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.

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.