Metatranscriptomic Analysis Reveals Synergistic Activities of Comammox and Anammox Bacteria in Full-Scale Attached Growth Nitrogen Removal System.

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.

Optimizing Ozone Disinfection in Water Reuse: Controlling Bromate Formation and Enhancing Trace Organic Contaminant Oxidation.

The use of ozone/biofiltration advanced treatment has become more prevalent in recent years, with many utilities seeking an alternative to membrane/RO based treatment for water reuse. Ensuring efficient pathogen reduction while controlling disinfection byproducts and maximizing oxidation of trace organic contaminants remains a major barrier to implementing ozone in reuse applications. Navigating these challenges is imperative in order to allow for the more widespread application of ozonation. Here, we demonstrate the effectiveness of ozone for virus, coliform bacteria, and spore forming bacteria inactivation in unfiltered secondary effluent, all the while controlling the disinfection byproduct bromate. A greater than 6-log reduction of both male specific and somatic coliphages was seen at specific ozone doses as low as 0.75 O3:TOC. This study compared monochloramine and hydrogen peroxide as chemical bromate control measures in high bromide water (Br- = 0.35 ± 0.07 mg/L). On average, monochloramine and hydrogen peroxide resulted in an 80% and 36% decrease of bromate formation, respectively. Neither bromate control method had any appreciable impact on virus or coliform bacteria disinfection by ozone; however, the use of hydrogen peroxide would require a non-Ct disinfection framework. Maintaining ozone residual was shown to be critical for achieving disinfection of more resilient microorganisms, such as spore forming bacteria. While extremely effective at controlling bromate, monochloramine was shown to inhibit TrOC oxidation, whereas hydrogen peroxide enhanced TrOC oxidation.

Co-Occurrence and Cooperation between Comammox and Anammox Bacteria in a Full-Scale Attached Growth Municipal Wastewater Treatment Process.

Cooperation between comammox and anammox bacteria for nitrogen removal has been recently reported in laboratory-scale systems, including synthetic community constructs; however, there are no reports of full-scale municipal wastewater treatment systems with such cooperation. Here, we report intrinsic and extant kinetics as well as genome-resolved community characterization of a full-scale integrated fixed film activated sludge (IFAS) system where comammox and anammox bacteria co-occur and appear to drive nitrogen loss. Intrinsic batch kinetic assays indicated that majority of the aerobic ammonia oxidation was driven by comammox bacteria (1.75 ± 0.08 mg-N/g TS-h) in the attached growth phase, with minimal contribution by ammonia-oxidizing bacteria. Interestingly, a portion of total inorganic nitrogen (∼8%) was consistently lost during these aerobic assays. Aerobic nitrite oxidation assays eliminated the possibility of denitrification as a cause of nitrogen loss, while anaerobic ammonia oxidation assays resulted in rates consistent with anammox stoichiometry. Full-scale experiments at different dissolved oxygen (DO = 2 - 6 mg/L) setpoints indicated persistent nitrogen loss that was partly sensitive to DO concentrations. Genome-resolved metagenomics confirmed the high abundance (relative abundance 6.53 ± 0.34%) of two Brocadia-like anammox populations, while comammox bacteria within the Ca. Nitrospira nitrosa cluster were lower in abundance (0.37 ± 0.03%) and Nitrosomonas-like ammonia oxidizers were even lower (0.12 ± 0.02%). Collectively, our study reports for the first time the co-occurrence and cooperation of comammox and anammox bacteria in a full-scale municipal wastewater treatment system.

Electron beam treatment for the removal of 1,4-dioxane in water and wastewater.

Electron beam (e-beam) treatment uses accelerated electrons to form oxidizing and reducing radicals when applied to water without the use of external chemicals. In this study, electron beam treatment was used to degrade 1,4-dioxane in several water matrices. Removal improved in the progressively cleaner water matrices and removals as high as 94% to 99% were observed at a dose of 2.3 kGy in secondary effluent. 1,4-dioxane removal was confirmed to be primarily through hydroxyl radical oxidation. The calculated electrical energy per order was found to be 0.53, 0.26, and 0.08 kWh/m3/order for secondary effluent (Avg. total organic carbon (TOC) 9.25 mg/L), granular activated carbon effluent (TOC 3.46 mg/L), and ultrapure water, respectively, with a 70% generation and transfer efficiency applied.

Using kinetics and modeling to predict denitrification fluxes in elemental-sulfur-based biofilms.

Elemental sulfur (S0 ) can serve as an electron donor for water and wastewater denitrification, but few researchers have addressed the kinetics of S 0 -based reduction of nitrate (NO 3- ), nitrite (NO 2- ), and nitrous oxide (N 2 O). In addition, S 0 -based denitrifying biofilms are counter-diffusional. This is because the electron donor (S 0 ) is supplied from the biofilm attachment surface while the acceptor, for example, NO 3- , is supplied from the bulk liquid. No existing mathematical model for S 0 -based denitrification considers this behavior. In this study, batch tests were used to determine the kinetic parameters for the reduction of NO 3- , NO 2- , and N 2 O. Additionally, a biofilm model was developed to explore the effects of counter-diffusion on overall fluxes, that is, the mass of NO 3- or NO 2- removed per unit biofilm support area per unit time. The maximum specific substrate utilization rates ( qˆ ) for NO 3- , NO 2- , and N 2 O were 3.54, 1.98, and 6.28 g N g COD -1 ·d -1 , respectively. The maximum specific growth rates ( µË† ) were 0.71, 1.21, and 1.67 d -1 for NO 3- to NO 2- , NO 2- to N 2 O, and N 2 O to N 2 , respectively. Results suggest that the observed NO 2- accumulation during S 0 -based denitrification results from a low qˆ for NO 2- relative to that for NO 3- . The high qˆ for N 2 O, relative to that for NO 3- and NO 2- , suggest that little N 2 O accumulation occurs during denitrification. A counter-diffusional biofilm model was used to predict trends for NO 3- fluxes, and confirmed NO 2- accumulation in S 0 -based denitrification biofilms. It also explains the observed detrimental effects of biofilm thickness on denitrification fluxes. This study allows a more accurate prediction of NO 3- , NO 2- , and N 2 O transformations in S 0 -based denitrification.

Impact of carbon source and COD/N on the concurrent operation of partial denitrification and anammox.

In this study, concurrent operation of anammox and partial denitrification within a nonacclimated mixed culture system was proposed. The impact of carbon sources (acetate, glycerol, methanol, and ethanol) and COD/NO3- -N ratio on partial denitrification selection under both short- and long-term operations was investigated. Results from short-term testing showed that all carbon sources supported partial denitrification. However, acetate and glycerol were preferred due to their display of efficient partial denitrification selection, which may be related to their different electron transport pathways in comparison with methanol. Long-term operation confirmed results of batch tests by showing the contribution of partial denitrification to nitrate removal above 90% after acclimation in both acetate and glycerol reactors. In contrast, methanol showed challenges of maintaining efficient partial denitrification. COD/NO3- -N ratio mainly controlled the rate of nitrate reduction and not directly partial denitrification selection; thus, it should be used to balance between denitrification rate and anammox rate. PRACTITIONER POINTS: The authors aimed to investigate the impact of carbon sources and COD/NO3-N ratio on partial denitrification selection. All the carbon sources supported partial denitrification as long as the nitrite sink was available. 90% partial denitrification could be achieved with both acetate and glycerol in long-term operations. COD/NO3-N ratio did not directly control partial denitrification but can be used to balance between denitrification rate and anammox rate.

Incorporating sulfur reactions and interactions with iron and phosphorus into a general plant-wide model.

Sulfur causes many adverse effects in wastewater treatment and sewer collection systems, such as corrosion, odours, increased oxygen demand, and precipitate formation. Several of these are often controlled by chemical addition, which will impact the subsequent wastewater treatment processes. Furthermore, the iron reactions, resulting from coagulant addition for chemical P removal, interact with the sulfur cycle, particularly in the digester with precipitate formation and phosphorus release. Despite its importance, there is no integrated sulfur and iron model for whole plant process optimization/design that could be readily used in practice. After a detailed literature review of chemical and biokinetic sulfur and iron reactions, a plant-wide model is upgraded with relevant reactions to predict the sulfur cycle and iron cycle in sewer collection systems, wastewater and sludge treatment. The developed model is applied on different case studies.

Status, Challenges, and Perspectives of Mainstream Nitritation-Anammox for Wastewater Treatment.

The nitritation-anammox process is an efficient and cost-effective approach for biological nitrogen removal, but its application in treating mainstream wastewater remains a great challenge. Mainstream nitritation-anammox processes could create opportunities for achieving energy self-sufficient, or energy-generating water resource recovery facilities. Significant advancements have been achieved via pilot- and full-scale trials to overcome the major obstacles under mainstream conditions, such as repression of nitrite-oxidizing bacteria, limiting the overgrowth of denitrifiers, and effective selection and retention of ammonia-oxidizing bacteria and anammox bacteria. This review paper intends to provide a detailed update of research progress on mainstream nitritation-anammox processes, discuss metabolic interactions, and examine major challenges and possible solutions towards the future development.

Thermal Degradation of Long Chain Fatty Acids.

The thermal hydrolysis of saturated (C16:0 and C18:0) and unsaturated fatty acids (C16:1, C18:1, and C18:2) was investigated at 90 °C to 160 °C for 30 min and 8 h durations. Hydrolysis efficiencies were calculated based on mass yield (i.e., mg/g parent compound), which accounted for all C2-C24 by-products. Very little degradation (less than 1%) of long chain fatty acids (LCFAs) was observed from 30 min thermal hydrolysis. At 140 and 160 °C for 8 h, saturated fatty acids degraded uniformly to C2 to C14. Saturated fatty acids tended to convert to alkanes (1.5-2.0% of total fatty acids) instead of fatty acids (8 h). Thermal hydrolysis did not significantly affect unsaturated LCFA degradation at any duration. The unsaturated by-products seen were the result of cleavage at the allylic or vinylic positions. Thermal hydrolysis of LCFAs with digested sludge was investigated. The amount of VFAs and LCFAs in primary and secondary sludge was increased at 140 and 160 °C as a result of lipid degradation in the sludge mixture. Thermal hydrolysis of fatty acids with different catalysts was also investigated. Whereas saturated LCFAs were stable under all catalytic conditions, unsaturated LCFAs were nearly completely degraded when hydrolyzed with hydrogen peroxide and activated carbon or copper sulfate.

Addressing the Issue of Microplastics in the Wake of the Microbead-Free Waters Act-A New Standard Can Facilitate Improved Policy.

The United States Microbead-Free Waters Act was signed into law in December 2015. It is a bipartisan agreement that will eliminate one preventable source of microplastic pollution in the United States. Still, the bill is criticized for being too limited in scope, and also for discouraging the development of biodegradable alternatives that ultimately are needed to solve the bigger issue of plastics in the environment. Due to a lack of an acknowledged, appropriate standard for environmentally safe microplastics, the bill banned all plastic microbeads in selected cosmetic products. Here, we review the history of the legislation and how it relates to the issue of microplastic pollution in general, and we suggest a framework for a standard (which we call "Ecocyclable") that includes relative requirements related to toxicity, bioaccumulation, and degradation/assimilation into the natural carbon cycle. We suggest that such a standard will facilitate future regulation and legislation to reduce pollution while also encouraging innovation of sustainable technologies.

Startup of a Partial Nitritation-Anammox MBBR and the Implementation of pH-Based Aeration Control.

The single-stage deammonification moving bed biofilm reactor (MBBR) is a process for treating high strength nitrogen waste streams. In this process, partial nitritation and anaerobic ammonia oxidation (anammox) occur simultaneously within a biofilm attached to plastic carriers. An existing tank at the James River Treatment Plant (76 ML/d) in Newport News, Virginia was modified to install a sidestream deammonification MBBR process. This was the second sidestream deammonification process in North America and the first MBBR type installation. After 4 months the process achieved greater than 85% ammonia removal at the design loading rate of 2.4 g /m2·d (256 kg /d) signaling the end of startup. Based on observations during startup and process optimization phases, a novel pH-based control system was developed that maximizes ammonium removal and results in stable aeration and effluent alkalinity.

Controlling the COD removal of an A-stage pilot study with instrumentation and automatic process control.

The pursuit of fully autotrophic nitrogen removal via the anaerobic ammonium oxidation (anammox) pathway has led to an increased interest in carbon removal technologies, particularly the A-stage of the adsorption/bio-oxidation (A/B) process. The high-rate operation of the A-stage and lack of automatic process control often results in wide variations of chemical oxygen demand (COD) removal that can ultimately impact nitrogen removal in the downstream B-stage process. This study evaluated the use dissolved oxygen (DO) and mixed liquor suspended solids (MLSS) based automatic control strategies through the use of in situ on-line sensors in the A-stage of an A/B pilot study. The objective of using these control strategies was to reduce the variability of COD removal by the A-stage and thus the variability of the effluent C/N. The use of cascade DO control in the A-stage did not impact COD removal at the conditions tested in this study, likely because the bulk DO concentration (>0.5 mg/L) was maintained above the half saturation coefficient of heterotrophic organisms for DO. MLSS-based solids retention time (SRT) control, where MLSS was used as a surrogate for SRT, did not significantly reduce the effluent C/N variability but it was able to reduce COD removal variation in the A-stage by 90%.

Nitrogen polishing in a fully anoxic anammox MBBR treating mainstream nitritation-denitritation effluent.

As nitrogen discharge limits are becoming more stringent, short-cut nitrogen systems and tertiary nitrogen polishing steps are gaining popularity. For partial nitritation or nitritation-denitritation systems, anaerobic ammonia oxidation (anammox) polishing may be feasible to remove residual ammonia and nitrite from the effluent. Nitrogen polishing of mainstream nitritation-denitritation system effluent via anammox was studied at 25°C in a fully anoxic moving bed bioreactor (MBBR) (V = 0.45 m(3) ) over 385 days. Unlike other anammox based processes, a very fast startup of anammox MBBR was demonstrated, despite nitrite limited feeding conditions (influent nitrite = 0.7 ± 0.59 mgN/L, ammonia = 6.13 ± 2.86 mgN/L, nitrate = 3.41 ± 1.92 mgN/L). The nitrogen removal performance was very stable within a wide range of nitrogen inputs. Anammox bacteria (AMX) activity up to 1 gN/m(2) /d was observed which is comparable to other biofilm-based systems. It is generally believed that nitrate production limits nitrogen removal through AMX metabolism. However, in this study, anammox MBBR demonstrated ammonia, nitrite, and nitrate removal at limited chemical oxygen demand (COD) availability. AMX and heterotrophs contributed to 0.68 ± 0.17 and 0.32 ± 0.17 of TIN removal, respectively. It was speculated that nitrogen removal might be aided by denitratation which could be due to heterotrophs or the recently discovered ability for AMX to use short-chain fatty acids to reduce nitrate to nitrite. This study demonstrates the feasibility of anammox nitrogen polishing in an MBBR is possible for nitritation-denitration systems.

Urine Bacterial Community Convergence through Fertilizer Production: Storage, Pasteurization, and Struvite Precipitation.

Source-separated human urine was collected from six public events to study the impact of urine processing and storage on bacterial community composition and viability. Illumina 16S rRNA gene sequencing revealed a complex community of bacteria in fresh urine that differed across collection events. Despite the harsh chemical conditions of stored urine (pH > 9 and total ammonia nitrogen > 4000 mg N/L), bacteria consistently grew to 5 ± 2 × 108 cells/mL. Storing hydrolyzed urine for any amount of time significantly reduced the number of operational taxonomic units (OTUs) to 130 ± 70, increased Pielou evenness to 0.60 ± 0.06, and produced communities dominated by Clostridiales and Lactobacillales. After 80 days of storage, all six urine samples from different starting materials converged to these characteristics. Urine pasteurization or struvite precipitation did not change the microbial community, even when pasteurized urine was stored for an additional 70 days. Pasteurization decreased metabolic activity by 50 ± 10% and additional storage after pasteurization did not lead to recovery of metabolic activity. Urine-derived fertilizers consistently contained 16S rRNA genes belonging to Tissierella, Erysipelothrix, Atopostipes, Bacteroides, and many Clostridiales OTUs; additional experiments must determine whether pathogenic species are present, responsible for observed metabolic activity, or regrow when applied.

Acidogenesis and Two-Phase Codigestion of Fats, Oils, and Greases and Municipal Biosolids.

Acidogenic codigestion of fats, oils, and greases (FOG) was studied using suspended growth sludge digesters operated as batch fed reactors that were fed twice daily. The digesters were maintained at a 2-day retention time and at 37 °C to mimic the acid phase of an acid-gas digestion system. As FOG loading rates increased, volatile fatty acid (VFA) production was found to increase, although the percentage of VFA production compared to theoretical values decreased exponentially to just 20% at the highest loading rates. FOG matter was found to have accumulated in the reactor vessel in semi-solid balls that floated near the liquid surface. Two-phase codigestion of FOG was studied at 37 °C using Continuously Stirred Tank Reactors (CSTRs) as acid phase digesters (APD) operated with 2-day retention times, followed by gas phase digesters (GPD) with 15-day retention times. The two-phase systems were compared by FOG addition to the APD versus GPD. FOG addition to the APD resulted in 88% destruction of LCFAs, whereas FOG addition to the GPD resulted in 95% destruction of LCFAs. Accumulated LCFAs were found in the APD receiving FOG and were primarily composed palmitic acid (16:0), followed by oleic acid (18:1) and stearic acid (18:0).

Evaluation of an Industrial Byproduct Glycol Mixture for Denitrification.

In this study, the effectiveness of an industrial byproduct that contained ethylene and propylene glycols to serve as a denitrification carbon source was investigated. Use of the byproduct was compared to methanol on the basis of denitrification rate and yield. Three sequencing batch reactors (SBR) were studied; one was fed methanol, the other two were fed with low and high dosages of the byproduct separately. The low dosage reactor (GLYL) exhibited the highest denitrification rate of 11.55 mg NOx-N/g MLVSS•h and the lowest yield of 0.21 mg VSS/mg COD, while the high dosage reactor (GLYH) had the lowest denitrification rate of 8.56 mg NOx-N/g MLVSS•h and the highest yield of 0.55 mg VSS/mg COD. The results of this study showed that the industrial byproduct can be used to effect efficient nitrogen removal, but excess dosage can cause poor performance.

Methods for increasing the rate of anammox attachment in a sidestream deammonification MBBR.

Deammonification (partial nitritation-anammox) is a proven process for the treatment of high-nitrogen waste streams, but long startup time is a known drawback of this technology. In a deammonification moving bed biofilm reactor (MBBR), startup time could potentially be decreased by increasing the attachment rate of anammox bacteria (AMX) on virgin plastic media. Previous studies have shown that bacterial adhesion rates can be increased by surface modification or by the development of a preliminary biofilm. This is the first study on increasing AMX attachment rates in a deammonification MBBR using these methods. Experimental media consisted of three different wet-chemical surface treatments, and also media transferred from a full-scale mainstream fully nitrifying integrated fixed-film activated sludge (IFAS) reactor. Following startup of a full-scale deammonification reactor, the experimental media were placed in the full-scale reactor and removed for activity rate measurements and biomass testing after 1 and 2 months. The media transferred from the IFAS process exhibited a rapid increase in AMX activity rates (1.1 g/m(2)/day NH(4)(+) removal and 1.4 g/m(2)/day NO(2)(-) removal) as compared to the control (0.2 g/m(2)/day NH(4)(+) removal and 0.1 g/m(2)/day NO(2)(-) removal) after 1 month. Two out of three of the surface modifications resulted in significantly higher AMX activity than the control at 1 and 2 months. No nitrite oxidizing bacteria activity was detected in either the surface modified media or IFAS media batch tests. The results indicate that startup time of a deammonification MBBR could potentially be decreased through surface modification of the plastic media or through the transfer of media from a mature IFAS process.

Ammonia-based intermittent aeration control optimized for efficient nitrogen removal.

This work describes the development of an intermittently aerated pilot-scale process (V = 0.45 m(3) ) operated for optimized efficient nitrogen removal in terms of volume, supplemental carbon and alkalinity requirements. The intermittent aeration pattern was controlled using a strategy based on effluent ammonia concentration set-points. The unique feature of the ammonia-based aeration control was that a fixed dissolved oxygen (DO) set-point was used and the length of the aerobic and anoxic time (anoxic time ≥25% of total cycle time) were changed based on the effluent ammonia concentration. Unlike continuously aerated ammonia-based aeration control strategies, this approach offered control over the aerobic solids retention time (SRT) to deal with fluctuating ammonia loading without solely relying on changes to the total SRT. This approach allowed the system to be operated at a total SRT with a small safety factor. The benefits of operating at an aggressive SRT were reduced hydraulic retention time (HRT) for nitrogen removal. As a result of such an operation, nitrite oxidizing bacteria (NOB) out-selection was also obtained (ammonia oxidizing bacteria [AOB] maximum activity: 400 ± 79 mgN/L/d, NOB maximum activity: 257 ± 133 mgN/L/d, P < 0.001) expanding opportunities for short-cut nitrogen removal. The pilot demonstrated a total inorganic nitrogen (TIN) removal rate of 95 ± 30 mgN/L/d at an influent chemical oxygen demand: ammonia (COD/NH4 (+) -N) ratio of 10.2 ± 2.2 at 25°C within the hydraulic retention time (HRT) of 4 h and within a total SRT of 5-10 days. The TIN removal efficiency up to 91% was observed during the study, while effluent TIN was 9.6 ± 4.4 mgN/L. Therefore, this pilot-scale study demonstrates that application of the proposed on-line aeration control is capable of relatively high nitrogen removal without supplemental carbon and alkalinity addition at a low HRT.

Source Separation of Urine as an Alternative Solution to Nutrient Management in Biological Nutrient Removal Treatment Plants.

Municipal wastewater contains a mixture of brown (feces and toilet paper), yellow (urine), and gray (kitchen, bathroom and wash) waters. Urine contributes approximately 70-80% of the nitrogen (N), 50-70% of the phosphorus (P) load and 60-70% of the pharmaceutical residues in normal domestic sewage. This study evaluated the impact of different levels of source separation of urine on an existing biological nutrient removal (BNR) process. A process model of an existing biological nutrient removal (BNR) plant was used. Increasing the amount of urine diverted from the water reclamation facilities, has little impact on effluent ammonia (NH3-N) concentration, but effluent nitrate (NO3-N) concentration decreases. If nitrification is necessary then no reduction in the sludge age can be realized. However, a point is reached where the remaining influent nitrogen load matches the nitrogen requirements for biomass growth, and no residual nitrogen needs to be nitrified. That allows a significant reduction in sludge age, implying reduced process volume requirements. In situations where nitrification is required, lower effluent nitrate (NO3-N) concentrations were realized due to both the lower influent nitrogen content in the wastewater and a more favorable nitrogen-to-carbon ratio for denitrification. The external carbon requirement for denitrification decreases as the urine separation efficiency increases due to the lower influent nitrogen content in the wastewater and a more favorable nitrogen-to-carbon ratio for denitrification. The effluent phosphorus concentration decreases when the amount of urine sent to water reclamation facilities is decreased due to lower influent phosphorus concentrations. In the case of chemical phosphate removal, urine separation reduces the amount of chemicals required.

Optimization of a mainstream nitritation-denitritation process and anammox polishing.

This paper deals with an almost 1-year long pilot study of a nitritation-denitritation process that was followed by anammox polishing. The pilot plant treated real municipal wastewater at ambient temperatures. The effluent of high-rate activated sludge process (hydraulic retention time, HRT=30 min, solids retention time=0.25 d) was fed to the pilot plant described in this paper, where a constant temperature of 23 °C was maintained. The nitritation-denitritation process was operated to promote nitrite oxidizing bacteria out-selection in an intermittently aerated reactor. The intermittent aeration pattern was controlled using a strategy based on effluent ammonia and nitrate+nitrite concentrations. The unique feature of this aeration control was that fixed dissolved oxygen set-point was used and the length of aerobic and anoxic durations were changed based on the effluent ammonia and nitrate+nitrite concentrations. The anaerobic ammonia oxidation (anammox) bacteria were adapted in mainstream conditions by allowing the growth on the moving bed bioreactor plastic media in a fully anoxic reactor. The total inorganic nitrogen (TIN) removal performance of the entire system was 75±15% during the study at a modest influent chemical oxygen demand (COD)/NH4+-N ratio of 8.9±1.8 within the HRT range of 3.1-9.4 h. Anammox polishing contributed 11% of overall TIN removal. Therefore, this pilot-scale study demonstrates that application of the proposed nitritation-denitritation system followed by anammox polishing is capable of relatively high nitrogen removal without supplemental carbon and alkalinity at a low HRT.