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The characteristics of enhanced biological phosphorus removal (EBPR) process under the combined actions of intracellular and extracellular polyphosphate (polyP) were investigated with the 31 P Nuclear Magnetic Resonance (NMR) and the fractionation extracting the loosely-bound and tightly-bound extracellular polymer substances (i.e., LB-EPS and TB-EPS) and bacterial cells in EBPR sludge. The hydrolysis/synthesis of extracellular and intracellular polyP was a key step of the phosphate migration and transformation in EBPR sludge. The orthophosphate (orthoP) produced from the intracellular and extracellular polyP anaerobic-hydrolysis was partially accumulated in the bacterial cells and TB-EPS, and then the accumulated orthoP was main composition for these polyP aerobic-synthesis. Importantly, the anaerobic-hydrolysis enhancement of intracellular and extracellular ployP could promote EBPR sludge to absorb volatile fatty acids (VFAs) followed by being transformed into intracellular poly-hydroxy-alkanoates (PHAs). The mechanism for VFAs passing through the LB-EPS and TB-EPS should be an anion-exchange action between orthoP and VFAs. The orthoP accumulation in the TB-EPS kept an orthoP concentration gradient among the TB-EPS, LB-EPS and bulk solution, driving orthoP and VFAs migrations. The orthoP accumulation in the bacterial cells could keep an orthoP concentration difference between the cell-membrane two sides of phosphorus accumulating organisms (PAOs) to promote VFAs passing through the cell membrane considered as an anion exchange membrane. The intracellular PHAs continuously hydrolyzed accompanied with the average chain-length increases of the extracellular and intracellular polyP during the whole aerobic stage. Additionally, the energy of the extracellular polyP synthesized in situ should came from the intracellular PHAs hydrolysis., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Intracellular polyphosphate (poly-P) plays important roles in Enhanced biological phosphorus removal (EBPR) process, but an effective and reliable protocol for extracting intracellular P and its poly-P in EBPR sludge without hydrolysis of poly-P has not been setup yet. In the study, it was revealed that the severe hydrolysis of intracellular poly-P occurred during the different extraction processes, such as acid (i.e., HClO 4 , H 2 SO 4 and HCl), basic (i.e., NaOH and KOH) and freezing-grind (under different solid-liquid ratios), but it did not occur during ultrasonic extraction process. The optimal extraction process of the ultrasonic protocol was 10 w/mL of ultrasonic power density and 15 min of ultrasonic time, when the extraction efficiency of intracellular P was 88.24 ± 1.56%. In addition, the extraction efficiency of intracellular P could be furtherly improved by that the 0.75 mol/L LiCl solution was used to resuspend the bacterial cell before ultrasonic extraction (i.e., LiCl-ultrasonic protocol). The ultrasonic protocol was more suitable to extract the intracellular P and its poly-P of EBPR sludge than the other 4 protocols (i.e., PCA-NaOH, EDTA-NaOH, freezing-grind and LiCl-ultrasonic), which had the technical characteristics of (i) with relatively high extraction efficiency of intracellular P, (ii) without hydrolysis of intracellular poly-P, (iii) with weak noise signal in 31 P NMR spectrum and (iv) with simple extraction process and short extraction time. It was founded by the ultrasonic protocol that there was the high content (82.88%-89.79% of intracellular P content) of intracellular poly-P with long average chain length (376.4-383.2) in the EBPR sludges. Importantly, it was confirmed that the EBPR process was related to the combined action of extracellular and intracellular poly-P using a new fractionation method of P in EBPR sludge, which included the ultrasonic protocol at high power density for extracting the intracellular P and its poly-P., (Copyright © 2019. Published by Elsevier B.V.)