Phosphate-accumulating Organisms Research Articles

Rapid start-up of partial nitritation and simultaneously phosphorus removal (PNSPR) granular sludge reactor treating low-strength domestic sewage

Obtaining desirable partial nitritation (PN) is crucial for successful application of the combined PN and anammox process. In this study, the partial nitritation and simultaneously phosphorus removal (PNSPR)1PNSPR: Partial nitritation and simultaneously phosphorus removal.1 granular sludge reactor treating low-strength domestic sewage was rapidly started up in 67days through seeding denitrifying phosphorus removal (DPR)2DPR: denitrifying phosphorus removal.2 sludge. The nitrite/ammonium ratio in effluent was approximately 1 and the nitrite accumulation rate (NAR) was more than 95%, about 93% of orthophosphate was removed. The DPR sludge rich in phosphate accumulating organisms (PAOs) with few nitrifying bacteria could promote the achievement of PNSPR. Quantitative microbial analysis showed that the ammonium oxidizing bacteria (AOB) gene ratio in sludge increased from 0.21% to 3.43%, while nitrite oxidizing bacteria (NOB) gradually decreased to 0.07%. The average particle size of sludge increased from 114 to 421μm, indicating the formation of PNSPR granules. The high phosphorus content in sludge and phosphorus removal facilitated rapid granulation.

Effects of electron acceptors on sulphate reduction activity in activated sludge processes

The concentration of sulphate present in wastewater can vary from 10 to 500 mg SO42−/L. During anaerobic conditions, sulphate is reduced to sulphide by sulphate-reducing bacteria (SRB). Sulphide generation is undesired in wastewater treatment plants (WWTPs). Previous research indicated that SRB are inhibited by the presence of electron acceptors (such as O2, NO3 and NO2). However, the contact times and concentrations used in those studies are by far higher than occur in WWTPs. Since sulphide can influence the biological nitrogen and phosphorus removal processes, this research aimed to understand how the different electron acceptors commonly present in biological nutrient removal (BNR) systems can affect the proliferation of SRB. For this purpose, a culture of SRB was enriched in a sequencing batch reactor (approx. 88% of the total bacteria population). Once enriched, the SRB were exposed for 2 h to typical concentrations of electron acceptors like those observed in BNR systems. Their activity was assessed using three different types of electron donors (acetate, propionate and lactate). Oxygen was the most inhibiting electron acceptor regardless the carbon source used. After exposure to oxygen and when feeding acetate, an inactivation time in the sulphate reduction activity was observed for 1.75 h. Once the sulphate reduction activity resumed, only 60% of the original activity was recovered. It is suggested that the proliferation of SRB is most likely to occur in BNR plants with an anaerobic fraction higher than 15% and operating at sludge retention times higher than 20 days (at a temperature of 20 °C). These results can be used to implement strategies to control the growth of sulphate reducers that might compete for organic carbon with phosphate-accumulating organisms.

Impact of phosphate-solubilizing bacteria inoculation methods on phosphorus transformation and long-term utilization in composting

This study aimed to assess the effect of phosphate-solubilizing bacteria (PSB) application and inoculation methods on rock phosphate (RP) solubilization and bacterial community during composting. The results showed that PSB inoculation in different stages of composting, especially both in the beginning and cooling stages, not only improved the diversity and abundance of PSB and bacterial community, but also distinctly increased the content of potential available phosphorus. Redundancy analysis indicated that the combined inoculation of PSB in the initial stage with higher inoculation amount and in the cooling stage with lower inoculation amount was the best way to improve the inoculation effect and increase the solubilization and utilization of RP during composting. Besides, we suggested three methods to improve phosphorus transformation and long-term utilization efficiency in composts based on biological fixation of phosphates by humic substance and phosphate-accumulating organisms.

Dynamics of microbial community structure and nutrient removal from an innovative side-stream enhanced biological phosphorus removal process

Biological phosphorous (P) and nitrogen (N) removal from municipal wastewater was studied using an innovative anoxic-aerobic-anaerobic side-stream treatment system. The impact of influent water quality including chemical oxygen demand (COD), ammonium and orthophosphate concentrations on the reactor performance was evaluated. The results showed the system was very effective at removing both COD (>88%) and NH4+-N (>96%) despite varying influent concentrations of COD, NH4+-N, and total PO43--P. In contrast, it was found that the removal of P was sensitive to influent NH4+-N and PO43--P concentrations. The maximum PO43--P removal of 79% was achieved with the lowest influent NH4+-N and PO43--P concentration. Quantitative PCR (qPCR) assays showed a high abundance and diversity of phosphate accumulating organisms (PAO), nitrifiers and denitrifiers. The MiSeq microbial community structure analysis showed that the Proteobacteria (especially β-Proteobacteria, and γ-Proteobacteria) were the dominant in all reactors. Further analysis of the bacteria indicated the presence of diverse PAO genera including Candidatus Accumulibacter phosphatis, Tetrasphaera, and Rhodocyclus, and the denitrifying PAO (DPAO) genus Dechloromonas. Interestingly, no glycogen accumulating organisms (GAOs) were detected in any of the reactors, suggesting the advantage of proposed process in term of PAO selection for enhanced P removal compared with conventional main-stream processes.

Conditions Favoring Proliferation of Glycogen Accumulating Organisms for Excess Biological Carbon Removal in Treating Nutrient Deficient Wastewater

Impact of marginal availability and severe deficiency of nitrogen on the growth of glycogen accumulating organisms (GAOs) and performance of anaerobic/aerobic activated sludge systems treating nutrient deficient wastewater was investigated at marginal availability and severe deficiency of phosphorus. Two continuous-flow lab-scale systems were operated simultaneously fed by model winery wastewater, one with marginal availability and the other one at severe deficiency of nitrogen. In the second experimental stage, marginal availability of P was converted into severe deficiency by interrupting external dosing.Common practice of dosing N- and P-sources to marginal availability caused enhanced proliferation of filamentous bacteria leading to poor biomass settling and instable operation. At marginal N-availability accumulation of GAOs started when conditions became deficient for phosphorus. In severe lack of nitrogen GAOs overgrew filaments, and outcompeted phosphorous accumulating organisms (PAOs) initially present in the seed. Stable and good performance could be maintained even after withdrawal of phosphorous dosing.

A fluorescence in situ hybridization method for the measurement of denitrifying phosphorus-accumulation organisms and study on the metabolisms of activated sludge

A new method using polyphosphate kinase (PPK) probe was developed in this study to measure the ratio of denitrifying phosphate-accumulating organisms (DNPAOs) to bacteria. Advantages and disadvantages of this method and previously published methods were evaluated. Biochemical characteristics and metabolic mechanisms of the bacterial community in activated sludge were analyzed. The result indicated that PPK probe targeting at the gene of polyphosphate kinase in bacterial cells could marked more PAOs than traditional PAO probes. The ratio of DNPAOs/bacteria reached 60.30% in activated sludge. The whole bacterial community in activated sludge, at the end of A/A stage, presented significant non-fermentative characteristics although the activated sludge still contained some fermentative bacteria. Study on the metabolic mechanisms suggested that intracellular PHB served as the major carbon source and energy source of bacterial community in activated sludge.

Looking for phosphate-accumulating bacteria in activated sludge processes: a multidisciplinary approach.

Over the past decades, an increasing need in renewable resources has progressively appeared. This trend concerns not only fossil fuels but also mineral resources. Wastewater and sewage sludge contain significant concentrations in phosphate and can be considered as a fertilizer source of the utmost importance. In wastewater treatment plants, the biological uptake of phosphate is performed by a specific microbiota: the phosphate-accumulating organisms. These microorganisms are recovered in sewage sludge. Here, we aimed to investigate the occurrence of phosphate accumulators in four wastewater treatment plants. A 16S metagenetic analysis identified the main bacterial phyla extracted from the aerobic treatment: α-Proteobacteria, β-Proteobacteria, and Sphingobacteria. An enrichment stage was performed to stimulate the specific growth of phosphate-accumulating bacteria in an acetate medium. An analysis of metabolic activities of sulfur and phosphorus highlighted strong modifications related to phosphorus and much less distinguishable effects with sulfur. A solid acetate medium containing 5-Br-4-Cl-3-indolyl phosphate was used to select potential phosphate-accumulating bacteria from the enriched consortia. The positive strains have been found to belong in the genera Acinetobacter, Corynebacterium, and Pseudomonas. Finally, electron microscopy was applied to the strains and allowed to confirm the presence of polyphosphate granules. Some of these bacteria contained granules the size of which exceeded 100nm.

Biological phosphorus removal in anoxic-aerobic sequencing batch reactor with starch as sole carbon source

In traditional biological phosphorus removal (BPR), phosphorus release in anaerobic stage is the prerequisite of phosphorus excessive uptake in aerobic conditions. Moreover, when low molecular weight of the organic substance such as volatile fatty acids (VFAs) is scarce in bulk liquid or anaerobic condition does not exist, phosphate accumulating organisms (PAOs) have difficulty removing phosphorus. However, in this work, phosphorus removal in two anoxic-aerobic sequencing batch reactors (SBRs) was observed when starch was supplied as a sole carbon source. The relations of the BPR with idle period were investigated in the two identical SBRs; the idle times were set to 0.5 hr (R1) and 4 hr (R2), respectively. Results of the study showed that, in the two SBRs, phosphorus concentrations of 0.26-3.11 mg/L in effluent were obtained after aeration when phosphorus concentration in influent was about 8 mg/L. Moreover, lower accumulations/transformations of polyhydroxyalkanoates (PHAs) and higher transformation of glycogen occurred in the SBRs, indicating that glycogen was the main energy source that was different from the traditional mechanism of BPR. Under the different idle time, the phosphorus removal was a little different. In R2, which had a longer idle period, phosphorus release was very obvious just as occurs in a anaerobic-aerobic regime, but there was a special phenomenon of chemical oxygen demand increase, while VFAs had no notable change. It is speculated that PAOs can assimilate organic compounds in the mixed liquor, which were generated from glycolysis by fermentative organisms, coupled with phosphorus release. In R1, which had a very short idle period, anaerobic condition did not exist; phosphorus removal rate reached 63%. It is implied that a new metabolic pathway can occur even without anaerobic phosphorus release when starch is supplied as the sole carbon source.

Denitrification of nitrate and nitrite by ‘Candidatus Accumulibacter phosphatis’ clade IC

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

Characterisation of Phosphate Accumulating Organisms and Techniques for Polyphosphate Detection: A Review.

Phosphate minerals have long been used for the production of phosphorus-based chemicals used in many economic sectors. However, these resources are not renewable and the natural phosphate stocks are decreasing. In this context, the research of new phosphate sources has become necessary. Many types of wastes contain non-negligible phosphate concentrations, such as wastewater. In wastewater treatment plants, phosphorus is eliminated by physicochemical and/or biological techniques. In this latter case, a specific microbiota, phosphate accumulating organisms (PAOs), accumulates phosphate as polyphosphate. This molecule can be considered as an alternative phosphate source, and is directly extracted from wastewater generated by human activities. This review focuses on the techniques which can be applied to enrich and try to isolate these PAOs, and to detect the presence of polyphosphate in microbial cells.

Improving phosphorus removal in aerobic granular sludge processes through selective microbial management

This study aimed to improve phosphorus removal in aerobic granular sludge sequential batch reactors (AGS-SBR) by a differential selection of the granules containing the highest proportion of phosphate accumulating organisms (PAOs). The abundance of PAOs in granules with different density was analyzed by PCR-DGGE, pyrosequencing and qPCR. Dense granules contained a higher proportion of Candidatus Accumulibacter (PAO) with a 16S rRNA gene frequency up to 45%. Starting with an AGS-SBR with low height/diameter ratio performing unstable P removal, two strategies of biomass removal were assessed. First, a high selective pressure (short settling time) was applied and second, an increase of the settling time was combined with a homogeneous purge of the sludge bed. The first strategy resulted in a reduction of P removal efficiency while the second improved and stabilized P removal over 90%. This study offers a new approach of biomass management in AGS-SBR.

Isolation of a non-fermentative bacterium, Pseudomonas aeruginosa, using intracellular carbon for denitrification and phosphorus-accumulation and relevant metabolic mechanisms

A newly designed pilot-scale system was developed to enrich denitrifying phosphate-accumulating organisms (DNPAOs) for nitrogen and phosphorus nutrient removal synchronously. A strain of DNPAOs was isolated and its biochemical characteristics and metabolic mechanisms of this bacterial strain were analyzed. The results showed that compared with previously reported system, this newly designed system has higher removal rates of nutrients. Removal efficiencies of NH3-N, TN, TP, and COD in actual wastewater were 82.64%, 79.62%, 87.22%, and 90.41%, respectively. Metabolic activity of DNPAOs after anoxic stage in this study even reached 94.64%. Pseudomonas aeruginosa is a strain of non-fermentative DNPAOs with strong nitrogen and phosphorus removal abilities. Study on the metabolic mechanisms suggested that intracellular PHB of P. aeruginosa plays dual roles, supplying energy for phosphorus accumulation and serving as a major carbon source for denitrification.

Simultaneous Carbon-Oxidation and Nutrient (N and P) Reduction in a Batch-Fed Reactor by Pseudomonas sp. P1 Isolated from a Small-scale Slaughterhouse Wastewater

Aims: Laboratory scale evaluation of phosphorus removal along with organic and nitrogen reduction in a batch reactor under anoxic-aerobic environment using Phosphorous Accumulating Organisms (PAOs). Study Design: Isolation of the predominant bacterial species through phenotypic and phylogenetic characterization of acclimatized sludge collected from a slaughterhouse wastewater treatment plant. Selected isolates able to utilize acetate compounds as superior carbon and energy source in sequential mode of operation.

The Effects of Secondary-Phosphorus Release on Biological-Phosphorus Removal in a Pre-anoxic Process

In this study, a sequencing batch reactor (SBR) with fill, anaerobic, oxic1, anoxic, oxic2, settle, decant and sludge pre-anoxic process, was used to investigate the performance efficiency of biological nutrient removal which occurs secondary- phosphorus release during different pre-anoxic hydraulic time (PAHRT). The results of the study showed that the processes of secondary-phosphorus release and secondary-phosphorus removal were significantly affected by PAHRT, whereas the removal of chemical oxygen demand (COD), ammonia nitrogen (NH4 -N) + and total nitrogen (TN) removal were only slightly affected. The research also showed that when PAHRT for 120 min., and MLVSS was 5330 mg/1, the secondary-phosphorus-release concentration was lower than 0.15 mg/L, and the removal efficiency of the total phosphorus (TP) was higher than 80.98%. Moreover, it was found that there is a negative correlation between secondaryphosphorus release and TP removal efficiency and between secondary-phosphorus release and the quantity of released anaerobic phosphorus. The study also showed that phosphate-accumulating organisms (PAOs) can be used to break down poly-p without PHA synthesis during the process of secondary- phosphorus release, resulting in a reduction of PHA content in PAOs. As a result, the rate of anaerobic-phosphorus release and oxic-phosphorous uptake decreases , leading to a duction in phosphate- removal efficiency. It is thus necessary to reduce or, avoid a secondary- phosphorus- release event in a pre-anoxic system. In summation, this project’s findings show that a decrease of secondary-phosphorus release in a pre-anoxic process is an effective way of enhancing biological- phosphorus removal.

Anoxic-oxic treatment of abattoir wastewater for simultaneous removal of carbon, nitrogen and phosphorous in a sequential batch reactor (SBR)

In the case of abattoir effluent, biological nutrient removal in sequential batch reactor (SBR) is achieved typically through pre-denitrification, which can be accomplished during the anoxic fill phase or an anoxic react phase between the fill and the aerobic react phase. The study includes phosphorus reduction from abattoir wastewater in a SBR under anoxic-oxic environment in presence of Phosphorous Accumulating Organisms (PAOs). The PAOs utilizing organic carbon for enabling simultaneous nitrification, denitrification and phosphorous uptake from the wastewater. The reactor was operated in three different variations of anoxic(A)-oxic(O) sequence, viz. (4+4,A/O), (3+3+2,A/O/A) and (3+4+2,A/O/A) hr of total react period with influent soluble chemical oxygen demand (SCOD), ammonia nitrogen (NH4+-N) level and phosphorous concentration of 2250±20 mg/L, 150±10 and 24±2 respectively. At (4+4,A/O) combination total phosphorous (TP) removal efficiency was found to be 84.34% at the end of settle period. Other combinations i.e. (3+3+2,A/O/A) and (3+4+2,A/O/A) were basically resorted to obtain nitrification and subsequent denitrification in a greater extent. The effective removal of SCOD, NH4+-N and TP were 95.14, 78.04 and 86.60%, at the initial concentration of SCOD, NH4+-N and TP 2265.52, 148.28 and 25.68 mg/L, respectively was achieved in the present SBR system under (3+4+2,A/O/A) combination operating condition which indicates that an intermittent aeration strategy in the form of (3+4+2,A/O/A) is distinctly advantageous compared to other two combinations (4+4,A/O) and (3+3+2,A/O/A).

Diameter control and stability maintenance of aerobic granular sludge in an A/O/A SBR

Diameter is the key factor that influencing the stability of granules. In this study, combined selective pressure generated by low strength wastewater (chemical oxygen demand, COD, 200±40mg/L), low hydrodynamic shear force (superficial gas velocity of 0.55cm/s and agitation of 180rpm), different sludge discharge mode and a sequencing batch bioreactor (SBR) with an alternating anaerobic/aerobic/anoxic (A/O/A) operational process were employed to limit the diameter of aerobic granules. Energy consumption was reduced as low superficial gas velocity and agitation were employed. Granulation process was completed after 40days. The matured granules with small diameter (d (0.9)<800μm) were stable without disaggregation during the experiment period (220days). The extracellular polysaccharide/extracellular protein (PS/PN) ratio increased along with granulation process, and maintained at a stable level in the following time. After granulation, the removal efficiencies of COD, NH4+–N, total nitrogen (TN) and phosphate (P) reached 99%, 98%, 90% and 99%, respectively. The majority of TN was consumed during aerobic stage after complete granulation. The effective TN removal was mainly due to the activities of denitrifying phosphate-accumulating organisms (DNPAOs) and denitrifying glycogen-accumulating organisms (DNGAOs).

Simultaneous biological nitrogen and phosphorus removal with a sequencing batch reactor–biofilm system

To achieve simultaneous biological nitrogen and phosphorus removal, a pilot-scale sequencing batch reactor–biofilm (SBR–BF) system was explored. SBR–BF system was started up with two phases, which were anaerobic/aerobic and anaerobic/aerobic/anoxic, to enrich phosphate accumulating organisms (PAOs) and denitrifying phosphate accumulating organisms (DNPAOs), and to form system of suspended activated sludge and attached biofilm. Operation cycle of SBR–BF system was adjusted as fill 5 min, anaerobic 90 min, aerobic 210 min, anoxic 90 min, settle 30 min, draw 15 min and idle 40 min, and average removal of chemical oxygen demand, total nitrogen and total phosphorus respectively reached 95%, 94%, and 97% during stable operation for 3 months. Analysis of a typical wastewater treatment cycle testified that DNPAOs played an important role for phosphate uptake and denitrification under aerobic and anoxic conditions. The SBR–BF system not only provided good living conditions for PAOs, DNPAOs, nitrifying bacteria and denitrifying bacteria which had different generation times, but also prevented the competition for carbon source between different bacteria.

Identification of the function of extracellular polymeric substances (EPS) in denitrifying phosphorus removal sludge in the presence of copper ion

Industrial wastewater containing heavy metals that enters municipal wastewater treatment plants inevitably has a toxic impact on biological treatment processes. In this study, the impact of Cu(II) (0, 1.5, 2, 2.5, 3 mg/L) on the performance of denitrifying phosphorus removal (DPR) and microbial community structures was investigated. Particularly, the dynamic change in the amount and composition of extracellular polymeric substances (EPS), and the role of EPS in P removal, were assessed using three-dimensional excitation–emission matrix fluorescence spectroscopy combined with parallel factor (PARAFAC) analysis. The results showed that, after long-term adjustment, the P removal efficiency was maintained at 95 ± 2.7% at Cu(II) addition up to 2.5 mg/L, but deteriorated when the Cu(II) addition was 3 mg/L. The EPS content, including proteins and humic substances, increased with increasing Cu(II) additions at concentrations ≤2.5 mg/L. This property of EPS was beneficial for protecting phosphate-accumulating organisms (PAOs) against heavy metals, as both proteins and humic substances are strong ligands for Cu(II). Therefore, the PAOs abundance was still relatively high (67 ± 3%) when Cu(II) accumulation in sludge was up to 10 mg/g SS. PARAFAC confirmed that aromatic proteins could be transformed into soluble microbial byproduct-like material when microorganisms were subjected to Cu(II) stress, owing to their strong metal ion complexing capacity. The increase in the percentage of humic-like substances enhanced the detoxification function of the sludge EPS. EPS accounted for approximately 26–47% of P removed by adsorption when Cu(II) additions were between 0 and 2.5 mg/L. The EPS function, including binding toxic heavy metals and P storage, enhanced the operating stability of DPR systems. This study provides us with a better understanding of (1) the tolerance of DPR sludge to copper toxicity and (2) the function of sludge EPS in the presence of heavy metals in biological P removal systems.

Impact of salinity on the aerobic metabolism of phosphate-accumulating organisms.

The use of saline water in urban areas for non-potable purposes to cope with fresh water scarcity, intrusion of saline water, and disposal of industrial saline wastewater into the sewerage lead to elevated salinity levels in wastewaters. Consequently, saline wastewater is generated, which needs to be treated before its discharge into surface water bodies. The objective of this research was to study the effects of salinity on the aerobic metabolism of phosphate-accumulating organisms (PAO), which belong to the microbial populations responsible for enhanced biological phosphorus removal (EBPR) in activated sludge systems. In this study, the short-term impact (hours) of salinity (as NaCl) was assessed on the aerobic metabolism of a PAO culture, enriched in a sequencing batch reactor (SBR). All aerobic PAO metabolic processes were drastically affected by elevated salinity concentrations. The aerobic maintenance energy requirement increased, when the salinity concentration rose up to a threshold concentration of 2 % salinity (on a W/V basis as NaCl), while above this concentration, the maintenance energy requirements seemed to decrease. All initial rates were affected by salinity, with the NH4- and PO4-uptake rates being the most sensitive. A salinity increase from 0 to 0.18 % caused a 25, 46, and 63 % inhibition of the O2, PO4, and NH4-uptake rates. The stoichiometric ratios of the aerobic conversions confirmed that growth was the process with the highest inhibition, followed by poly-P and glycogen formation. The study indicates that shock loads of 0.18 % salt, which corresponds to the use or intrusion of about 5 % seawater may severely affect the EBPR process already in wastewater treatment plants not exposed regularly to high salinity concentrations.

Understanding the impact of influent nitrogen concentration on granule size and microbial community in a granule-based enhanced biological phosphorus removal system

To better understand the effect of influent nitrogen concentration on granule size and microbial community in a granule-based enhanced biological phosphorus removal system, three influent nitrogen concentrations were tested while carbon concentration was an unlimited factor. The results show that although ammonium and phosphate were well removed in the tested nitrogen concentration range (20–50mgL−1), granule size, the amount of phosphate accumulating organisms (PAOs) and microbial activity were affected significantly. A possible mechanism for the effect of influent nitrogen concentration on granule size is proposed based on the experimental results. The increase in proteins/polysaccharides ratio caused by high influent nitrogen concentration plays a crucial role in granule breakage. The small granule size then weakens simultaneous nitrification–denitrification, which further causes higher nitrate concentration in the effluent and lower amount of PAOs in sludge. Consequently, phosphate concentration in the anaerobic phase decreases, which plays the secondary role in granule breakage.