Simultaneous nitrification,denitrification, and phosphorus removal in a lab-scale sequencing batch reactor

Simultaneous nitrification and denitrification (SND) via the nitrite pathway and anaerobic-anoxic-enhanced biological phosphorus removal (EBPR) are two processes that can significantly reduce the energy and COD demand for nitrogen and phosphorus removal. The combination of these two processes has the potential of achieving simultaneous nitrogen and phosphorus removal with a minimal requirement for COD. A lab-scale sequencing batch reactor (SBR) was operated in alternating anaerobic-aerobic mode with a low dissolved oxygen (DO) concentration (0.5 mg/L) during the aerobic period, and was demonstrated to accomplish nitrification, denitrification, and phosphorus removal. Under anaerobic conditions, COD was taken up and converted to polyhydroxyalkanoates (PHAs), accompanied by phosphorus release. In the subsequent aerobic stage, PHA was oxidized and phosphorus was taken up to <0.5 mg/L by the end of the cycle. Ammonia was also oxidized during the aerobic period, but without accumulation of nitrite or nitrate in the system, indicating the occurrence of simultaneous nitrification and denitrification. However, off-gas analysis showed that the final denitrification product was mainly nitrous oxide (N(2)O), not N(2). Further experimental results demonstrated that nitrogen removal was via nitrite, not nitrate. These experiments also showed that denitrifying glycogen-accumulating organisms (DGAOs), rather than denitrifying polyphosphate-accumulating organisms (DPAOs), were responsible for the denitrification activity.     

Short-term temperature effects on the anaerobic metabolism of glycogen accumulating organisms

Proliferation of glycogen accumulating organisms (GAO) has been identified as a potential cause of enhanced biological phosphorus removal (EBPR) failure in wastewater treatment plants (WWTP). GAO compete for substrate with polyphosphate accumulating organisms (PAO) that are the microorganisms responsible for the phosphorus removal process. In the present article, the effects of temperature on the anaerobic metabolism of GAO were studied in a broad temperature range (from 10 to 40 degrees C). Additionally, maximum acetate uptake rate of PAO, between 20 and 40 degrees C, was also evaluated. It was found that GAO had clear advantages over PAO for substrate uptake at temperatures higher than 20 degrees C. Below 20 degrees C, maximum acetate uptake rates of both microorganisms were similar. However, lower maintenance requirements at temperature lower than 30 degrees C give PAO metabolic advantages in the PAO-GAO competition. Consequently, PAO could be considered to be psychrophilic microorganisms while GAO appear to be mesophilic. These findings contribute to understand the observed stability of the EBPR process in WWTP operated under cold weather conditions. They may also explain the proliferation of GAO in WWTP and thus, EBPR instability, observed in hot climate regions or when treating warm industrial effluents. It is suggested to take into account the observed temperature dependencies of PAO and GAO in order to extend the applicability of current activated sludge models to a wider temperature range.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.     

反硝化聚磷菌及其脱氮除磷机理研究进展

水体富营养化是当前水环境保护工作的重点关注问题,微生物修复富营养化水体具有高效、低耗且不产生二次污染等特点,已经成为富营养化水体生态修复的一种重要方式。近年来,对反硝化聚磷菌的研究及其在污水处理工艺中的应用越来越广泛。不同于传统的反硝化细菌联合聚磷菌去除氮磷工艺,反硝化聚磷菌在交替厌氧、缺氧/好氧条件下能同时进行脱氮除磷而被广泛关注与研究。值得注意的是,近几年报道的部分微生物仅在好氧条件下就可进行同时脱氮除磷,但是其脱氮除磷机理仍未理清。基于此,文中总结了目前发现的反硝化聚磷菌和同时硝化反硝化聚磷微生物的种类及特点,并对其脱氮与除磷的关系及其机理进行了系统性分析,对目前反硝化除磷存在的问题进行了梳理,最后对今后的研究方向进行了展望,以期为完善反硝化聚磷菌的脱氮除磷机理及工艺改进提供参考。     

Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm

A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia-oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%.     

Enhanced biological phosphorus removal in a sequencing batch reactor using propionate as the sole carbon source

An enhanced biological phosphorus removal (EBPR) system was developed in a sequencing batch reactor (SBR) using propionate as the sole carbon source. The microbial community was followed using fluorescence in situ hybridization (FISH) techniques and Candidatus 'Accumulibacter phosphatis' were quantified from the start up of the reactor until steady state. A series of SBR cycle studies was performed when 55% of the SBR biomass was Accumulibacter, a confirmed polyphosphate accumulating organism (PAO) and when Candidatus 'Competibacter phosphatis', a confirmed glycogen-accumulating organism (GAO), was essentially undetectable. These experiments evaluated two different carbon sources (propionate and acetate), and in every case, two different P-release rates were detected. The highest rate took place while there was volatile fatty acid (VFA) in the mixed liquor, and after the VFA was depleted a second P-release rate was observed. This second rate was very similar to the one detected in experiments performed without added VFA.A kinetic and stoichiometric model developed as a modification of Activated Sludge Model 2 (ASM2) including glycogen economy, was fitted to the experimental profiles. The validation and calibration of this model was carried out with the cycle study experiments performed using both VFAs. The effect of pH from 6.5 to 8.0 on anaerobic P-release and VFA-uptake and aerobic P-uptake was also studied using propionate. The optimal overall working pH was around 7.5. This is the first study of the microbial community involved in EBPR developed with propionate as a sole carbon source along with detailed process performance investigations of the propionate-utilizing PAOs.     

Effect of oxic and anoxic conditions on nitrous oxide emissions from nitrification and denitrification processes

A lab-scale sequencing batch reactor fed with real municipal wastewater was used to study nitrous oxide (N(2)O) emissions from simulated wastewater treatment processes. The experiments were performed under four different controlled conditions as follows: (1) fully aerobic, (2) anoxic-aerobic with high dissolved oxygen (DO) concentration, (3) anoxic-aerobic with low DO concentration, and 4) intermittent aeration. The results indicated that N(2)O production can occur from both incomplete nitrification and incomplete denitrification. N(2)O production from denitrification was observed in both aerobic and anoxic phases. However, N(2)O production from aerobic conditions occurred only when both low DO concentrations and high nitrite concentration existed simultaneously. The magnitude of N(2) O produced via anoxic denitrification was lower than via oxic denitrification and required the presence of nitrite. Changes in DO, ammonium, and nitrite concentrations influenced the magnitude of N(2)O production through denitrification. The results also suggested that N(2)O can be produced from incomplete denitrification and then released to the atmosphere during aeration phase due to air stripping. Therefore, biological nitrogen removal systems should be optimized to promote complete nitrification and denitrification to minimize N(2)O emissions.     

Simultaneous COD, nitrogen, and phosphate removal by aerobic granular sludge

Aerobic granular sludge technology offers a possibility to design compact wastewater treatment plants based on simultaneous chemical oxygen demand (COD), nitrogen and phosphate removal in one sequencing batch reactor. In earlier studies, it was shown that aerobic granules, cultivated with an aerobic pulse-feeding pattern, were not stable at low dissolved oxygen concentrations. Selection for slow-growing organisms such as phosphate-accumulating organisms (PAO) was shown to be a measure for improved granule stability, particularly at low oxygen concentrations. Moreover, this allows long feeding periods needed for economically feasible full-scale applications. Simultaneous nutrient removal was possible, because of heterotrophic growth inside the granules (denitrifying PAO). At low oxygen saturation (20%) high removal efficiencies were obtained; 100% COD removal, 94% phosphate (P-) removal and 94% total nitrogen (N-) removal (with 100% ammonium removal). Experimental results strongly suggest that P-removal occurs partly by (biologically induced) precipitation. Monitoring the laboratory scale reactors for a long period showed that N-removal efficiency highly depends on the diameter of the granules.     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of nitrate in biological phosphorus removal in a sequencing batch reactor

Summary The effects of nitrate on phosphorus release and uptake in a sequencing batch reactor for biological phosphorus removal was investigated. The addition of nitrate decreased phosphorus release in the anaerobic stage. The synthesis of poly(hydroxyalkanoates) was decreased with the presence of nitrate, possibly due to the competitive utilization of the carbon source by PHA synthesis and denitrification of nitrate. Instead of oxygen, nitrate could be used as an electron acceptor for phosphorus removal. However, the simultaneous addition of nitrate and acetate greatly reduced the phosphorus removal rate. Phosphate and nitrate could be removed simultaneously with nitrate as the electron acceptor, and the continuous and steady feeding of nitrate was beneficial to phosphate removal.     

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass . h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis.     

Effect of anaerobic reaction time on denitrifying phosphorus removal and N2O production

Nitrous oxide (N₂O) is a highly potent greenhouse gas; however, the characteristics of N₂O production during denitrification using poly-β-hydroxyalkanoates (PHA) as a carbon source are not well understood. In this study, effects of anaerobic reaction time (AnRT) on PHA formation, denitrifying phosphorus removal and N₂O production were investigated using a laboratory-scale anaerobic/anoxic/oxic sequencing batch reactor (An/A/O SBR). The results showed that operation of the An/A/O SBR for 0.78 SRT (47 cycles) after the AnRT was shortened from 90 min to 60 min resulted in anaerobically synthesized PHA improving by 1.8 times. This improvement was accompanied by increased phosphorus removal efficiency and denitrification. Accordingly, the N₂O-N production was reduced by 6.7 times. Parallel batch experiments were also conducted with AnRTs of 60, 90 and 120 min. All results indicated that in addition to the amount of anaerobically synthesized PHA, the kinetics of PHA degradation also regulated denitrifying phosphorus removal and N₂O production.     

Long-term population dynamics and in situ physiology in activated sludge systems with enhanced biological phosphorus removal operated with and without nitrogen removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.     

A structured metabolic model for anaerobic and aerobic stoichiometry and kinetics of the biological phosphorus removal process

A structured metabolic model is developed that describes the stoichiometry and kinetics of the biological P removal process. In this approach all relevant metabolic reactions underlying the metabolism, considering also components like adenosine triphosphate (ATP) and nic-otinamide-adenine dinucleotide (NADH(2)) are describedbased on biochemical pathways. As a consequence of the relations between the stoichiometry of the metabolic reactions and the reaction rates of components, the required number of kinetic relations to describe the process is reduced. The model describes the dynamics of the storage compounds which are considered separately from the active biomass. The model was validated in experiments at a constant sludge retention time of 8 days, over the anaerobic and aerobic phases in which the external oncentrations as well as the internal fractions of the relevant components involved in the P-removal process were monitored. These measurements include dissolved acetate, phosphate, and ammonium; oxygen consumption; poly-beta-hydroxybutyrate (PHB); glycogen; and active biomass. The model satisfactorily describes the dynamic behavior of all components during the anaerobicand aerobic phases.(c) 1995 John Wiley & Sons, Inc.     

Temperature effects on the aerobic metabolism of glycogen-accumulating organisms

Short-term temperature effects on the aerobic metabolism of glycogen-accumulating organisms (GAO) were investigated within a temperature range from 10 to 40 degrees C. Candidatus Competibacter Phosphatis, known GAO, were the dominant microorganisms in the enriched culture comprising 93 +/- 1% of total bacterial population as indicated by fluorescence in situ hybridization (FISH) analysis. Between 10 and 30 degrees C, the aerobic stoichiometry of GAO was insensitive to temperature changes. Around 30 degrees C, the optimal temperature for most of the aerobic kinetic rates was found. At temperatures higher than 30 degrees C, a decrease on the aerobic stoichiometric yields combined with an increase on the aerobic maintenance requirements were observed. An optimal overall temperature for both anaerobic and aerobic metabolisms of GAO appears to be found around 30 degrees C. Furthermore, within a temperature range (10-30 degrees C) that covers the operating temperature range of most of domestic wastewater treatment systems, GAOs aerobic kinetic rates exhibited a medium degree of dependency on temperature (theta = 1.046-1.090) comparable to that of phosphorus accumulating organisms (PAO). We conclude that GAO do not have metabolic advantages over PAO concerning the effects of temperature on their aerobic metabolism, and competitive advantages are due to anaerobic processes.     

等氮滴灌对宿根蔗产量及土壤氧化亚氮排放的影响

为得到合理的水肥管理措施,研究等氮量下不同滴灌施肥比例对宿根蔗产量以及不同生育期蔗田土壤氧化亚氮(N2 O)通量和无机氮含量的影响,并分析蔗田土壤N2 O通量与无机氮含量之间的关系.该文以自然降雨W0为对照,设置2种滴灌灌水量水平W1(田间持水量的75％)和W2(田间持水量的85％),等量氮肥(N 300 kg·hm-...     

Factors Affecting Nitrous Oxide Production: A Comparison of Biological Nitrogen Removal Processes with Partial and Complete Nitrification

Nitrous oxide (N₂O) emission from biological nitrogen removal (BNR) processes has recently received more research attention. In this study, two lab-scale BNR systems were used to investigate the effects of various operating parameters including the carbon to nitrogen (C/N) ratio, ammonia loading, and the hydraulic retention time on N₂O production. The first system was operated in a conventional BNR mode known as the Ludzack–Ettinger (LE) process, consisting of complete denitrification and nitrification reactors, while the second one was operated in a shortcut BNR (SBNR) mode employing partial nitrification and shortcut denitrification, which requires less oxygen and carbon sources. As the C/N ratio was decreased, a significant increase in N₂O production was observed only in the anoxic reactor of the LE process, indicating that N₂O was released as an intermediate of the denitrification reaction under the carbon-limited condition. However, the SBNR process did not produce significant N₂O even at the lowest C/N ratio of 0.5. When the SBNR process was subjected to increasing concentrations of ammonia, N₂O production from the aerobic reactor was rapidly increased. Furthermore, the increasing production of N₂O was observed mostly in the aerobic reactor of the SBNR process with a decline in hydraulic retention time. These experimental findings indicated that the increase in N₂O production was closely related to the accumulation of free ammonia, which was caused by an abrupt increase of the ammonium loading. Consequently, the partial nitrification was more susceptible to shock loading conditions, resulting in a high production of N₂O, although the SBNR process was more efficient with respect to nitrogen removals as well as carbon and oxygen requirements.     

Effect of influent nutrient ratios and hydraulic retention time (HRT) on simultaneous phosphorus and nitrogen removal in a two-sludge sequencing batch reactor process

A laboratory-scale anaerobic–anoxic/nitrification sequencing batch reactor (A₂N-SBR) fed with domestic wastewater was operated to examine the effect of varying ratios of influent COD/P, COD/TN and TN/P on the nutrient removal. With the increased COD/P, the phosphorus removals exhibited an upward trend. The influent TN/P ratios had a positive linear correlation with the phosphorus removal efficiencies, mainly because nitrates act as electron acceptors for the phosphorus uptake in the A₂N-SBR. Moreover, it was found that lower COD/TN ratio, e.g. 3.5, did not significantly weaken the phosphorus removal, though the nitrogen removal first decreased greatly. The optimal phosphorus and nitrogen removals of 94% and 91%, respectively were achieved with influent COD/P and COD/TN ratios of 19.9 and 9.9, respectively. Additionally, a real-time control strategy for A₂N-SBR can be undertaken based on some characteristic points of pH, redox potential (ORP) and dissolved oxygen (DO) profiles in order to obtain the optimum hydraulic retention time (HRT) and improve the operating reliability.