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.

Revisiting the role of Acinetobacter spp. in side-stream enhanced biological phosphorus removal (S2EBPR) systems.

We piloted the incorporation of side-stream enhanced biological phosphorus removal (S2EBPR) with A/B stage short-cut nitrogen removal processes to enable simultaneous carbon-energy-efficient nutrients removal. This unique configuration and system conditions exerted selective force on microbial populations distinct from those in conventional EBPR. Interestingly, effective P removal was achieved with the predominance of Acinetobacter (21.5 ± 0.1 %) with nearly negligible level of known conical PAOs (Ca. Accumulibacter and Tetrasphaera were 0.04 ± 0.10 % and 0.47 ± 0.32 %, respectively). Using a combination of techniques, such as fluorescence in situ hybridization (FISH) coupled with single cell Raman spectroscopy (SCRS), the metabolic tracing of Acinetobacter-like cells exerted PAO-like phenotypic profiling. In addition, comparative metagenomics analysis of the closely related Acinetobacter spp. revealed the EBPR relevant metabolic pathways. Further oligotyping analysis of 16s rRNA V4 region revealed sub-clusters (microdiversity) of the Acinetobacter and revealed that the sub-group (oligo type 1, identical (100 % alignment identity) hits from Acinetobacter_midas_s_49494, and Acinetobacter_midas_s_55652) correlated with EBPR activities parameters, provided strong evidence that the identified Acinetobacter most likely contributed to the overall P removal in our A/B-shortcut N-S2EBPR system. To the best of our knowledge, this is the first study to confirm the in situ EBPR activity of Acinetobacter using combined genomics and SCRS Raman techniques. Further research is needed to identify the specific taxon, and phenotype of the Acinetobacter that are responsible for the P-removal.

Toward Integrating EBPR and the Short-Cut Nitrogen Removal Process in a One-Stage System for Treating High-Strength Wastewater.

Enhanced biological phosphorus removal (EBPR) is an economical and sustainable process for phosphorus removal from wastewater. Despite the widespread application of EBPR for low-strength domestic wastewater treatment, limited investigations have been conducted to apply EBPR to the high-strength wastewaters, particularly, the integration of EBPR and the short-cut nitrogen removal process in the one-stage system remains challenging. Herein, we reported a novel proof-of-concept demonstration of integrating EBPR and nitritation (oxidation of ammonium to nitrite) in a one-stage sequencing batch reactor to achieve simultaneous high-strength phosphorus and short-cut nitrogen removal. Excellent EBPR performance of effluent 0.8 ± 1.0 mg P/L and >99% removal efficiency was achieved fed with synthetic high-strength phosphorus wastewater. Long-term sludge acclimation proved that the dominant polyphosphate accumulating organisms (PAOs), Candidatus Accumulibacter, could evolve to a specific subtype that can tolerate the nitrite inhibition as revealed by operational taxonomic unit (OTU)-based oligotyping analysis. The EBPR kinetic and stoichiometric evaluations combined with the amplicon sequencing proved that the Candidatus Competibacter, as the dominant glycogen accumulating organisms (GAOs), could well coexist with PAOs (15.3-24.9% and 14.2-33.1%, respectively) and did not deteriorate the EBPR performance. The nitrification activity assessment, amplicon sequencing, and functional-based gene marker quantification verified that the unexpected nitrite accumulation (10.7-21.0 mg N/L) in the high-strength EBPR system was likely caused by the nitritation process, in which the nitrite-oxidizing bacteria (NOB) were successfully out-selected (<0.1% relative abundance). We hypothesized that the introduction of the anaerobic phase with high VFA concentrations could be the potential selection force for achieving nitritation based on the literature review and our preliminary batch tests. This study sheds light on developing a new feasible technical route for integrating EBPR with short-cut nitrogen removal for efficient high-strength wastewater treatment.

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.

Selectivity in Enzymatic Phosphorus Recycling from Biopolymers: Isotope Effect, Reactivity Kinetics, and Molecular Docking with Fungal and Plant Phosphatases.

Among ubiquitous phosphorus (P) reserves in environmental matrices are ribonucleic acid (RNA) and polyphosphate (polyP), which are, respectively, organic and inorganic P-containing biopolymers. Relevant to P recycling from these biopolymers, much remains unknown about the kinetics and mechanisms of different acid phosphatases (APs) secreted by plants and soil microorganisms. Here we investigated RNA and polyP dephosphorylation by two common APs, a plant purple AP (PAP) from sweet potato and a fungal phytase from Aspergillus niger. Trends of δ18O values in released orthophosphate during each enzyme-catalyzed reaction in 18O-water implied a different extent of reactivity. Subsequent enzyme kinetics experiments revealed that A. niger phytase had 10-fold higher maximum rate for polyP dephosphorylation than the sweet potato PAP, whereas the sweet potato PAP dephosphorylated RNA at a 6-fold faster rate than A. niger phytase. Both enzymes had up to 3 orders of magnitude lower reactivity for RNA than for polyP. We determined a combined phosphodiesterase-monoesterase mechanism for RNA and terminal phosphatase mechanism for polyP using high-resolution mass spectrometry and 31P nuclear magnetic resonance, respectively. Molecular modeling with eight plant and fungal AP structures predicted substrate binding interactions consistent with the relative reactivity kinetics. Our findings implied a hierarchy in enzymatic P recycling from P-polymers by phosphatases from different biological origins, thereby influencing the relatively longer residence time of RNA versus polyP in environmental matrices. This research further sheds light on engineering strategies to enhance enzymatic recycling of biopolymer-derived P, in addition to advancing environmental predictions of this P recycling by plants and microorganisms.

Machine Learning-Based Determination of Sampling Depth for Complex Environmental Systems: Case Study with Single-Cell Raman Spectroscopy Data in EBPR Systems.

Rapid progress in various advanced analytical methods, such as single-cell technologies, enable unprecedented and deeper understanding of microbial ecology beyond the resolution of conventional approaches. A major application challenge exists in the determination of sufficient sample size without sufficient prior knowledge of the community complexity and, the need to balance between statistical power and limited time or resources. This hinders the desired standardization and wider application of these technologies. Here, we proposed, tested and validated a computational sampling size assessment protocol taking advantage of a metric, named kernel divergence. This metric has two advantages: First, it directly compares data set-wise distributional differences with no requirements on human intervention or prior knowledge-based preclassification. Second, minimal assumptions in distribution and sample space are made in data processing to enhance its application domain. This enables test-verified appropriate handling of data sets with both linear and nonlinear relationships. The model was then validated in a case study with Single-cell Raman Spectroscopy (SCRS) phenotyping data sets from eight different enhanced biological phosphorus removal (EBPR) activated sludge communities located across North America. The model allows the determination of sufficient sampling size for any targeted or customized information capture capacity or resolution level. Promised by its flexibility and minimal restriction of input data types, the proposed method is expected to be a standardized approach for sampling size optimization, enabling more comparable and reproducible experiments and analysis on complex environmental samples. Finally, these advantages enable the extension of the capability to other single-cell technologies or environmental applications with data sets exhibiting continuous features.

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.

Oligotyping and metagenomics reveal distinct Candidatus Accumulibacter communities in side-stream versus conventional full-scale enhanced biological phosphorus removal (EBPR) systems.

Candidatus Accumulibacter phosphatis (CAP) and its clade-level micro-diversity has been associated with and implicated in functional differences in phosphorus removal performance in enhanced biological phosphorus removal (EBPR) systems. Side-stream EBPR (S2EBPR) is an emerging process that has been shown to present a suite of advantages over the conventional EBPR design, however, large knowledge gaps remain in terms of its underlying ecological mechanisms. Here, we compared and revealed the higher-resolution differences in microbial ecology of CAP between a full-scale side-stream EBPR configuration and a conventional A2O EBPR process that were operated in parallel and with the same influent feed. Even though the relative abundance of CAP, revealed by 16S rRNA gene amplicon sequencing, was similar in both treatment trains, a clade-level analysis, using combined 16S rRNA-gene based amplicon sequencing and oligotyping analysis and metagenomics analysis, revealed the distinct CAP microdiversity between the S2EBPR and A2O configurations that likely attributed to the improved performance in S2EBPR in comparison to conventional EBPR. Furthermore, genome-resolved metagenomics enabled extraction of three metagenome-assembled genomes (MAGs) belonging to CAP clades IIB (RCAB4-2), IIC (RC14) and II (RC18), from full-scale EBPR sludge for the first time, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. The results also revealed the temporally increasing predominance of RC14, which belonged to Clade IIC, during the implementation of the S2EBPR configuration. Finally, we also show the existence of previously uncharacterized diversity of clades of CAP, namely the clades IIB and as yet unidentified clade of type II, in full-scale EBPR communities, highlighting the unknown diversity of CAP communities in full-scale EBPR systems.

Molecular Coordination, Structure, and Stability of Metal-Polyphosphate Complexes Resolved by Molecular Modeling and X-ray Scattering: Structural Insights on the Biological Fate of Polyphosphate.

Polyphosphate-accumulating organisms (PAOs), which can store high levels of phosphate (Pi) in the form of polyphosphate (polyP), are employed to engineer enhanced biological P removal (EBPR) from wastewaters. Co-localization of Mg and K in polyP granules of PAOs has been reported, and higher abundance of Mg-polyP granules relative to other metal complexes was correlated positively with EBPR performance stability. However, the underlying mechanism remains unknown. Here, we obtained molecular structural information of hydrated polyP complexes with four physiologically relevant metal cations (Na+, K+, Ca2+, and Mg2+) using computational and experimental techniques. Molecular dynamics simulations revealed that Mg-polyP and K-polyP complexes were the most and least stable of the complexes, respectively, suggesting that the co-occurrence of these complexes facilitates variable polyP bioavailability. The relative thermodynamic stability reflected the strength of metal chelation whereby the coordination distance between the polyP ligand O and the metal was 1.71-2.01 Å for Mg2+ but this distance was 2.64-2.70 Å for K+. Pair distribution function analysis of X-ray scattering data obtained with a Mg-polyP solution corroborated the theoretical Mg-polyP coordination geometry. These findings implied a possible mechanistic role of metal complexation in the P cycling traits of PAOs in engineered and natural systems.

Status and advances in technologies for phosphorus species detection and characterization in natural environment- A comprehensive review.

Poor recovery of phosphorus (P) across natural environment (water, soil, sediment, and biological sources) is causing rapid depletion of phosphate rocks and continuous accumulation of P in natural waters, resulting in deteriorated water quality and aquatic lives. Accurate detection and characterization of various P species using suitable analytical methods provide a comprehensive understanding of the biogeochemical cycle of P and thus help its proper management in the environment. This paper aims to provide a comprehensive review of the analytical methods used for P speciation in natural environment by dividing them into five broad categories (i.e., chemical, biological, molecular, staining microscopy, and sensors) and highlighting the suitability (i.e., targeted species, sample matrix), detection limit, advantages-limitations, and reference studies of all methods under each category. This can be useful in designing studies involving P detection and characterization across environmental matrices by providing insights about a wide range of analytical methods based on the end user application needs of individual studies.

Unrevealed roles of polyphosphate-accumulating microorganisms.

We first review current knowledge on PAOs, with a focus on bacteria, in terms of their phylogenetic identities, metabolic pathways and detection methods. We further discuss the evidence that suggests the ubiquitous presence of PAOs in nature and point out the unrevealed roles of the PAOs that warrant future investigation.

Impact of oxidation processes on the composition and biodegradability of soluble organic nutrients in wastewater effluents.

The characteristics and bioavailability of wastewater derived organic nutrients and their susceptibility to removal technologies have implications in nutrient loading to aquatic environments and their contributions to eutrophication. Therefore, a better understanding of treatability of effluent organic nutrients is of interest for water resource recovery facilities (WRRFs) and regulators. Oxidation processes (OPs) can reduce concentrations of soluble organic nutrients and convert them into more biodegradable forms. In this study, three WRRF effluents were treated with low-pressure ultraviolet (UV) irradiation, hydrogen peroxide (H2 O2 ), and combined UV/H2 O2 . Untreated and treated effluents were subjected to nitrogen and phosphorus speciation analyses and soluble organic nitrogen (SON) biodegradability assays. The OP treatments did not change SON concentrations significantly. For two WRRFs, OP treatments decreased soluble organic phosphorus (SOP) and seemed to convert it into soluble acid hydrolyzable phosphorus (SAHP), indicating possible increases in phosphorus bioavailability. Fingerprinting and quantification of dissolved organic matter (DOM) using fluorescence spectroscopy with parallel factor analysis revealed changes in DOM pool composition in response to OPs treatments, which suggests likely organic nutrients composition changes. Based on biodegradability assessments, OP treatments likely changed the composition and biodegradability of effluent SON compounds. Combined UV/H2 O2 treatment seemed more effective than other OPs at oxidizing some of the organic nutrients. PRACTITIONER POINTS: Treatment of secondary or tertiary effluent with UV, H2 O2 , or UV/ H2 O2 was generally not effective at mineralizing SON and SOP, when applied at the doses used in this study. Treatment resulted in observable changes in DOM compositions, likely including SON and SOP compounds. Combined UV/H2 O2 treatment was more effective than UV or H2 O2 alone at oxidizing some DOM compounds. The BSON (bioavailable SON) assay indicated that the composition of the SON pool in the effluents was likely changed by the OP treatments. This was supported by fluorescence spectroscopy analysis.

Phenotypic dynamics in polyphosphate and glycogen accumulating organisms in response to varying influent C/P ratios in EBPR systems.

This study employed molecular tools and single cell Raman micro-spectroscopy techniques to reveal the single cell- and population-level phenotypic dynamics and changes in functionally relevant organisms, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), in response to influent loading readily biodegradable carbon to phosphorus ratio (C/P) changes in enhanced biological phosphorus removal (EBPR) systems. The results, for the first time, provided direct and cellular evidence confirming the adaptive anaerobic metabolic pathway shifts in PAOs in response to influent loading variations. Increase in influent readily biodegradable carbon to phosphorus (C/P) ratio from 20 to 50 led to nearly 50% decline in polyphosphate content and drastic rise of intracellular polyßhydroxybutyrate (PHB) to polyphosphate (polyP) ratio by nearly 6 times in PAOs, indicating corresponding diminishing reliance on polyP hydrolysis for energy as P becomes limiting. Influent carbon availability surge also impacted the intracellular carbon polymers in GAOs, with significant increase in the mean PHB content level but no observed changes in the intracellular glycogen level. Furthermore, the Raman-based quantification of differentiated intracellular polymer content associated with PAOs and GAOs, revealed new insights into the quantitative shift in intracellular carbon storage distribution between the two populations and their variations between the two carbon polymers (PHB, Glycogen). In summary, this investigation revealed high-resolution cellular level information regarding the metabolic flexibility in PAOs, phenotypic stoichiometry changes and carbon flux and distribution among PAOs and GAOs, in response to influent loading conditions. The new information will contribute to improvement in mechanistic EBPR modeling and design.

Impact of solid residence time (SRT) on functionally relevant microbial populations and performance in full-scale enhanced biological phosphorus removal (EBPR) systems.

Investigations of the impact of solid residence time (SRT) on microbial ecology and performance of enhanced biological phosphorus removal (EBPR) process in full-scale systems have been scarce due to the challenges in isolating and examining the SRT from other complex plant-specific factors. This study performed a comprehensive evaluation of the influence of SRT on polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) dynamics and on P removal performance at Clark County Water Reclamation District Facility in Las Vegas, USA. Five parallel treatment trains with separated clarifiers were operated with five different SRTs ranging from 6 to 40 days. Microbial community analysis using multiple molecular and Raman techniques suggested that the relative abundances and diversity of PAOs and GAOs in EBPR systems are highly affected by the SRT. The resultant EBPR system stability and performance can be potentially controlled and optimized by manipulating the system SRT, and shorter SRT (<10 days) seems to be preferred. PRACTITIONER POINTS: Phosphorus removal performance and kinetics are highly affected by the operational solid residence time (SRT), with lower and more stable effluent P level achieved at SRT < 10 days. Excessive long SRTs above that needed for nitrification may harm EBPR performance; additionally, excessive long SRT may favor GAOs to dominate over PAOs and thus further reducing efficient use of rbCOD for EBPR. Microbial population abundance and diversity, especially those functionally relevant PAOs and GAOs, can impact the P removal performances, and they are highly dependent on the operational solid residence time. EBPR performance can be potentially controlled and optimized by manipulating the system SRT, and shorter SRT (≤10 days) seems to be preferred at the influent rbCOD/P ratio and environmental conditions as in the plant studied.

Survey of full-scale sidestream enhanced biological phosphorus removal (S2EBPR) systems and comparison with conventional EBPRs in North America: Process stability, kinetics, and microbial populations.

Sidestream EBPR (S2EBPR) is an emerging alternative process to address common challenges in EBPR related to weak wastewater influent and may improve EBPR process stability. A systematic evaluation and comparison of the process performance and microbial community structure was conducted between conventional and S2EBPR facilities in North America. The statistical analysis suggested higher performance stability in S2EBPR than conventional EBPR, although possible bias associated with other plant-specific factors might have affected the comparison. Variations in stoichiometric values related to EBPR activity and discrepancies between the observed values and current model predictions suggested a varying degree of metabolic versatility of PAOs in S2EBPR systems that warrant further investigation. Microbial community analysis using various techniques suggested comparable known candidate PAO relative abundances in S2EBPR and conventional EBPR systems, whereas the relative abundance of known candidate GAOs seemed to be consistently lower in S2EBPR facilities than conventional EBPR facilities. 16S rRNA gene sequencing analysis revealed differences in the community phylogenetic fingerprints between S2EBPR and conventional facilities and indicated statistically higher microbial diversity index values in S2EBPR facilities than those in conventional EBPRs. PRACTITIONER POINTS: Sidestream EBPR (S2EBPR) can be implemented with varying and flexible configurations, and they offer advantages over conventional configurations for addressing the common challenges in EBPR related to weak wastewater influent and may improve EBPR process stability. Survey of S2EBPR plants in North America suggested statistically more stable phosphorus removal performance in S2EBPR plants than conventional EBPRs, although possible bias might affect the comparison due to other plant-specific factors. The EBPR kinetics and stoichiometry of the S2EBPR facilities seemed to vary and are associated with metabolic versatility of PAOs in S2EBPR systems that warrant further investigation. The abundance of known candidate PAOs in S2EBPR plants was similar to those in conventional EBPRs, and the abundance of known candidate GAOs was generally lower in S2EBPR than conventional EBPR facilities. Further finer-resolution analysis of PAOs and GAOs, as well as identification of other unknown PAOs and GAOs, is needed. Microbial diversity is higher in S2EBPR facilities compared with conventional ones, implying that S2EBPR microbial communities could show better resilience to perturbations due to potential functional redundancy.

Side-stream enhanced biological phosphorus removal (S2EBPR) process improves system performance - A full-scale comparative study.

To address the common challenges in enhanced biological phosphorus removal (EBPR) related to stability and unfavorable influent carbon to phosphorus ratio, a side-stream EBPR (S2EBPR) process that involves a side-stream anaerobic biological sludge hydrolysis and fermentation reactor was proposed as an emerging alternative. In this study, a full-scale pilot testing was performed with side-by-side operation of a conventional anaerobic-anoxic-aerobic (A2O) process versus a S2EBPR process. A comparison of the performance, activity and microbial community between the two configurations was performed. The results demonstrated that, with the same influent wastewater characteristics, S2EBPR configuration showed improved P removal performance and stability than the conventional A2O configuration, especially when the mixers in the side-stream anaerobic reactor were operated intermittently. Mass balance analysis illustrated that both denitrification and EBPR were enhanced in S2EBPR configuration, where return activated sludge was diverted into the anaerobic zone to promote fermentation and enrichment of polyphosphate accumulating organisms (PAOs), and the influent was bypassed to the anoxic zone for enhancing denitrification. A relatively higher PAO activity and total PAO abundance were observed in S2EBPR than in A2O configuration, accompanied by a higher degree of dependence on glycolysis pathway than tricarboxylic acid cycle. No significant difference in the relative abundances of putative PAOs, including Ca. Accumulibacter and Tetrasphaera, were observed between the two configurations. However, higher microbial community diversity indices were observed in S2EBPR configuration than in conventional one. In addition, consistently lower relative abundance of known glycogen accumulating organisms (GAOs) was observed in S2EBPR system. Extended anaerobic retention time and conditions that generate continuous and more complex volatile fatty acids in the side-stream anaerobic reactor of S2EBPR process likely give more competitive advantage for PAOs over GAOs. PAOs exhibited sustained EBPR activity and delayed decay under extended anaerobic condition, likely due to their versatile metabolic pathways depending on the availability and utilization of multiple intracellular polymers. This study provided new insights into the effects of implementing side-stream EBPR configuration on microbial populations, EBPR activity profiles and resulted system performance.

The Composition and Implications of Polyphosphate-Metal in Enhanced Biological Phosphorus Removal Systems.

The individual cellular level and quantitative Polyphosphate (PolyP)-metal compositions in EBPR (enhanced biological phosphorus removal) systems have hardly been investigated and its potential link to EBPR performance therefore remain largely unknown. In this study, we applied scanning electron microscopy combined with energy dispersive X-ray spectroscopy (SEM/EDX) method that enabled detection and semiquantification of metal elemental compositions in intact intracellular PolyP granules in individual PAO (polyphosphate accumulating organism) cells. We, for the first time, revealed diverse and dynamic distributions of different metals ions in the PolyP-metal granules in different EBPR systems operated with the same influent metal composition but varying SRT of 5-30 days. We further demonstrated that the PolyP-metal composition diversity correlated with 16S rRNA gene based PAO phylogenetic diversity, suggesting the possible phylogeny-dependent PolyP-metal composition variation. The impact of PolyP metal composition in EBPR system, especially the Mg content in PolyP granules, was evidenced by the significant and strong positive correlation between PolyP-Mg content and the long-term stability of the four EBPR systems with varying SRTs. The PolyP-Mg content can therefore possibly serve as an indicator for EBPR performance monitoring. The results demonstrated that phenotyping techniques, such as PolyP-metal-based profiling, in compliment, or combined with genotyping techniques such as phylogenetic and functional gene sequencing, can provide more insights into the mechanisms and performance prediction of this important microbial ecosystem.

Toward Better Understanding of EBPR Systems via Linking Raman-Based Phenotypic Profiling with Phylogenetic Diversity.

This study reports a proof-of concept study to demonstrate the novel approach of phenotyping microbial communities in enhanced biological phosphorus removal (EBPR) systems using single cell Raman microspectroscopy and link it with phylogentic structures. We use hierarchical clustering analysis (HCA) of single-cell Raman spectral fingerprints and intracellular polymer signatures to separate and classify the functionally relevant populations in EBPR systems, namely polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), as well as other microbial populations. We then investigated the link between Raman-based community phenotyping and 16S rRNA gene-based phylogenetic characterization of four lab-scale EBPR systems with varying solid retention time (SRT) to gain insights into possible genotype-function relationships. Combined and simultaneous phylogenetic and phenotypic evaluation of EBPR ecosystems revealed SRT-dependent phylogenetic and phenotypic characteristics of the PAOs and GAOs, and their association with EBPR performance. The phenotypic diversity and plasticity of PAO populations, which otherwise could not be obtained with phylogenetic analysis alone, showed complex but potentially crucial association with EBPR process stability.

Recent advances in bio-P modelling - a new approach verified by full-scale observations.

This paper summarizes recent developments in biological phosphorus removal modelling, with special attention to side-stream enhanced biological phosphorus removal (S2EBPR) systems on which previous models proved to be ineffective without case-by-case parameter adjustments. Through the research and experience of experts and practitioners, a new bio-kinetic model was developed including an additional group of biomass (glycogen accumulating organisms - GAOs) and new processes (such as aerobic and anoxic maintenance for PAO and GAO; enhanced denitrification processes; fermentation by PAOs which - along with PAO selection - is driven by oxidation-reduction potential (ORP)). This model successfully described various conditions in laboratory measurements and full plant data. The calibration data set is provided by Clean Water Services from Rock Creek Facility (Hillsboro, OR) including two parallel trains: conventional A2O and Westbank configurations, allowing the model to be verified on conventional and side-stream EBPR systems as well.

Life-Cycle Assessment of Advanced Nutrient Removal Technologies for Wastewater Treatment.

Advanced nutrient removal processes, while improving the water quality of the receiving water body, can also produce indirect environmental and health impacts associated with increases in usage of energy, chemicals, and other material resources. The present study evaluated three levels of treatment for nutrient removal (N and P) using 27 representative treatment process configurations. Impacts were assessed across multiple environmental and health impacts using life-cycle assessment (LCA) following the Tool for the Reduction and Assessment of Chemical and Other Environmental Impacts (TRACI) impact-assessment method. Results show that advanced technologies that achieve high-level nutrient removal significantly decreased local eutrophication potential, while chemicals and electricity use for these advanced treatments, particularly multistage enhanced tertiary processes and reverse osmosis, simultaneously increased eutrophication indirectly and contributed to other potential environmental and health impacts including human and ecotoxicity, global warming potential, ozone depletion, and acidification. Average eutrophication potential can be reduced by about 70% when Level 2 (TN = 3 mg/L; TP = 0.1 mg/L) treatments are employed instead of Level 1 (TN = 8 mg/L; TP = 1 mg/L), but the implementation of more advanced tertiary processes for Level 3 (TN = 1 mg/L; TP = 0.01 mg/L) treatment may only lead to an additional 15% net reduction in life-cycle eutrophication potential.