygen ...(DO) by obtaining higher oxygen affinity•Incorporation of nitrite-scavengers (anammox) overcame FA- and low DO-adaptation•The combo-strategy delivered stable mainstream nitrogen removal via PN/A.
Stable suppression of nitrite-oxidizing bacteria (NOB) is still a major challenge for the implementation of partial nitritation and anammox (PN/A) in mainstream treatment. Despite numerous suppression strategies demonstrated, it is increasingly recognized that NOB could develop resistance to these strategies, threatening the long-term stability of the mainstream PN/A process. This study aims to understand adaption mechanisms and develop counter-strategies to overcome the adaptation. To this end, three previously-demonstrated suppression strategies, including NOB inactivation via side stream sludge treatment with free ammonia (FA), the use of low dissolved oxygen (DO), and the use of anammox to scavenge nitrite, were stepwise applied, over a period of 800 days, to a laboratory-scale reactor treating effluent from a high-rate activated sludge (HRAS) plant. FA sludge treatment alone sustained nitrite accumulation for about two months, after which NOB adaptation occurred causing PN to fail. The FA adaptation was induced by a shift in the NOB community from Nitrospira to Ca. Nitrotoga. The latter was found to have higher resistance to FA and also a higher maximum specific growth rate. Low DO at 0.2–0.4 mg O2 L−1 was then applied, in conjunction with FA treatment, which successfully eliminated Ca. Nitrotoga and re-established PN. However, new and unidentified NOB with a higher apparent oxygen affinity emerged in three months, again leading to PN failure. Lastly, as the third strategy for NOB suppression, anammox was introduced as an in-situ nitrite-scavenger. The combo-strategy delivered reliable NOB suppression with no further adaptation in the remaining experimental period (eight months). The resulted one-stage PN/A reactor achieved a nitrogen removal efficiency of 84.2 ± 5.37%. A control reactor, operated in parallel under the same conditions but without FA treatment, only achieved 10.4 ± 4.6% nitrogen removal, with anammox completely outcompeted by NOB in the last phase.
Sludge treatment using free ammonia (FA) is an innovative approach that was recently reported effective achieving stable mainstream nitrogen removal via the nitrite pathway. This study aims to ...investigate the adaptation of nitrifying community and the response of nitrification performance to high-level of FA exposure under real wastewater conditions. Two parallel lab-scale sequencing batch reactors were operated and fed with real municipal wastewater, with one receiving sludge treatment by FA and another as a control. While the FA approach rapidly achieved partial nitrification with a nitrite accumulation ratio (NAR) of approximately 60%, the partial nitrification eventually failed due to nitrite-oxidizing bacteria (NOB) adaptation to FA inactivation. NOB activity in the inoculum was suppressed by 82% after exposure to FA at ~220 mg NH3-N/L. However, towards the end of the experiments, significantly higher NOB activities were observed after exposure to the same level of FA. Distinct behaviours of NOB observed in batch tests during the study supported the reactor operational data and strongly suggested the adaptation of NOB under the FA stress. Furthermore, microbial community analysis revealed the underlying mechanism of the observed adaptation: the dominant NOB changed from Nitrospira to Candidatus Nitrotoga. It is for the first time shown that Ca. Nitrotoga are highly resistant to FA inhibition and inactivation in comparison to Nitrospira and Nitrobacter. In addition, while the Nitrosomonas genus was always the dominant ammonia-oxidizing bacteria (AOB) throughout the study, different shift in a species level was observed.
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•Inactivation and adaptation of nitrifiers exposed to FA were investigated.•Dominant NOB genus shifted from Nitrospira to Ca. Nitrotoga under FA stress.•Ca. Nitrotoga are resistant to FA inhibition/inactivation compared with other NOB.•Nitrosomonas were always dominant AOB genus under FA stress.
The nitritation step is the fundament for the biological nitrogen removal regardless of the traditional nitrification and denitrification process, the nitrite shunt process or the anammox process. ...Thus, exploring the effective nitritation performance is an important aspect of biological nitrogen removal. This study explored the upper limit of nitritation rate by increasing hydraulic residence time with the well-mixed and continuous granular sludge-type reactor characterized with low complexity and easy operation. The results showed that with the nitrogen loading rate of 1.0 kg/m3/d, the nitrite production rate could reach up to 0.65 kg/m3/d with the nitrite production efficiency of 63.49%, which is remarkable compared to that in the previously similar research. The microbial analysis indicated that ammonia-oxidizing bacteria was successfully enriched (13.27%) and genus Nitrosomonas was the dominant bacteria type. Besides, the activity of ammonium oxidizing bacteria in the continuous flow reactor was higher than that of other reactor types. The growth of vorticella on the sludge was also found in the reactor. The test of specific sludge activity and the microbe analysis both indicated that the nitrite-oxidizing bacteria was well inhibited during the whole experiment, which indicated the strategy of simply adjusting the dissolved oxygen is effective for running of nitritation process. The phosphorus removal performance was also achieved with a removal efficiency of 23.53%. The functional composition of the microbial community in the samples was predicted and finally transformation mechanism of nitrogen in sludge was drawn. In sum, this study indicated the superior performance of the granule sludge-type nitritation process and give a reference for the application of biological nitrogen removal technology.
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•A strategy for recovering partial nitritation in a mainstream PN/A system is proposed.•Selectively reviving AOB after thoroughly inhibiting AOB and NOB by FNA is applied.•NOB reduced ...largely whereas AOB declined merely after 1.35 mg/L FNA treatment.•Ca. Kuenenia and Ca. Brocadia formed important links with other N cycle processes.
Starting up or recovering partial nitritation is a major challenge for achieving or maintaining stable partial nitritation/anammox (PN/A) during mainstream wastewater treatment. This study presents a novel strategy for recovering the nitrite pathway by selectively reviving ammonium oxidizing bacteria (AOB) after thoroughly inhibiting AOB and nitrite oxidizing bacteria (NOB) using free nitrous acid (FNA). A sequencing batch reactor was operated for PN/A to treat real domestic wastewater for 423 days, during which twice FNA treatment was temporarily implemented. Results showed that with a single 0.45 mg/L FNA treatment on flocculent sludge, the NO3−-N concentration during the aerobic period showed an uptrend again and the partial nitritation performance was deteriorated. In contrast, 1.35 mg/L FNA treatment induced the inhibition of both AOB and NOB leading to regressive ammonium oxidation, but a subsequently higher DO (1.5 mg/L) and longer aeration duration recovered partial nitritation. For the relative abundances of the acquired biomass related to nitrogen conversion, Nitrosomonas, Nitrospira and Nitrolancea increased to 9.65%, 10.27% and 4.35%, respectively, at the beginning of the 1.35 mg/L FNA treatment, and Nitrospira and Nitrolancea decreased to 2.80% and 0.03% whereas Nitrosomonas declined to 8.71% after 76 days. Ca. Brocadia showed less resilience after the 1.35 mg/L FNA treatment, with the relative abundance decreasing from 13.38% to 0.62% due to insufficient nitrite. Molecular ecological network analysis indicates that among anammox taxa, Ca. Kuenenia and Ca. Brocadia formed important links with other N cycle processes. Moreover, the proposed strategy shows operational flexibility because it can be easily used to control NOB in mainstream PN/A applications offered by flocculent sludge systems.
A granular airlift reactor enriched in ammonia oxidizing bacteria (AOB) was operated at 10 °C performing stable partial nitritation in the long-term. The reactor treated a synthetic low-strength ...influent during 250 days with an average nitrogen loading rate of 0.63 ± 0.06 g N L−1 d−1. Nitrate production was barely detected, being the average concentration in the effluent of 0.6 ± 0.3 mg N-NO3 L−1. Furthermore, a suitable effluent for a subsequent reactor performing the anammox process was achieved. A maximum specific growth rate as high as 0.63 ± 0.05 d−1 was determined by performing kinetic experiments with the granular sludge in a chemostat and fitting the results to the Monod model. Pyrosequencing analysis showed a high enrichment in AOB (41 and 65% of the population were identified as Nitrosomonas genus on day 98 and 233, respectively) and an effective repression of nitrite oxidizing bacteria in the long-term. Pyrosequencing analysis also identified the coexistence of nitrifying bacteria and heterotrophic psychrotolerant microorganisms in the granular sludge. Some psychrotolerant microorganisms are producers of cryoprotective extracellular polymeric substances that could explain the better survival of the whole consortia at cold temperatures.
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•Long-term partial nitritation was maintained at 10 °C in a granular reactor.•High μmax explains high nitrification rates and effective NOB repression.•The bacterial diversity in the sludge decreased after long-term operation at 10 °C.•Granular sludge was mainly composed by AOB and psychrotolerant microorganisms.•Partial nitritation at 10 °C can be operated with low N2O emissions.
•Current molecular technologies to identify and detect AOB/NOB are summarized.•Strategies for partial nitrification via nitrite are critically reviewed.•Combination of partial nitrification and ...Anammox benefits wastewater treatment.•Existing problems and directions for future research in partial nitrification are suggested.
Partial nitrification has gained broad interests in the biological nitrogen removal (BNR) from wastewater, since it alleviates carbon limitation issues and acts as a shortcut nitrogen removal system combined with anaerobic ammonium oxidation (Anammox) process. The occurrence and maintenance of partial nitrification relies on various conditions, which favor ammonium oxidizing bacteria (AOB) but inhibit or limit nitrite oxidizing bacteria (NOB). The studies of the AOB and NOB activities have been conducted by state-of-the-art molecular techniques, such as Polymerase Chain Reaction (PCR), Quantitative PCR, denaturing gradient gel electrophoresis (DGGE), Fluorescence in situ hybridization (FISH) technique, Terminal Restriction Fragment Length Polymorphism (T-RFLP), Live/Dead BacLight, and quinone profile. Furthermore, control strategies for obtaining partial nitrification are mainly focused on the pH, temperature, dissolved oxygen concentration, real-time aeration control, sludge retention time, substrate concentration, alternating anoxic and aerobic operation, inhibitor and ultrasonic treatment. Existing problems and further perspectives for the scale-up of partial nitrification are also proposed and suggested.
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•PN/A-EPD/A process was developed in an IFAS-SBR treating real municipal wastewater.•High NRE of 90.1% was achieved under aerobic HRT of 8 h and DO of 0.4 ± 0.1 mg/L.•89.9% of TN was ...removed by anammox pathway in typical cycle.•Relative abundance of Candidatus Brocadia rose from 0% to 0.79% in suspended sludge.
In this study, an innovative partial nitrification-anammox (PN/A) and endogenous partial denitrification-anammox (EPD/A) process was developed in a single-stage integrated fixed film activated sludge sequencing batch reactor (IFAS-SBR) treating real municipal wastewater with C/N ratio below 3.2. Enhanced efficiency of total nitrogen (TN) removal reached 90.1% with low HRT of 12 h and DO of 0.4 ± 0.1 mg/L. Detailed nitrogen removal mechanism analysis of typical cycle revealed that 89.9% of TN was eliminated through anammox pathway. Anammox bacteria (Candidatus Brocadia) and endogenous denitrifying bacteria (Candidatus Competibacter) were abundant both in biofilms and suspended sludge, meanwhile ammonium-oxidizing bacteria has outcompeted nitrite-oxidizing bacteria, which all favored the synergistic effect of anammox with PN and EPD and contributed to the improvement of nitrogen removal. Overall, the above results confirmed that combined PN/A and EPD/A process is a reliable and efficient alternative for mainstream anammox process.
•Balancing AOB and AnAOB rates is the key factor for mainstream PNA at μM DO.•Stable PNA after temperature decrease from 25 to 15 °C without acclimation.•Heterotrophs growth on dissolved organic ...carbon from cell lysis decreases effluent NO3−.•Nitrospira was the only NOB and their control was assisted by AnAOB activity.•Candidatus Kuenenia was the predominant AnAOB throughout the study.
This study aimed at assessing the performance and microbial community in a granular one-stage partial nitritation-anammox sequencing batch reactor (PNA-SBR) subjected to temperature transition from 25 to 15 °C without biomass acclimation. The PNA-SBR was operated by controlling the oxygen transfer rate (OTR) according to the ammonium loading rate (ALR), which resulted in micromolar (µM) bulk dissolved oxygen (DO) concentration. The applied strategy proved to be feasible to operate the one-stage PNA-SBR at mainstream conditions because it was possible to control nitritation according to anammox rate. Nitrogen removal rate (NRR) of 330.24 ± 25.36 mg N·L−1·d−1 was achieved at 25 °C. Nitratation control by µM bulk DO limited the NO3−production:NH4+removed at 0.28 ± 0.04. No instability was experienced by decreasing the temperature to 15 °C, but removal rates were adapted to the resulting anammox activity, which decreased at low temperature. After temperature transition, nitratation was kept controlled and the NO3−production:NH4+removed molar ratio remained at 0.33 ± 0.05, although anammox activity deteriorated and higher nitrate production was obtained. Sequencing analysis revealed the dominant bacterial groups in the microbial community that clustered within the phyla Planctomycetes, Proteobacteria, Chloroflexi, and Bacteroidetes. Temperature drop only affected bacterial abundance, but the main bacteria involved in nitrification and anammox processes did not change during the study. Candidatus Kuenenia was the main anammox genus. Moreover, the presence of bacterial groups associated with heterotrophic metabolism indicates denitrification might be supported by the release of dissolved organic carbon due to bacterial lysis, and lower nitrate effluent concentration could be reached in PNA reactors.
Intermittent aeration is favorable for maintaining a long-term sewage partial nitrification-anammox (PN/A) process but the underlying mechanism is not yet fully understood. In this study, mainstream ...PN/A was established in an integrated fixed film activated sludge (IFAS) PN/A reactor and nitrite oxidization bacteria (NOB) activity was continuously suppressed. The suppression of NOB was significantly affected by the dissolved oxygen (DO) concentration during the aeration period as well as the duration of anoxic period. NOB was more suppressed in the hybrid system under a low DO level (0.5 mg/L) than under a high DO level (1.5–1.8 mg/L). Meanwhile, shortening the anoxic time from 40 to 20 min and keeping low DO during the intermittent aeration cycle could still suppress NOB activity, increasing the nitrogen removal rate by 40%. Biomass segregation was also enhanced by low DO, which favors the NOB inhibition in IFAS PN/A system. Overall, under an optimized intermittent aeration, a stable and high nitrogen removal efficiency (80–89%) with a nitrogen removal rate of 0.101 kg-N/(m3·d). This study is useful to supports the application of PN/A in sewage treatment.
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•High TIN removal efficiency of 85 ± 4% was achieved mainstream PN/A system.•DO was more important than anoxic time for NOB inhibition by intermittent aeration.•Nitrogen removal rate was increased 40% by optimizing intermittent aeration model.•Biomass segregation contributed to the stability of hybrid PN/A system.
•A concept of AnMBR-PN/A process was proposed for mainstream treatment.•In-situ FA/FNA exposure eliminated Nitrospira while retained Nitosonomas community.••DO limitation prevented switch of the NOB ...community in the mainstream conditions.•The effluent TN of the mainstream PN/A system was lower than 10 mg-N/L.•Enhanced hydrodynamic shear force resulted in sludge settleability deterioration.
In this pilot-scale study, an innovative mainstream treatment process that couples the anaerobic membrane reactor (AnMBR) with a one-stage PN/A system was proposed for advancing the concept of carbon neutrality in the municipal wastewater treatment plant. This work demonstrates the start-up procedure of a pilot-scale one-stage PN/A system for mainstream treatment. The 255-day start-up of the one-stage PN/A system involved the cultivation of ammonium-oxidizing bacteria (AOB) from the activated sludge, suppression of nitrite-oxidizing bacteria (NOB), investigation of in-situ growth kinetics of anammox bacteria (AnAOB), and the 50-day operation of the pilot-scale AnMBR-PN/A process for natural mainstream treatment. It is verified in the pilot-scale system for the first time that the in-situ free ammonia (FA) and free nitrous acid (FNA) exposure could effectively eliminate the Nitrospira (the NOB genus) while retaining the Nitosonomas (the AOB genus) community in the suspended sludge. NOB community rebounding was not detected even at the mainstream conditions with low nitrogen concentrations (Influent ammonium concentration=38±6 mg-NH4+-N/L) by intermittent aeration to control the system dissolved oxygen (DO) below 0.5 mg/L. The results of the mainstream treatment showed that the average effluent total nitrogen (TN) in the coupled process was generally lower than 10 mg-N/L, which meets the discharge limits of most prefectures in Japan. The investigated results of the in-situ anammox bacteria (AnAOB) growth kinetics suggested that the promoted start-up strategy of taking advantage of the warm months with higher mainstream temperature to achieve the rapid in-situ growth of the AnAOB is applicable in the investigated regions. From the perspective of the removal performance of the TN and organic substance, the AnMBR-PN/A process has great potential as the layouts of the carbon-neutral mainstream wastewater treatment plants.
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