Side-stream EBPR process (S2EBPR) is a new alternative to address the common challenges in EBPR related to weak wastewater influent and to improve EBPR process stability. A systematic evaluation and comparison of the process performance and microbial community structure between four S2EBPR with conventional EBPR configurations in US was conducted. The statistical analysis suggested higher performance stability in S2EBPR than the conventional EBPRs, although possible bias is recognized due to variations in the target permit levels and plant-specific factors among the plants. Total and known PAOs and GAOs abundance and identities were investigated with FISH, DAPI, 16S rRNA gene sequencing and Raman microspectroscopy. The results suggested comparable relative PAO and Candidatus Accumulibacter abundances in S2EBPR and conventional EBPR systems. Tetrasphaera, a putative PAO, was also found at similar abundance in S2EBPR as in conventional facilities, whereas the relative abundance of known GAOs was lower in S2EBPR than those typically seen at conventional EBPRs. Microbial community analyses via 16S rRNA gene amplicon sequencing revealed differences in the community phylogenetic fingerprints between S2EBPR and conventional plants. Shannon and Inverse Simpson indices, which are combined measures of richness and evenness evaluation of the microbial communities, suggested that the microbial diversity in S2EBPR plants were higher than those in conventional EBPRs.

Thorium (Th) is a natural radioactive element present in the environment and has a potential to be used as a nuclear fuel. Relatively little is known about the influence and toxicity of Th in the environment. In the present study, the toxicity of Th to the green algae Chlorella pyrenoidosa (C. Pyrenoidosa) was evaluated by algal growth inhibition, biochemical assays and morphologic observations. In the cultural medium (OECD TG 201), Th(NO₃)₄ was transformed to amorphous precipitation of Th(OH)₄ due to hydrolysis. Th was toxic to C. Pyrenoidosa, with a 96 h half maximum effective concentration (EC₅₀) of 10.4 μM. Scanning electron microscopy shows that Th-containing aggregates were attached onto the surface of the algal cells, and transmission electron microscopy indicates the internalization of nano-sized Th precipitates and ultrastructural alterations of the algal cells. The heteroagglomeration between Th(OH)₄ precipitation and alga cells and enhanced oxidative stress might play important roles in the toxicity of Th. To our knowledge, this is the first report of the toxicity of Th to algae with its chemical species in the exposure medium. This finding provides useful information on understanding the fate and toxicity of Th in the aquatic environment.

In this study, a full-scale pilot testing was performed with side-by-side operation of a conventional enhanced biological phosphorus removal (EBPR) process and a side-stream EBPR (S2EBPR) process. A comparison of the performance, activities and population dynamics of key functionally relevant populations between the two configurations were carried out. The results demonstrated that, with the same influent wastewater characteristics, S2EBPR configuration showed more effective and stable orthophosphate (PO₄-P) removal performance (up to 94% with average effluent concentration down to 0.1 mg P/L) than conventional EBPR, especially when the mixers in side-stream reactor were operated intermittently. Mass balance analysis illustrated that both denitrification and EBPR performance have been enhanced in S2EBPR configuration through diverting primary effluent to anoxic zone and producing additional carbon (~40%) via fermentation in side-stream reactor. Microbial characterization showed that there was no significant difference in the relative abundances of Ca. Accumulibacter (~5.9%) and Tetrasphaera (~16%) putative polyphosphate-accumulating organisms (PAOs) between the two configurations. However, lower relative abundance of known GAOs was observed in S2EBPR configuration (1.1%) than the conventional one (2.7%). A relatively higher PAO activity and increased degree of dependence on glycolysis pathway than TCA cycle was observed in S2EBPR configuration using P release and uptake batch test. Adequate anaerobic solid retention time (SRT) and conditions that generate continuous and slow feeding/production of volatile fatty acid (VFA) with higher composition percentage of propionate in the side-stream reactor of S2EBPR process likely provide a competitive advantage for PAOs over GAOs.

A large number of microplastics (MPs) have been found in various stages of wastewater treatment plants, which may affect the functional microbial activity in activated sludge and lead to unstable pollutant removal performance. In this study, the effects of different concentrations of polylactic acid microplastics (PLA MPs) on system performance, nitrification and phosphorus (P) removal activities, and extracellular polymeric substances (EPS) were evaluated. The results showed that under the same influent conditions, low concentrations (50 particles/(g TS)) of PLA MPs had no significant effect on effluent quality. The average removal efficiencies of chemical oxygen demand, phosphate and ammonia were all above 80%, and the average removal efficiencies of total nitrogen remained above 70%. High concentrations (200 particles/(g TS)) of PLA MPs inhibited the activities of polyphosphate accumulating organisms (PAOs) and nitrifying bacteria. The specific anaerobic P release rate decreased from 37.7 to 23.1 mg P/(g VSS·h), and the specific aerobic P uptake rate also decreased significantly. The specific ammonia oxidation rate decreased from 0.67 to 0.34 mg N/(g VSS·h), while the change in specific nitrite oxidation rate was not significant. The dosing of PLA MPs decreased the total EPS and humic acid content. As the concentration of PLA MPs increased, microbial community diversity increased. The relative abundance of potential PAOs (i.e., Acinetobacter) increased from 0.08% to 12.57%, while the relative abundance of glycogen accumulating organisms (i.e., Competibacter and Defluviicoccus) showed no significant changes, which would lead to improved P removal performance. The relative abundance of denitrifying bacteria (i.e., Pseudomonas) decreased from 95.43% to 58.98%, potentially contributing to the decline in denitrification 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.

To meet the growing interest in the development of innovative biological nutrient removal (BNR) alternatives for energy neutrality, resource recovery, and decarbonization, the adsorption/bio-oxidation (A/B) process has been widely studied for carbon capture and shortcut nitrogen (N) removal, while limited research has focused on incorporating enhanced biological phosphorus (P) removal (EBPR), mainly due to the differential carbon usage characteristics of functionally relevant microorganisms. Here, a full-scale pilot testing with an integrated system combining A-stage high-rate activated sludge (HRAS) 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 low influent carbon load, the B-stage P(D)N-S2EBPR system achieved effective and stable P removal performance, especially when the volatile fatty acid (VFA) load from A-stage was elevated. Sludge fermentation in both A-stage and B-stage promoted carbon redistribution and likely provided more competitive advantages for ammonium-oxidizing bacteria and polyphosphate accumulating organisms, leading to carbon-efficient shortcut N removal mainly through partial nitrification pathway and influent carbon-independent EBPR simultaneously. Exposure to high VFA levels was considered a potential selection factor for the suppression of nitrite-oxidizing bacteria in the system. The involvement of internal carbon-accumulating organisms would potentially play an important role in endogenous denitrification. This study provided new insights into the effects of incorporating side-stream EBPR into the A/B process on microbial ecology, metabolic activities, and system performance.