Phosphorus Removal in an Enhanced Biological Phosphorus

Article pubs.acs.org/est

Phosphorus Removal in an Enhanced Biological Phosphorus Removal Process: Roles of Extracellular Polymeric Substances Hai-Ling Zhang, Wei Fang, Yong-Peng Wang, Guo-Ping Sheng,* Raymond J. Zeng, Wen-Wei Li, and Han-Qing Yu Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China S Supporting Information *

ABSTRACT: Phosphorus-accumulating organisms are considered to be the key microorganisms in the enhanced biological phosphorus removal (EBPR) process. A large amount of phosphorus is found in the extracellular polymeric substances (EPS) matrix of these microorganisms. However, the roles of EPS in phosphorus removal have not been fully understood. In this study, the phosphorus in the EBPR sludge was fractionated and further analyzed using quantitative 31P nuclear magnetic resonance spectroscopy. The amounts and forms of phosphorus in EPS as well as their changes in an anaerobic−aerobic process were also investigated. EPS could act as a reservoir for phosphorus in the anaerobic−aerobic process. About 5−9% of phosphorus in sludge was reserved in the EPS at the end of the aerobic phase and might further contribute to the phosphorus removal. The chain length of the intracellular long-chain polyphosphate (polyP) decreased in the anaerobic phase and then recovered under aerobic conditions. However, the polyP in the EPS had a much shorter chain length than the intracellular polyP in the whole cycle. The migration and transformation of various forms of phosphorus among microbial cells, EPS, and bulk liquid were also explored. On the basis of these results, a model with a consideration of the roles of EPS was proposed, which is beneficial to elucidate the mechanism of phosphorus removal in the EBPR system.

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INTRODUCTION Phosphorus is recognized to be one of the main culprits for the eutrophication in natural waters.1,2 The enhanced biological phosphorus removal (EBPR) process is the widely implemented approach to remove phosphorus in wastewater. The enhanced phosphorus removal is attributed mainly to a group of selectively enriched heterotrophic bacteria, i.e., phosphorusaccumulating organisms (PAOs). PAOs store carbon sources as intracellular polymers [i.e., poly-β-hydroxyalkanoates (PHAs)] using the energy of polyphosphate (polyP) and glycogen degradation and then release orthophosphate (orthoP) into the outside under anaerobic conditions. In the subsequent aerobic phase, PAOs use the stored PHAs as an energy source for “luxury uptake” of orthoP, transforming orthoP to polyP, replenishing glycogen and self-growth.1−4 Net phosphorus removal is achieved by discharging the phosphorus-rich excess sludge. Extensive studies have been conducted toward understanding the physiochemical and microbiological properties of the EBPR process.1,5 However, information about extracellular polymeric substances (EPS) and their roles in the EBPR process is very limited. As a major component of activated sludge flocs, EPS are usually present outside cells or form a matrix where microbial cells are enclosed. Their properties substantially affect the physicochemical characteristics and mass transfer of activated sludge.6−8 A significant level of phosphorus accumulation in EPS has been reported,9−12 implying that © 2013 American Chemical Society

EPS might play an important role in the EBPR process and should not be ignored. However, thus far, the roles of EPS have not yet been clearly understood. To explore the roles of EPS in the EBPR process, the dynamic migration and transformation of phosphorus among microbial cells, EPS, and bulk liquid in a typical anaerobic− aerobic process should be investigated. However, the major obstacle to do so is the lack of proper methods for isolating and examining the actual forms of phosphorus in microbial cells and the EPS matrix. Phosphorus in EBPR sludge is reported to be present mainly in the form of polyP, with a content ranging from 5 to 15% by weight.3 Effective isolation and accurate determination of the high-content polyP inside cells are crucial to understand the transformation of phosphorus in the EBPR process. Furthermore, long-chain polyP is labile and can be hydrolyzed to short-chain polyP under acidic or alkaline conditions,13−15 even to more stable forms of orthoP and pyrophosphate (pyroP).16 The interference of the high-content intracellular polyP to the analysis of extracellular phosphorus in EPS also causes a large difficulty in elucidating the roles of EPS. 31 P nuclear magnetic resonance (31P NMR) spectroscopy has been proven to be an efficient tool for characterizing the forms Received: Revised: Accepted: Published: 11482

February 4, 2013 September 9, 2013 September 10, 2013 September 25, 2013 dx.doi.org/10.1021/es403227p | Environ. Sci. Technol. 2013, 47, 11482−11489

Environmental Science & Technology

Article

Figure 1. Schematic diagrams for the fractionation and characterization of various forms of (a) phosphorus in sludge (mainly intracellular phosphorus) and (b) extracellular phosphorus in the EBPR sludge.

2.5 μg/L ZnCl2, 1.5 μg/L CuCl2, 5.1 μg/L MnCl2·4H2O, 1.1 μg/L (NH4)6Mo7O24·4H2O, 50 μg/L ethylenediaminetetraacetic acid (EDTA), 2.5 μg/L AlCl3, 1.6 μg/L CoCl2·6H2O, and 2.5 μg/L NiCl2. Typical Phosphorus Release and Uptake Test of the EBPR Sludge. The phosphorus release and uptake test was conducted in a batch mode to investigate the dynamic behaviors of phosphorus in microbial cells and EPS. A total of 2 L of concentrated sludge taken from the reactor at the end of the aerobic phase was placed in a 4 L vessel and fed with 2 L of synthetic wastewater and then operated in an anaerobic/ aerobic mode as for the parent reactor. In the cycle, a 30 mL mixed solution was taken at 0, 10, 60, 120, 200, and 290 min. A 10 mL solution was used for the extraction of phosphorus in sludge and 20 mL for EPS extraction. The structures and contents of both extracelluar phosphorus in EPS and intracellular polyP were analyzed. Each batch test was repeated for three cycles. Extraction and Species Analysis of Phosphorus in the EBPR Sludge. Among the methods for fractionating the phosphorus in sludge (mainly in microbial cells),21−25 the cold perchloric acid (PCA) fractionation and subsequent NaOH extraction approach has been widely used because of its effectiveness.23 The schematic diagram for the fractionation and characterization of phosphorus in sludge using the PCA− NaOH extraction procedure is shown in Figure 1a and Table S1 of the Supporting Information. After extraction, neutralizing was implemented immediately to minimize the phosphorus transformation. Because of the severe degradation of polyP in the NaOH extraction process according to the preliminary test, only the structure information of the PCA-extractable phosphorus was analyzed and the contents of various phosphorus species were not calculated. After that, all of the PCA extracts were lyophilized at −50 °C for 48 h and stored at −20 °C before 31P NMR analysis. In addition, 0.1 g of polyP model compound (sodium hexametaphosphate) was also extracted with PCA to examine the potential hydrolysis of polyP in the extraction process. A

of phosphorus in environmental samples.12,17−19 It can distinguish among similar compounds and provide structure information of various forms of phosphorus.17,20 The structure transformation of extracellular and intracellular phosphorus could be evaluated by this technique, and more detailed information about the roles of EPS in phosphorus removal in EBPR systems could be obtained. In this study, the phosphorus in microbial cells and the EPS matrix in an EBPR process was extracted and further analyzed using 31P NMR spectroscopy, to elucidate the possible roles of EPS in phosphorus removal. The migration and transformation of phosphorus among microbial cells, EPS, and bulk liquid were investigated. On the basis of these results, EPS were introduced into the conventional EBPR metabolic model to better understand the phosphorus removal mechanism in EBPR systems.

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MATERIALS AND METHODS Operation of the EBPR Reactor. A laboratory-scale sequencing batch reactor with a working volume of 8 L was operated for 6 months to enrich PAOs. The EBPR reactor was inoculated with the activated sludge from Wangtang Wastewater Treatment Plant, Hefei, China. The cycle of the reactor operation consisted of a 120 min anaerobic period, 170 min aerobic period, and 70 min settle/decant period. Sodium acetate was used as the sole carbon source, and the mixed liquor suspended solids (SS) concentration was around 4.0 g/L. The pH was maintained around 7.0 by two automatic titration units dosing HCl and NaOH to avoid the phosphate precipitation in the operation. The temperature was controlled at 20 ± 1 °C. The sludge mixture of 250 mL was discharged at the end of the aerobic phase in each cycle. The hydraulic and solids retention times were 12 h and 8 days, respectively. Synthetic wastewater was fed to the reactor in the initial 6 min, which included 200 mg/L acetate (in chemical oxygen demand), 38.2 mg/L NH4Cl, 43.9 mg/L KH2PO4, 73.6 mg/ L K2HPO4·3H2O, 40 mg/L CaCl2, 75 mg/L MgSO4 and a trace element solution of 5.0 μg/L FeSO4·7H2O, 2.5 μg/L H3BO3, 11483

dx.doi.org/10.1021/es403227p | Environ. Sci. Technol. 2013, 47, 11482−11489


Article

Table 1. Phosphorus Fractionation and Their Contents (mg of P/g of SS) in the EBPR Sludge at Various Operating Times PCA extracts time (min) 0 10 60 120 200 290 a

orthoP 1.40 1.17 1.59 1.72 1.49 1.24

± ± ± ± ± ±

0.05 0.02 0.05 0.03 0.01 0.03

complex Pa 78.2 61.2 46.5 42.9 71.6 80.6

± ± ± ± ± ±

1.2 0.6 0.6 0.9 0.6 0.6

NaOH extracts TP 79.6 62.4 48.1 44.6 73.1 81.8

± ± ± ± ± ±

complex Pa

orthoP 1.2 0.5 0.6 0.9 0.6 0.6

1.15 0.33 0.15 0.19 0.90 1.56

± ± ± ± ± ±

0.06 0.08 0.09 0.14 0.05 0.15

15.1 11.4 8.3 7.8 10.2 10.6

± ± ± ± ± ±

0.8 0.1 0.4 0.5 1.1 0.1

TP 16.2 11.7 8.4 8.0 11.1 12.2

± ± ± ± ± ±

TPsludge 0.8 0.2 0.34 0.4 1.0 0.2

92.1 81.4 58.1 57.6 83.8 93.2

± ± ± ± ± ±

5.5 3.1 5.1 4.4 0.3 0.3

recovery (%) 103.9 91.1 97.2 91.4 100.4 100.9

± ± ± ± ± ±

2.0 0.7 1.6 2.2 1.9 0.8

Complex P = TP − orthoP.

and an external 85% H3PO4 standard. The delay time used here allows for sufficient spin−lattice relaxation between scans. The number of scans was set to at least 128 (PCA or EPS extracts) or 8 (polyP model compounds). To express the phosphorus forms accurately to the greatest extent, spectra were collected immediately after preparation and the process was finished within 2 h to avoid transformation of phosphorus forms. Peak identification was based on the literature.20,29 Chemical shift and area of individual signals were determined with the NMR data processing software (MestReNova, version 6.0.2-5475, Mestrelab Research S.L., Spain).

total of 0.1 g of polyP model compound dissolved in deionized water was used as the control. EPS Extraction. In this work, EPS were extracted from the EBPR sludge using the cation-exchange resin (CER) method (Figure 1b), which has been proven to be suitable for EPSassociated phosphorus extraction from the EBPR sludge in our previous study because of its low cell lysis extent and high extracellular phosphorus extraction efficiency.7,26 Before extraction, a certain amount of sludge solution taken at each given interval was centrifuged at 8000 rpm for 5 min at 4 °C. Then, the sludge pellets were washed twice with 100 mM NaCl solution and resuspended in 100 mM NaCl solution to the original volume. The sludge mixture was stirred afterward for 6 h at 500 rpm and 4 °C in a 200 mL beaker with an appropriate amount of resin (Dowex Marathon C, Na+ form, 20−50 mesh, Sigma-Aldrich, Inc., St. Louis, MO). Thereafter, the suspensions were centrifuged at 12 000 rpm for 20 min, and the supernatant was filtered through 0.45 μm acetate cellulose membranes. After that, all of the EPS solution were lyophilized at −50 °C for 48 h and stored at −20 °C before 31P NMR analysis. Similarly, 0.1 g of the polyP model compound (sodium hexametaphosphate) was also used to examine the potential hydrolysis of polyP in the CER extraction process. Chemical Analysis. All chemicals used in this work were of analytical grade. The SS, orthoP and total phosphorus (TP) in the PCA or NaOH extracts (expressed as orthoPPCA, TPPCA, orthoPNaOH, and TPNaOH, respectively), and TP in supernatant (TPsupernatant), EPS solutions (TPEPS), or sludge (TPsludge) were all measured according to the Standard Methods.27 The volatile fatty acid (VFA) concentration was measured by gas chromatography (7890A, Agilent Technologies, Inc., Santa Clara, CA) equipped with a flame ionization detector. The concentrations of carbohydrates, proteins, and humic substances in EPS extracts were determined as described previously.28 The total EPS content was measured using a total organic carbon (TOC) analyzer (TOC-VCPN, Shimadzu Co., Japan). 31 P NMR Analysis. To collect the 31P NMR spectrum, the freeze-dried PCA extracts, EPS powders, or polyP compound were redissolved in 0.2 mL of D2O and 0.2 mL of 100 mM EDTA solution and then 0.4 mL of NaOH solution (1 M) was added. The dose of EDTA and NaOH solutions was to avoid the interference of divalent cations and to adjust pH above 12.0, respectively, to ensure consistent chemical shifts and optimal spectral resolution during the NMR measurement.20 A quantitative 31P NMR spectrum using an inverse-gated proton-decoupling process was acquired at 162.02 MHz with an Avance AV400 spectrometer (Bruker Co., Germany) at 25 °C. The acquisition parameters were as follows: 90° pulse width, 9.6 μs; acquisition time, 0.51 s; relaxation delay, 50 s;

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RESULTS AND DISCUSSION Content and Speciation of Phosphorus in the EBPR Sludge in the Anaerobic−Aerobic Process. The phosphorus fractions in the EBPR sludge extracted by the PCA−NaOH method are shown in Table 1 and Table S2 of the Supporting Information. The high phosphorus recovery indicates the effectiveness of this method. Although extracellular phosphorus was partially extracted in the PCA extracts, its proportion was relatively lower compared to that of the intracellular phosphorus (see Table S3 of the Supporting Information). Thus, the PCA-extractable phosphorus was mainly intracellular phosphorus. The complex phosphorus (i.e., non-orthoP; calculated by the difference between TP and orthoP) in the PCA extracts (complexPPCA) decreased greatly in the anaerobic phase and later recovered under aerobic conditions. This confirmed that the PCA-extractable intracellular phosphorus was involved in the cyclic process of anaerobic degradation and aerobic synthesis.21,23 With the analysis of 31P NMR spectroscopy, the signals of orthoP and polyP (including both the end and middle groups) were both observed in the spectra of the sludge extracted by the PCA method in the anaerobic and aerobic phases (Figure 2). While in the spectra of the sludge harvested in the anaerobic phase, a small amount of pyroP was also detected. The proportions of polyP in the two spectra were both higher than 90%, indicating that polyP was the dominant form of phosphorus in the PCA extracts (Table 2). Incidentally, the signal of the degradation products of nucleic acids (4.39 ppm, assigned to orthoP monoesters or mononucleotide) was not calculated in the total phosphorus forms because of its low proportion. Other forms of phosphorus, such as nucleic acids and lipids, were undetectable. A higher PCA concentration and a longer extraction time might be needed for nucleic acid extraction, while organic solvent was required for lipid extraction.15,21 From the ratio of the peak area between the middle and end groups in the NMR spectra,18 the mean chain length of polyP in the PCA extracts was estimated to be 11 and 16 for the 11484



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Figure 3. EPS concentrations and compositions in the anaerobic− aerobic process.

Figure 2. Solution 31P NMR spectra of the PCA extracts from the EBPR sludge taken at the end of the (a) anaerobic phase (t = 120 min) and (b) aerobic phase (t = 290 min).

sludge at the end of the anaerobic and aerobic periods, respectively (Table 2). However, the average chain length of the polyP model compound was reduced from 49 to about 18 after the PCA extraction (see Table S4 of the Supporting Information), indicating that polyP degradation could occur in the PCA extraction process. Then, considering the polyP degradation during extraction, the original polyP in the sludge should be long-chain, which was reduced in the anaerobic phase and recovered under aerobic conditions. Differing from other methods, such as high-performance liquid chromatography and gel electrophoresis, which offer the distribution range of intracellular specific-chain polyP only,13,30,31 31P NMR spectroscopy can quantitatively provide the average chain length of polyP. The analysis of the chain length of polyP indicates that the long-chain intracellular polyP in the PCA extracts is responsible for the cycles of phosphorus release and uptake in the typical EBPR process. Variations of EPS Contents and Compositions in the Anaerobic−Aerobic Process. Figure 3 shows the production of EPS in the anaerobic−aerobic cycle. VFAs were absorbed quickly after feeding (Figure 4a), coupling with a significant increase in the EPS content from 9.98 to 13.45 mg of TOC/g of SS. After that, EPS gradually decreased during the later anaerobic period. Under aerobic conditions, EPS increase slightly. The EPS component changes in the anaerobic−aerobic process were also investigated (Figure 3). Proteins and humic substances varied slightly in the process, while the carbohydrate content changed substantially with a similar trend of EPS. Contents and Forms of Phosphorus in EPS in the Anaerobic−Aerobic Process. Figure 4a shows the typical

Figure 4. Typical profiles of (a) VFAs, TPsupernatant, TPEPS, TPsludge, and TPcell and (b) TPEPS/TOCEPS in the anaerobic−aerobic process.

profiles of VFAs, TP in the aqueous phase (TPsupernatant), TP in EPS (TPEPS), TP in sludge samples (TPsludge), and TP in sludge cells (TPcell; calculated by the difference between TPsludge and TPEPS) in the anaerobic−aerobic process (normalized to the sludge SS content). Because of the very rapid absorption of

Table 2. Chemical Shifts and Relative Peak Areas of Identified Phosphorus Forms in the 31P NMR Spectra of the PCA Extracts of the EBPR Sludge orthoP

a

pyroP

end groups of polyP

middle groups of polyP

chemical shift (ppm)

peak area (%)


peak area (%)


peak area (%)


peak area (%)

sludgeANa

5.59

5.92

−5.10

2.23

−4.33 −4.46

17.29

74.56

sludgeAEb

5.32

1.85

−4.42 −4.54

12.37

−19.52 −20.60 −21.15 −19.76 −20.77 −21.25

85.78

SludgeAN = sludge taken at the end of the anaerobic phase. bSludgeAE = sludge taken at the end of the aerobic phase. 11485



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VFAs (Figure 4a; about 60% uptake within 10 min) at the initial stage of the anaerobic phase, only the sample taken at t = 10 min was analyzed. Profiles of VFAs and TPsupernatant exhibited a typical EBPR pattern, with the absorption of VFAs and the release of phosphorus in the anaerobic phase and phosphorus uptake in the aerobic phase. TPsludge and TPcell exhibited the same variation trends: decreasing anaerobically because of the degradation of polyP and increasing aerobically with the synthesis of polyP. In the anaerobic−aerobic process, TPEPS slightly increased after feeding, then decreased significantly in the anaerobic phase, and then increased in the subsequent aerobic phase. The dynamic change of the phosphorus content in EPS (expressed as TPEPS/TOCEPS) in the EBPR process is shown in Figure 4b. At the initial anaerobic stage, the phosphorus content in the EPS decreased, which might be attributed to the higher amount of EPS production (Figure 3) than the phosphorus adsorption by EPS. In the later anaerobic phase, most phosphorus in EPS was desorbed and released into solution, leading to a low phosphorus content in EPS. Under the aerobic conditions, the EPS matrix around PAOs began to adsorb phosphorus gradually, resulting in an increasing phosphorus content. The phosphorus in the pools of the cells (Pcell) and EPS (PEPS) were also determined, and the respective typical profiles are shown in Figure S1 of the Supporting Information. Pcell presents a similar trend to TPcell (Figure 4a). Besides, PEPS varied similarly to TPEPS/TOCEPS (Figure 4b) and also had a similar changing trend to TPEPS (Figure 4a). Thus, the variation of phosphorus in EPS in the anaerobic−aerobic process showed a dynamic change and might be closely related to the production/hydrolysis of EPS and the adsorption/desorption of phosphorus by EPS. After an entire cycle, TPEPS increased from initial 5.9 ± 0.8 to 6.4 ± 1.0 mg of P/g of SS (Figure 4a). Then, about 6.7 ± 1.1% of TP in the sludge was reserved in the EPS at the end of the aerobic phase and could contribute to the phosphorus removal through sludge discharge. This contribution was calculated by averaging the results of the three individual experiments. Fivecycle continuous operation was also implemented, and the contribution was calculated to be among 5−9% (see Table S5 of the Supporting Information). These results indicate that the contribution of EPS to the phosphorus removal in the EBPR process could not be neglected. The 31P NMR spectra of EPS extracted during different periods in operation are shown in Figure 5, and the chemical shifts and relative percentage of identified phosphorus forms are presented in Table 3. With the help of 31P NMR spectroscopy, the complex and diverse forms of phosphorus in the EPS matrix were clarified. OrthoP, pyroP, and polyP were all clearly detected by NMR spectroscopy. Multichain phosphorus (pyroP and polyP) was richer in the EPS than orthoP, indicating a stronger affinity between EPS and multichain phosphorus. Notably, the average chain length of polyP in EPS varied little in the cycle, implying little ability of PAOs to synthesis polyP extracellularly. EPS have plenty of functional groups (e.g., hydroxyl, sulfuric acid, and humic substances) and huge specific surface, which facilitate their adsorption of phosphorus.32 OrthoP in EPS originated mainly from its adsorption by EPS when it was released to the outside under anaerobic conditions or absorbed into microbial cells under aerobic conditions in the EBPR process. PolyP with a shorter chain was observed in EPS rather than inside cells. This might be attributed to the fact that polyP degradation or

Figure 5. Solution 31P NMR spectra of EPS extracts from the EBPR sludge taken at (a) t = 10 min, (b) t = 120 min, (c) t = 200 min, and (d) t = 290 min.

cleavage could occur under extracellular conditions by exopolyphosphatases.33 Thus, in the EBPR operation, longchain polyP leaked from cells could be hydrolyzed to short chains or finally to pyroP and orthoP in the EPS matrix by extracellular enzymes.31 With TPEPS and the proportions of various phosphorus forms in EPS during different periods obtained from the NMR spectra, the profiles of the contents of orthoP, pyroP, and polyP in EPS are shown in Figure S2 of the Supporting Information. OrthoP and pyroP in EPS were minor and changed slightly in the process, while the content of polyP, the major phosphorus form in EPS, varied substantially with a similar trend of TPEPS. Possible Roles of EPS in the EBPR Process. The results above clearly show that the intracellular biochemical processes and extracellular reaction processes are different and relatively independent. Some reactions are deduced to be involved in the dynamic changes of the amounts and forms of the extracellular phosphorus in the EBPR process, including the production and hydrolysis of EPS, phosphorus adsorption and desorption by EPS, and polyP hydrolysis in the EPS matrix. Thus, the current widely accepted EBPR metabolic model was improved with an introduction of probable transformation of various phosphorus in the EPS matrix. The metabolic model for the EBPR process with a consideration of the roles of EPS (Figure 6) is proposed as follows: In the anaerobic phase, PAOs assimilate VFAs and store them as PHAs using the energy of degradation of glycogen and long-chain polyP,34−37 accompanying with the release of orthoP into EPS then into the bulk solution. Under aerobic conditions, PAOs use stored PHAs as an energy source to take up orthoP in the bulk solution into microbial cells through the EPS matrix,35−37 then synthesize polyP, and replenish glycogen and self-growth. In the EBPR process, the 11486



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Table 3. Solution 31P NMR Chemical Shifts and Relative Peak Areas of Identified Phosphorus Forms in the 31P NMR Spectra of the EPS at Various Operating Times orthoP

pyroP

end groups of polyP

middle groups of polyP

time (min)


peak area (%)


peak area (%)


peak area (%)


peak area (%)

10

5.71

8.11

−4.83

12.23

−4.46 −4.58

51.47

28.20

120

5.66

3.23

−4.92

14.91

−4.55 −4.66

55.40

200

5.79

7.03

−4.68

6.47

−4.38 −4.49

56.71

290

5.60

6.15

−5.01

8.85

−4.59 −4.71

53.05

−18.62 −18.73 −18.85 −19.39 −20.90 −18.53 −18.65 −18.76 −18.41 −18.52 −18.63 −19.13 −18.72 −18.83 −18.95 −19.43

26.46

29.80

31.95

Figure 6. (a) Current metabolic model for EBPR and (b) supposed metabolic model for EBPR with a consideration of the roles of EPS. Processes I and IV, intacellular long-chain polyP leaked from PAO cells and was adsorbed by EPS; processes II and V, long-chain polyP in EPS was hydrolyzed to short-chain polyP and even to pyroP; process III, most phosphorus in EPS was further finally hydrolyzed to orthoP and released into solution because of EPS hydrolysis in the anaerobic phase; and process VI, part of phosphorus in EPS was further hydrolyzed to orthoP and then absorbed into PAO cells in the aerobic phase.

further finally to pyroP or orthoP by extracellular enzymes (processes III and VI). Various forms of phosphorus will be adsorbed or intercepted by EPS when they pass through the

autolysis of microbial cells will also release intracellular polyP into EPS (processes I and IV) and polyP with long chains can be hydrolyzed to short-chain polyP (processes II and V) and 11487



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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (NSFC) (51178443 and 51322802) and the Key Special Program on the S&T for the Pollution Control (2012X07103001 and 2011ZX07303-002-04) for the partial support of this study.

EPS matrix, and part of phosphorus in EPS will also be released into solution in the anaerobic phase (mainly in the form of orthoP; process III) and absorbed into cells under aerobic conditions (in the form of orthoP; process VI). EPS could act as a dynamic reservoir for phosphorus, and the strong phosphorus-accumulating ability of EPS may have a contribution to the phosphorus removal of the EBPR process. Nevertheless, more investigations are needed to justify the supposed model and further explore the important roles of EPS in the EBPR process, e.g., the potential hydrolysis of long-chain polyP outside microbial cells, the usage of the energy released through polyP hydrolysis in EPS and EPS themselves, polyphosphatase activity in EPS, whether and how the extracellular processes are regulated, in situ determination of the contents and forms of the phosphorus in cells and EPS, etc. Besides, the conventional thermodynamic EBPR models may also need to be modified with the information of polyP in EPS, to explore the effect of EPS-associated polyP on the system energy balance. In addition, the phosphate precipitation has been effectively restrained in this study, while colloidal phosphorus or tiny particles of phosphate deposits could be often formed and adsorbed by the EPS matrix in real wastewater treatment plants.9 Thus, the roles of EPS in these processes could be more important and complex. In summary, the intracellular polyP extracted by PCA is confirmed to be involved in the cyclic degradation and synthesis in the EBPR process. Their chain lengths decrease in the anaerobic phase and recover under aerobic conditions, evidenced with the analysis of 31P NMR spectroscopy. EPS could act as a reservoir for phosphorus in the anaerobic− aerobic process, and the high content of phosphorus in EPS at the end of the aerobic phase would contribute about 5−9% to phosphorus removal. An EBPR metabolic model with the recognition of EPS contribution to phosphorus removal is proposed. The results imply that EPS may play a significant role in the EBPR process and deserve to be given more attention.

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NOMENCLATURE 31 P NMR P nuclear magnetic resonance CER cation-exchange resin complexPPCA complex phosphorus (i.e., non-orthoP) in PCA extracts complexPNaOH complex phosphorus in NaOH extracts EBPR enhanced biological phosphorus removal EPS extracellular polymeric substances orthoP orthophosphate orthoPNaOH orthoP in the NaOH extracts orthoPPCA orthoP in the PCA extracts PAO phosphorus-accumulating organism PCA cold perchloric acid PHA poly-β-hydroxyalkanoate Pcell phosphorus in the pools of the cells (mg of P/g of cell) PEPS phosphorus in the pools of the EPS (mg of P/g of EPS) polyP polyphosphate pyroP pyrophosphate SS suspended solids TOC total organic carbon TOCEPS TOC of the EPS solution TP total phosphorus TPsludge TP in sludge TPcell TP in sludge cells (mg of P/g of SS) TPEPS TP in EPS solutions (mg of P/g of SS) TPsupernatant TP in supernatant TP in the PCA extracts TPPCA TPNaOH TP in the NaOH extracts VFA volatile fatty acid VSS volatile suspended solids 31

ASSOCIATED CONTENT

S Supporting Information *

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PCA−NaOH fractionation procedures (Table S1), percentage of respective phosphorus portion (percentage of mixed liquor TP) (Table S2), total phosphorus amount in the EPS and the PCA extracts from the EBPR sludge at the end of aerobic phase before and after the extraction of EPS (mg of P/g of SS) (Table S3), chemical shifts and relative peak areas of various phosphorus forms in 31P NMR spectra of polyP model compounds after various extractions (Table S4), content of phosphorus in EPS and their contribution to phosphorus removal after the five-cycle consecutive test (Table S5), typical profiles of Pcell and PEPS in the anaerobic−aerobic process (Figure S1), and typical profiles of TPEPS and orthophosphate (orthoPEPS), pyrophosphate (pyroPEPS), and polyphosphate (polyPEPS) in the EPS in the anaerobic−aerobic process (Figure S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +86-551-63607453. Fax: +86-551-63601592. Email: [email protected]. Notes

The authors declare no competing financial interest. 11488



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Phosphorus Removal in an Enhanced Biological Phosphorus

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