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.

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.

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.

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.

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.

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.

Ammonia-based feedforward and feedback aeration control in activated sludge processes.

Aeration control at wastewater treatment plants based on ammonia as the controlled variable is applied for one of two reasons: (1) to reduce aeration costs, or (2) to reduce peaks in effluent ammonia. Aeration limitation has proven to result in significant energy savings, may reduce external carbon addition, and can improve denitrification and biological phosphorus (bio-P) performance. Ammonia control for limiting aeration has been based mainly on feedback control to constrain complete nitrification by maintaining approximately one to two milligrams of nitrogen per liter of ammonia in the effluent. Increased attention has been given to feedforward ammonia control, where aeration control is based on monitoring influent ammonia load. Typically, the intent is to anticipate the impact of sudden load changes, and thereby reduce effluent ammonia peaks. This paper evaluates the fundamentals of ammonia control with a primary focus on feedforward control concepts. A case study discussion is presented that reviews different ammonia-based control approaches. In most instances, feedback control meets the objectives for both aeration limitation and containment of effluent ammonia peaks. Feedforward control, applied specifically for switching aeration on or off in swing zones, can be beneficial when the plant encounters particularly unusual influent disturbances.

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) enables effective phosphorus removal in a pilot-scale A-B stage shortcut nitrogen removal system for mainstream municipal wastewater treatment.

While the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, its integration with enhanced biological phosphorus (P) removal (EBPR) has been considered challenging and thus unexplored. Here, full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge with B-stage partial (de)nitrification/anammox and side-stream EBPR (HRAS-P(D)N/A-S2EBPR) was conducted treating real municipal wastewater. The results demonstrated that, despite the relatively low influent carbon load, the B-stage P(D)N-S2EBPR system could achieve effective P removal performance, with the carbon supplement and redirection of the A-stage sludge fermentate to the S2EBPR. The novel process configuration design enabled a system shift in carbon flux and distribution for efficient EBPR, and provided unique selective factors for ecological niche partitioning among different key functionally relevant microorganisms including polyphosphate accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs). The combined nitrite from B-stage to S2EBPR and aerobic-anoxic conditions in our HRAS-P(D)N/A-S2EBPR system promoted DPAOs for simultaneous internal carbon-driven denitrification via nitrite and P removal. 16S rRNA gene-based oligotyping analysis revealed high phylogenetic microdiversity within the Accumulibacter population and discovered coexistence of certain oligotypes of Accumulibacter and Competibacter correlated with efficient P removal. Single-cell Raman micro-spectroscopy-based phenotypic profiling showed high phenotypic microdiversity in the active PAO community and the involvement of unidentified PAOs and internal carbon-accumulating organisms that potentially played an important role in system performance. This is the first pilot study to demonstrate that the P(D)N-S2EBPR system could achieve shortcut N removal and influent carbon-independent EBPR simultaneously, and the results provided insights into the effects of incorporating S2EBPR into A/B process on metabolic activities, microbial ecology, and resulted system performance.

Evaluation of gasoline-denatured ethanol as a carbon source for denitrification.

In this study concerning denitrification, the performance of three carbon sources, methanol (MeOH), ethanol (EtOH) and gasoline-denatured ethanol (dEtOH), was compared and evaluated on the basis of treatment efficiency, inhibition potential and cost. The gasoline denaturant considered here contained mostly aliphatic compounds and little of the components that typically boost the octane rating, such as benzene, toluene, ethylbenzene and xylenes. Results were obtained using three lab-scale SBRs operated at SRT of 12.0 +/- 0.9 days. After biomass was acclimated, denitrification rates with dEtOH were similar to those of EtOH (201 +/- 50 and 197 +/- 28 NO3-N/g MLVSS x d, respectively), and higher than those of MeOH (165 +/- 49 mg NO3-N/g MLVSS x d). The denaturant did not affect biomass production, nitrification or denitrification. Effluent soluble COD concentrations were always less than the analytical detection limit. Although the cost of dEtOH ($2.00/kg nitrate removed) was somewhat higher than that of methanol ($1.63/kg nitrate removed), the use of dEtOH is very promising and utilities will have to decide if it is worth paying a little extra to take advantage of its benefits.

In vitro bioassays to monitor complex chemical mixtures at a carbon-based indirect potable reuse plant.

Potable water reuse technologies are used to treat wastewater to drinking water quality to help sustain a community's water resources. California has long led the adoption of potable water reuse technologies in the United States and more states are exploring these technologies as water resources decline. Reuse technologies also need to achieve adequate reductions in microbial and chemical contaminant risks to meet public health goals and secure public acceptance. In vitro bioassays are a useful tool for screening if reuse treatment processes adequately reduce toxicity associated with a range of chemical classes that are contaminants of concern. In this study, we used an aryl hydrocarbon receptor (AhR) and an estrogen receptor luciferase bioassay to detect the presence of dioxin-like and estrogenic compounds across a 3800 m3/d carbon-based indirect potable reuse plant that uses carbon-based treatment (SWIFT-RC). Our results demonstrate significant removal of dioxin-like compounds across the SWIFT-RC treatment train. Estrogenicity declined across the treatment train for some months but was extremely variable and low with many samples falling below the method quantification level; consequently, we were not able to reliably determine estrogenicity trends for SWIFT-RC. Comparing the bioanalytical equivalent concentrations detected in the SWIFT-RC water with established monitoring trigger levels from the state of California suggests that SWIFT-RC produced water that met the bioassay guidelines. The log total organic carbon concentration and AhR assay equivalent concentrations are weakly correlated when data across all SWIFT-RC processes are included. Overall, this research demonstrates the performance of in vitro bioassays at a demonstration-scale carbon-based IPR system and highlights both the potential utility and challenges associated with these methods for assessing system performance.

Sidestream bio-P and mainstream anammox in a BNR process with upstream carbon capture.

The integration of biological phosphorus removal (bio-P) and shortcut nitrogen removal (SNR) processes is challenging because of the conflicting demands on influent carbon: SNR allows for upstream carbon diversion, but this reduction of influent carbon (especially volatile fatty acids [VFAs]) prevents or limits bio-P. The objective of this study was to achieve SNR, either via partial nitritation/anammox (PNA) or partial denitrification/anammox (PdNA), simultaneously with biological phosphorus removal in a process with upstream carbon capture. This study took place in a pilot scale A/B process with a sidestream bio-P reactor and tertiary anammox polishing. Despite low influent rbCOD concentrations from the A-stage effluent, bio-P occurred in the B-stage thanks to the addition of A-stage WAS fermentate to the sidestream reactor. Nitrite accumulation occurred in the B-stage via partial denitrification and partial nitritation (NOB out-selection), depending on operational conditions, and was removed along with ammonia by the tertiary anammox MBBR, with the ability to achieve effluent TIN less than 2 mg/L. PRACTITIONER POINTS: A sidestream reactor with sufficient fermentate addition enables biological phosphorus removal in a B-stage system with little-to-no influent VFA. Enhanced biological phosphorus removal is not inhibited by intermittent aeration and is stable at a wide range of process SRTs. Partial nitritation and partial denitrification are viable routes to produce nitrite within an A/B process with sidestream bio-P, for downstream anammox in a polishing MBBR.

Coupling physical selection with biological selection for the startup of a pilot-scale, continuous flow, aerobic granular sludge reactor without treatment interruption.

This study removes two technical constraints for transitioning full-scale activated sludge infrastructure to continuous flow, aerobic granular sludge (AGS) facilities. The first of these is the loss of treatment capacity as a result of the rapid washout of flocculent sludge inventory and in turn the potential loss of nitrification during initial AGS reactor startup. The second is the physical selector design which currently is limited to either the complex sequencing batch reactor selection or sidestream hydrocyclones. Briefly, real wastewater data collected from this study suggested that by increasing the surface overflow rate (SOR) of an upflow clarifier to 10 m h - 1, the clarifier can be taken advantage of as a physical selector to separate flocculant sludge from AGS. Redirecting the physical selector underflow and overflow sludge to the feast and famine zones of a treatment train, respectively, can create a biological selection that not only promotes AGS formation but also safeguards the effluent quality throughout the AGS reactor startup period. This study provides a novel concept for economically implementing continuous flow AGS within existing full-scale, continuous flow treatment trains.

An advanced synergy of partial denitrification-anammox for optimizing nitrogen removal from wastewater: A review.

Anammox is a widely adopted process for energy-efficient removal of nitrogen from wastewater, but challenges with NOB suppression and NO3- accumulation have led to a deeper investigation of this process. To address these issues, the synergy of partial denitrification and anammox (PD-anammox) has emerged as a promising solution for sustainable nitrogen removal in wastewater. This paper presents a comprehensive review of recent developments in the PD-anammox system, including stable performance outcomes, operational parameters, and mathematical models. The review categorizes start-up and recovery strategies for PD-anammox and examines its contributions to sustainable development goals, such as reducing N2O emissions and saving energy. Furthermore, it suggests future trends and perspectives for improving the efficiency and integration of PD-anammox into full-scale wastewater treatment system. Overall, this review provides valuable insights into optimizing PD-anammox in wastewater treatment, highlighting the potential of simultaneous processes and the importance of improving efficiency and integration into full-scale systems.

WEF/WERF study of BNR plants achieving very low N and P limits: evaluation of technology performance and process reliability.

The Water Environment Research Foundation (WERF) funded a two-year comprehensive study of nutrient removal plants designed and operated to meet very low effluent total nitrogen (TN) and total phosphorus (TP) concentrations. WERF worked with the Water Environment Federation (WEF) to solicit participation of volunteers and provide a forum for information exchange at workshops at its annual conferences. Both existing and new technologies are being adapted to meet requirements that are as low as 3.0 mg/L TN and 0.1 mg/L TP, and there is a need to define their capabilities and reliabilities in the real world situation of wastewater treatment plants. A concern over very low daily permits for ammonia caused the work to be extended to include nitrification reliability. This effort focused on maximizing what can be learned from existing technologies in order to provide a database that will inform key decision makers about proper choices for both technologies and rationale bases for statistical permit writing. To this end, managers of 22 plants, 10 achieving low effluent TP, nine achieving low effluent TN, and three achieving low effluent NH(3)-N, provided three years of operational data that were analyzed using a consistent statistical approach. Technology Performance Statistics (TPSs) were developed as three separate values representing the ideal, median, and reliably achievable performance. Technological conclusions can be drawn from the study in terms of what can be learned by comparing the different nutrient removal and nitrification processes employed at these 22 plants.

Investigating the use of anaerobically stored carbon in post-anoxic denitrification.

Significant methanol savings are hypothesized to result from anaerobic storage of internal carbon that is used for post-anoxic denitrification. An investigation into this internal carbon-driven denitrification was performed via a series of batch tests using biomass from Hampton Roads Sanitation District's (HRSD's) water resource recovery facilities (WRRFs): the Virginia Initiative Plant (VIP), Nansemond Plant (NP), and Army Base (AB) Treatment Plant. Internal carbon specific denitrification rates (SDNRs) increased during winter, by as much as 1 mg N/g MLVSS/h for VIP. Increasing the aeration time by 2-4 h lowered the SDNR by an average of 0.21-0.35 mg N/g MLVSS/h. No internal carbon denitrification was observed for biomass from non-nitrifying/denitrifying, biological phosphorus removal (bio-P) WRRFs. The increase in internal carbon SDNRs when the anaerobic acetate dose increased from 20 to 100 mg COD/L ranged from 0.06 to 0.28 mg N/g MLVSS/h. Higher phosphorus uptake rates were found to correlate to higher internal carbon SDNRs, but no significant post-anoxic P uptake was observed. The first steps are taken towards developing a strategy for full-scale implementation of this relatively novel type of denitrification by evaluating how some factors affect its occurrence. PRACTITIONER POINTS: Significant methanol savings at a full-scale facility may result from use of internally stored carbon for post-anoxic denitrification. Short aerobic HRTs and high anaerobic zone VFA loading increase the post-anoxic internal carbon-driven denitrification. Non-nitrifying, bio-P biomass is not capable of internal carbon-driven denitrification. Internal carbon-driven denitrification is correlated with the activity of polyphosphate accumulating organisms.

Free ammonia resistance of nitrite-oxidizing bacteria developed in aerobic granular sludge cultivated in continuous upflow airlift reactors performing partial nitritation.

Free ammonia (FA) inhibition has been taken advantage as a strategy to suppress the growth of nitrite-oxidizing bacteria (NOB) in aerobic granules stabilized in a continuous upflow airlift reactor to achieve partial nitritation. However, after nearly 18 months of continuous exposure of aerobic granules to FA in the reactor, the FA inhibition of NOB was proven ineffective, and the partial nitritation gradually shifted to partial nitrification even though the long-term granule structural stability remained excellent under the continuous-flow mode. The extent of NOB resistance to FA inhibition was quantified based on the kinetic response of NOB to various FA concentrations in the form of an uncompetitive inhibition coefficient. It was confirmed that the NOB immobilized in larger granules under longer term exposure to FA tend to become more resistant to FA. Thereby, it was concluded that NOB can develop strong resistance to FA after continuous exposure, and thus, FA inhibition is not a reliable strategy to achieve partial nitritation in mainstream wastewater treatment. PRACTITIONER POINTS: Nitrifying aerobic granules can remain structurally stable in continuous-flow bioreactors. NOB developed free ammonia resistance after 6-month continuous exposure. Larger aerobic granules tended to develop stronger free ammonia resistance. Free ammonia inhibition is not a reliable strategy for mainstream anammox.

Intracellular polyphosphate length characterization in polyphosphate accumulating microorganisms (PAOs): Implications in PAO phenotypic diversity and enhanced biological phosphorus removal performance.

Polyphosphate (polyP) accumulating organisms (PAOs) are the key agent to perform enhanced biological phosphorus removal (EBPR) activity, and intracellular polyP plays a key role in this process. Potential associations between EBPR performance and the polyP structure have been suggested, but are yet to be extensively investigated, mainly due to the lack of established methods for polyP characterization in the EBPR system. In this study, we explored and demonstrated that single-cell Raman spectroscopy (SCRS) can be employed for characterizing intracellular polyPs of PAOs in complex environmental samples such as EBPR systems. The results, for the first time, revealed distinct distribution patterns of polyP length (as Raman peak position) in PAOs in lab-scale EBPR reactors that were dominated with different PAO types, as well as among different full-scale EBPR systems with varying configurations. Furthermore, SCRS revealed distinctive polyP composition/features among PAO phenotypic sub-groups, which are likely associated with phylogenetic and/or phenotypic diversity in EBPR communities, highlighting the possible resolving power of SCRS at the microdiversity level. To validate the observed polyP length variations via SCRS, we also performed and compared bulk polyP length characteristics in EBPR biomass using conventional polyacrylamide gel electrophoresis (PAGE) and solution 31P nuclear magnetic resonance (31P-NMR) methods. The results are consistent with the SCRS findings and confirmed the variations in the polyP lengths among different EBPR systems. Compared to conventional methods, SCRS exhibited advantages as compared to conventional methods, including the ability to characterize in situ the intracellular polyPs at subcellular resolution in a label-free and non-destructive way, and the capability to capture subtle and detailed biochemical fingerprints of cells for phenotypic classification. SCRS also has recognized limitations in comparison with 31P-NMR and PAGE, such as the inability to quantitatively detect the average polyP chain length and its distribution. The results provided initial evidence for the potential of SCRS-enabled polyP characterization as an alternative and complementary microbial community phenotyping method to facilitate the phenotype-function (performance) relationship deduction in EBPR systems.