Elucidating sludge characteristic, substrate transformation and microbial evolution in a two-sludge denitrifying...

Granule formation has been recognized as a promising biotechnology in denitrifying phosphorus removal (DPR) systems by facilitating phosphorus accumulation organisms (PAOs) especially denitrifying PAOs (DPAOs), and hydraulic selection made this a more difficult task in continuous operation. This study aimed at exploring the microscopic mechanism and putting forward an effective strategy for DPR granulation under the impact of hydraulic retention time (HRT) (12 h, 10 h, 8 h) in a novel Anaerobic Anoxic Oxic - Moving Bed Biofilm Reactor (A²/O - MBBR) system. With the reduction of intracellular carbon storage (COD_intra) efficiency (88.58%-78.53%), nitrogen (N) (85.45%-79.11%) and phosphorus (P) (96.55%-92.47%) removals both dropped, but it exhibited a growth of anoxic phosphorus uptake rate (PUR_A) (3.79-5.68 mg P/(gMLVSS·h)). The batch tests associating with substrate transformation of poly-β-hydroxyalkanoates (PHA), glycogen (Gly) agreed well with the corresponding stoichiometry of phosphorus release rate (PRR) (4.83-7.53 mg P/(gMLVSS·h)), PUR_A (3.55-5.43 mg P/(gMLVSS·h)), oxic phosphorus uptake rate (PUR_O) (6.08-6.21 mg P/(gMLVSS·h)), and DPAOs/PAOs ratios (57.17%-89.31%), indicating a shift of microbial community. DPR granules gradually stabilized with low sludge volume index (SVI₅/SVI₃₀ ratio = 1.1-1.2), dense and compact structure, higher P content (11.63%), more extracted extracellular polymeric substances (EPS) (111.40-160.31 mg/gMLVSS) as proteins/polysaccharides (PN/PS) ratios (1.70-3.47) increased, leading to better sludge settleability and cell hydrophobicity. Fluorescence in situ hybridization (FISH) results showed that PAOs (mainly Cluster I: 20.20%) were the dominant bacteria in the A²/O reactor although a small amount of Defluviicoccus (3.18-3.48%) was responsible for nitrite accumulation, while ammonium-oxidizing bacteria (AOB) (mainly Nitrosomonas: 10.75%) and nitrite-oxidizing bacteria (NOB) (mainly Nitrospira: 15.06%) were enriched in the MBBR.