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.

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.

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.

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.

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.