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The Effect of Extracellular Polyphosphate in the Process of Enhanced Biological Phosphorus Removal (EBPR): from the Perspective of Transformations of Extracellular Polyphosphate and Intracellular Energy-storing Substances

The distributions and cycle changes of orthophosphate (ortho-P), polyphosphate (poly-P), glycogen and polyhydroxyalkanoates (PHAs) in the 2 reactors fed with acetate at 20°C or 35°C were investigated to understand the effect of extracellular polyphosphate (poly-P) in enhanced biological phosphorus removal (EBPR) process. The specific anaerobic-uptake acetate rate in the 20°C EBPR reactor was about 2.24 times that in the 35°C non-EBPR reactor. The anaerobic-hydrolysis and the aerobic-synthesis amounts of intracellular glycogen in the former were only half those in the latter, with the comparable corresponding-transformation amounts of intracellular PHAs between the 2 reactors. The anaerobic-hydrolysis / aerobic-synthesis of extracellular poly-P and the anaerobic-release / aerobic-uptake of intracellular ortho-P occurred in the 20°C EBPR sludge, corresponding to the lower transformation level of intracellular glycogen and the higher anaerobic-uptake rate of acetate. A hypothesis that the anaerobic-hydrolysis of extracellular poly-P could accelerate the acetate migration though anion-exchange was proposed. In addition, phosphorus accumulating organisms (PAOs) was the microbial population basis for the existence and transformation of extracellular poly-P.


Introduction
Enhanced biological phosphorus removal (EBPR) is applied in global to remove phosphorus (P) from wastewater [1,2]. The success operation of EBPR system is mostly relied on the phosphorus accumulating organisms (PAOs), which excessively absorb P under the aerobic/anoxic condition and then store intracellular polyphosphat (poly-P) that is an important energy substance for PAOs to anaerobically absorb carbon source [3][4][5][6] and is benifical for PAOs to gain advantage over glycogen accumulating organisms (GAOs) [7]. PAOs also can behave as GAOs under low poly-P condition [8,9] or at high influent Ca concentration [10,11], because intracellular glycogen is also used as an energy source for PAOs to absorb carbon source and synthesize polyhydroxyalkanoates (PHAs) [5,6]. The bacterial cells in biological aggregate are enclosed by extracellular polymer substances (EPS), which contained 27% -57% extracellular P of the sludge P content [12][13][14][15], illustrating that the role of NEPEE 2020 IOP Conf. Series: Earth and Environmental Science 696 (2021) 012031 IOP Publishing doi: 10.1088/1755-1315/696/1/012031 2 EPS in biological P removal could not be ignored. Furthermore, the 31 P-NMR (nuclear magnetic resonance) spectra results illustrated that poly-P, pyrophosphate (pyro-P) and orthophosphate (ortho-P) were coexisted in the EPS of EBPR sludge [16,17]. Importantly, the anaerobic-decrease and aerobicincrease profile of the extracellular total P (TP) content was accompanied with the aerobic-hydrolysis and aerobic-synthesis of the extracellular poly-P [17,18], indicating extracellular poly-P was involved in EBPR mechanism. EPS can be divided into outer loosely-bound EPS (LB-EPS) and inner tightlybound EPS (TB-EPS) [19][20][21]. Recently, the authors found that LB-EPS and TB-EPS played different roles in EBPR process [19,22], in which the anaerobic-hydrolysis and aerobic-synthesis of the poly-P in TB-EPS occured [19]. In the EBPR process, intracellular glycogen and PHAs are important energystoring substances of PAOs and GAOs, of which the transformation processes can reveal some microbial metabolism characteristics [8 -11]. However, the transformation relationships between extracellular poly-P and intracellular energy-storing substances are still unclear, thus it is essential to understand the effect of extracellular poly-P in EBPR process. Temperature is an important factor on the EBPR performance, influencing the competition between PAOs and GAOs and the transformation of intracellular energy-storing substances [1]. Temperatures higher than 25°C are generally unfavorable to EBPR process due to the domination of GAOs without accumulating poly-P [2,23]. The effect of extracellular poly-P in the EBPR process can be discovered, through comparing the structures of microbial population and the transformations of intracellular energy-storing substances in different temperaturet systems. This aim of the study is to understand the effect of extracellular poly-P in EBPR process, taking 2 labscale anaerobic/oxic reactors respectively at 20°C and 35°C as the objects. The contents, distributions and cycle changes of the ortho-P, poly-P, glycogen and PHAs in the 2 reactors were firstly compared by fractionating sludge floc into LB-EPS, TB-EPS and bacterial cell. The effects of the microbial population structures on the transformations of the poly-P in TB-EPS and the intracellular energystoring substances as well as the transformation relationships between extracellular poly-P and intracellular energy-storing substances were discussed, in order to understand the effect of extracellular poly-P in EBPR process.

Culture of Activated Sludge
The 2 A/O-sequencing batch reactors (SBR) at 20 ± 1 °C and 35 ± 1 °C were fed with sodium acetate, running for more than one year with 750 mg/L of the influent COD concentration and 100: 5: 5 of the COD: N: P ratio. The reactors were operated with 2 cycles per day with 15 L working volume, including instantaneous filling wastewater, 4-h anaerobic and 7-h aerobic conditions and 50-min settling, 5-min decanting and about 5-min idling stages. The approximately 20 d of solid retention time (SRT), the 7.4-8.8 of pH during running cycle and 2.5 -4.5 mg/L of dissolved oxygen (DO) concentrations at the end of aeration phase were controlled. The concentrations of the influent phosphrous and nitrogen were detailed in the previous reports [19,22]. The volatile suspended solids (VSS), sludge volume index (SVI) and total phosphorus (TP) and orthophosphate (ortho-P) of influent and effluent were monitored every week. The tests were carried out after these parameter values kept relatively stable for more than 3 months.

31 P NMR Analysis
These extractions were freeze-dried and then were kept at -20°C before 31 P NMR analysis. The procedures of re-dissolution of the lyophilized LB-EPS and TB-EPS and lysis of the lyophilized  [19]. A 600 MHz NMR spectrometer (Agilent Technologies, Santa Clara, USA) was utilized to record the 31 P NMR spectrum with the 0.56-μs 90 ° pulse width, 25 °C regulated temperature and 128 scan number, followed by the analysis with the NMR data processing software (MestReNova v6.1.0-6224) [16,19].

Measurement of Acetate, PHAs and Glycogen
The acetate concentration in bulk solution and the PHAs contents (including PHB and PHV) of activated sludge or bacterial cell were respectively determined by gas chromatograph (GC, 7890A, Agilent Technologies, Santa Clara, USA) equipped with a HP-FFAP [24] or DB-5 column [25]. The contents of glycogen or polysaccharide in activated sludge, LB-EPS and TB-EPS were determined with the anthranone sulfate method [25]. The glycogen content in bacterial cell (intracellular glycogen) was calculated by subtraction.

16S rRNA Gene Amplicon Sequencing
The DNA of sludge sample collected at the end of aerobic phase was extracted with soil genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing, China) and then was stored at -20°C. The extract were amplified through PCR using primer set 338F (5/-ACTCCTACGGGAGGCAGCAG-3/) and 806R (5/-GGACTACHVGGGTWTCTAAT-3/) for the V3-V4 region of 16S rRNA gene. The 20 μL mixture was employed to carry out PCR, which was detailed in the previous literature [22]. The PCR was performed in a Gene Amp_9700 (Applied Biosystems, U.S.) with the procedure reported in the previous literature [26], followed by sequencing on an Illumina MiSeq PE300 platform according to the standard protocols. The PCR amplification and sequencing were carried out by Meiji Biomedical Technology Co., LTD., (Shanghai, China) and the data were analyzed with Meiji Biocloud platform.

Other Analyses
The DO concentration was measured through a DO analyzer (Pro20, YSI, Ohio, USA). The SVI, the concentrations of suspended solids (SS) and volatile suspended solids (VSS), the ortho-P concentration (ortho-P solution ) and the TP concentration (TP solution ) in bulk solution as well the TP contents of LB-EPS (TP LB-EPS ) and TB-EPS (TP TB-EPS ) were determined by the standard methods [27]. The TP content of sludge (TP sludge ) was determined through the sonication dispersion, and the content of TP in bacterial cell (TP cell ) was calculated with subtraction. All measurements were conducted in triplicate. non-EBPR reactors during steady cycle. The 2 reactors at 20°C and 35°C had steadily runned more than 3 months before cycle experiments. From Figure 1a, the cycle profile of ortho-P solution in the 20°C reactor presented a typical EBPR pattern, while the 35°C reactor had non-EBPR performance. In addition, the ortho-P solution made up 98.5 -99.6% of the TP solution (The data not shown), illustrating that the 20°C EBPR sludge anaerobically released or aerobically took up ortho-P. From Figure 1b,  reactors at 30 min and 45 min, respectively, when the VSS concentration (2450 ± 69 mg VSS/L) in the former was lower than that (3661 ± 75 mg VSS/L) in the latter, indicating that the specific anaerobicuptake acetate rate in the former was about 2.24 times that in the latter.

P Content, Distribution and Species of Sludge
From Figure 2, the TP content of sludge (TP sludge ) in the 20°C EBPR reactor was greater than that in the 35°C non-EBPR reactor. The TP content of TB-EPS (TP TB-EPS ) of the 20°C sludge was slightly more than that of cell (TP cell ), while the TP TB-EPS of the 35°C sludge was slightly less than the TP cell . The steady-cycle profiles of the TP sludge , TP cell and TP TB-EPS in the 20°C EBPR reactor all presented anaerobicdecrease and aerobic-increase, while these in the 35°C non-EBPR reactor kept relatively stable, indicating that the anaerobic P-release and aerobic P-uptake of the EBPR sludge was due to the combined actions of TB-EPS and bacterial cell [19]. According to the 31 P NMR spectra (Figure 3), ortho-P was the main P species in the bacterial cell and LB-EPS, while poly-P, pyro-P and ortho-P co-existed in the TB-EPS of 20°C EBPR sludge, but only ortho-P in that of 35°C non-EBPR sludge. From Table 1, the contents of poly-P, pyro-P and ortho-P (poly-P TB-EPS , pyro-P TB-EPS and ortho-P TB-EPS ) in TB-EPS of the 20°C sludge respectively accounted for 2.09% -60.18%, 0.09% -2.86% and 39.40% -96.42% of TP TB-EPS , thus poly-P and ortho-P were the main P species. In addition, the apoptosis / death of bacterial cell was very weak induced by sonication extraction of LB-EPS and CER extraction of TB-EPS and there were no bacterial cells in the LB-EPS and TB-EPS extracts (data not shown). The cycle profiles of the TP cell and the ortho-P content (ortho-P cell ) of bacterial cell in the 20°C reactor presented anaerobic-decrease and aerobic-increase, indicating that the bacterial cell directly released or took up ortho-P, when the cycle profiles of poly-P TB-EPS and TP TB-EPS also exhibited anaerobic-decrease and aerobic-increase. Furthermore, the average chain length of poly-P in TB-EPS for the 20°C reactor reduced from 286.4 ± 16.3 to 17.28 ± 3.46 at the early anaerobic stage (0 -1 h) and increased from 6.44 ± 2.63 to 286.0 ± 23.9 at the early aerobic stage (4 -6 h), illuminating that the extracelluar poly-P played an important role in the EBPR process.

Glycogen Content and Distribution of Sludge
From Figure 4, the glycogen content of sludge (glycogen sludge ) in the 20°C EBPR reactor was obviously lower than that in the 35°C non-EBPR reactor.    Figure 5, the PHAs content of sludge (PHA sludge ) in the 20°C EBPR reactor was obviously lower than that in the 35°C non-EBPR reactor. In addition, the PHAs also mainly existed in bacterial cell, similarily to the glycogen distributions. The intracellular PHAs contents (PHA cell ) of the 20°C sludge and 35°C sludge were 23.67 -67.80 mg/g VSS and 112.9 -149.2 mg/g VSS, respectively, which accounted for 90.02 % -96.12 % and 83.28 -96.11% of the PHA sludge . The extracellular PHAs existed in LB-EPS and TB-EPS was the difference between the PHA sludge and the PHA cell . In general, the PHAs and the glycogen of sludge in the 2 reactors were mainly intracellular, while the poly-P of the 20°C sludge was almost extracellular. The cycle profiles of the PHA sludge and the PHA cell in the 2 reactors exhibited anaerobic-increase and aerobic-decrease, which were opposite to those of the glycogen sludge and the glycogen cell . In addition, the cycle changes of the PHA sludge and the glycogen sludge were respectively attributed to those of the PHA cell and the glycogen cell . There was obviouse difference in the component of intracellular PHAs between the 2 reactors ( Figure  5). The intracellular PHB of the 20°C sludge was the dominant component of intracellular PHAs, of which the content (PHB cell ) was 14.59 -49.47 mg/g VSS, while the intracellular PHV content (PHV cell ) was 9.07 -18.33 mg/g VSS. On the contrary, the intracellular PHV of the 35°C sludge was the main component of intracellular PHAs. Specifically, The PHV cell and the PHB cell were 86.10 -97.69 mg/g VSS and 15.26 -62.48 mg/g VSS, respectively. The PHB cell of the 20°C EBPR sludge was close to that of the 35°C non-EBPR sludge, while the PHV cell of the former was much lower than that of the latter. The cycle profiles of the PHB cell and the PHV cell in the 20°C reactor presented anaerobic-increase and aerobic-decrease. But, the change of the PHV cell in the 35°C reactor was anaerobic-decrease and aerobicincrease, which was opposite to that of the PHB cell .   4 h), the change values of these intracellular energy-storing substances and /or extracellular poly-P in the 2 reactors were less. In addition, the VSS concentrations in the 20°C EBPR reactor and 35°C non-EBPR reactor were 2450 ± 69 mg VSS/L and 3661 ± 75 mg VSS/L, respectively. During the anaerobic phase, the anaerobic-synthesis amounts of intracellular PHAs for the 20°C and 35°C reactors were about 108.1 mg/L and 101.1 mg/L, respectively, while the anaerobic-hydrolysis amounts of intracellular glycogen were about 156.3 mg /L and 272.3 mg /L. At the early aerobic stage (4 -6 h), the PHB cell and the PHV cell in the 20°C EBPR reactor decreased by 27.46 ± 2.73 mg/g VSS and 6.28 ± 0.69 mg/g VSS, respectively, while the poly-P TB-EPS and the glycogen cell increased by 9.31 ± 0.85 mg/g VSS and 69.75 ± 5.86 mg/g VSS. At the stage, the PHB cell of the 35°C non-EBPR reactor decreased by 35.48 ± 3.01 mg/g VSS, while the glycogen cell and the PHV cell increased by 72.55 ± 3.56 mg/g VSS and 8.81 ± 0.94 mg/g VSS, respectively. At the middle and later aerobic stage (6 -11h), the change values of these intracellular energy-storing substances and /or extracellular poly-P in the 2 reactors were less. During the aerobic phase, the aerobic-hydrolysis amounts of the intracellular PHAs in the 20°C and 35°C reactors were about 114.0 mg /L and 130.4 mg /L, respectively, while the corresponding aerobic-synthesis amounts of the intracellular glycogen were about 196.8 mg/L and 386.1 mg/L. In brief, the transformation amounts of intracellular glycogen in the 35°C non-EBPR reactor were almost twice as much as those in 20°C EBPR reactor, but those of the intracellular PHAs between the 2 reactors were close.

Microbial Population Structure of Sludge
Temperature is an important factor to influence EBPR performance and microbial population structure [2,23]. From Figure 6, there were obvious differences in the microbial population structure between the 2 reactors. In the 20°C EBPR sludge, Candidatus_Competibacter and Candidatus_Accumulibacter were abundantly co-existed, of which the abundances were 33.77% and 30.48%, respectively, while the abundances of Anaerolineaceae and Defluviicoccus all were very low. In the 35°C EBPR sludge, the abundances of Anaerolineaceae, Defluviicoccus and Candidatus_Competibacter were 28.20%, 27.13% and 15.21%, respectively, but Candidatus_Accumulibacter was not observed. It is well known that Candidatus_Accumulibacter has been recognized as PAOs, and Defluviicoccus and Candidatus_Competibacter has been considered as GAOs [1,5]. Thus, the Candidatus_Accumulibacter was the key PAOs in the 20°C EBPR sludge and the Defluviicoccus was the dominant GAOs in the 35°C non-EBPR sludge. The differences of transformation characteristics of the extracellular poly-P and the intracellular glycogen and PHAs between the 2 reactors were related to the microbial population structures. Firstly, the anaerobic-hydrolysis and aerobic-synthesis of extracellular poly-P in the 20°C EBPR sludge occurred, while the extracellular poly-P in the 35°C non-EBPR sludge was not detected. In addition, Candidatus_Accumulibacter was abundant in the 20°C sludge and not existed in the 35°C sludge ( Figure  6b), which was the microbial population basis for the existence and the transformation of the poly-P in TB-EPS of the 20°C EBPR sludge. Secondly, the anaerobic-hydrolysis and aerobic-synthesis amounts of intracellular glycogen in the 35°C reactor were almost twice those in the 20°C reactor, respectively. Although the VSS concentration (3661 ± 75 mg/L) in the 35°C non-EBPR reactor was more than that (2450 ± 69 mg/L) in the 20°C EBPR reactor, the glycogen-accumulating metabolism (GAM) level of microorganism in the former was 1.16-1.31 times that in the latter (Figure 4 and Table 2), corresponding to that the total abundance of GAOs in the former was greater than that in the later. Specifically, the total abundance of Defluviicoccus (27.13%) and Candidatus_Competibacter (15.21%) in the 35°C sludge was about 1.25 times the abundance of Candidatus_Competibacter (33.77%) in the 20°C sludge. Thirdly, the anaerobic-hydrolysis and aerobic-synthesis of intracellular PHV in the 35°C non-EBPR sludge occurred, which was contrary to the change of intracellular PHV in the 20°C EBPR sludge. It had been reported that PAOs anaerobically produced mainly PHB with little PHV production (< 10 %) when fed with acetate [28,29], while GAOs anaerobically produced about 75 % PHB and 25 % PHV when fed with acetate [30,31]. The formation amounts of PHB cell and PHV cell in the 20°C EBPR sludge at the anaerobic phase were respectively about 79 % and 21 % of the produced PHA cell , , because the Candidatus_Accumulibacter and Candidatus_Competibacter abundantly co-existed ( Figure 5 and Table  2). The different transformation characteristics of intracellular PHV in the 35°C non-EBPR sludge might be related to a great deal of Defluviicoccus and Anaerolineaceae as well as some different Candidatus_Competibacter at species level.   In the EBPR theory, intracellular poly-P is assumed as a main energy source for PAOs to anaerobically absorb carbon source [5,6] and a key substance for PAOs to gain advantage over GAOs [7], which can promote PAOs to take up carbon sources, such as acetate and propionate [6]. In this study, the specific anaerobic-uptake acetate rate in the 20°C EBPR reactor was about 2.24 times that in the 35°C non-EBPR reactor (Figure 1). However, the 31 P NMR resonance signals of intracellular poly-P (i.e., the end and the middle groups of poly-P) of the 20°C sludge were also weak (Figure 3c), which could not explain the difference of the specific anaerobic-uptake acetate rate between the 2 reactors. The proton motive force (PMF) by efflux of proton and ortho-P is also related to promoting carbon source uptake [5,6]. From Figure 3 and Table 1, the bacterial cell in the 20°C EBPR sludge directly anaerobically released, indicating that the release of intracellular ortho-P into the cell outer surface produced PMF to promote acetate pass through cell membrane. There was a great deal of extracellular poly-P in the 20°C EBPR sludge, which produced ortho-P by anaerobic hydrolysis. These ortho-P all passed through TB-EPS and LB-EPS before entering bulk solution. In addition, the anion-exchange performance of ortho-P is superior to that of acetate, thus the ortho-P both released from the intracellular ortho-P and came from the hydrolyzed extracellular poly-P could accelerate acetate pass through LB-EPS and TB-EPS by the anion-exchange between ortho-P and acetate (Figure 7). From Table 2, the decrease amount (-8.77 ± 1.19 mg/g VSS) of the poly-P TB-EPS was almost 1.44 times that (-6.10 ± 1.32 mg/g VSS) of the ortho-P cell in the 20°C EBPR sludge at the early anaerobic stage (0 -1 h), suggesting that the hydrolysis of extracellular poly-P played an important role in the acetate migration from the bulk solution to the cell surface (passing through LB-EPS and TB-EPS). During the aerobic phase, the aerobic-hydrolysis amount of intracellular PHAs in the 20°C EBPR reactor was comparable to that in the 35°C non-EBPR reactor, but the aerobic-synthesis amount of intracellular glycogen in the former was about half that in the latter. Meanwhile, the aerobic-synthesis amount of extracellular poly-P and the aerobic-uptake amount of intracellular ortho-P in the former were far greater than that in the latter, of which the energy should came from the hydrolysis of intracellular PHAs. In the future, it is very necessary to verify the anion exchange hypothesis and understand the different routes of P migration and transformation in EBPR process.

Conclusions
The anaerobic-hydrolysis and aerobic-synthesis amounts of intracellular glycogen in the 20°C EBPR reactor were only half those in the 35°C non-EBPR reactor, while the corresponding-transformation amounts of intracellular PHAs between them were comparable. Notably, the anaerobic-hydrolysis / aerobic-synthesis of extracellular poly-P (in tightly-bound EPS (TB-EPS)) and the anaerobic-release / aerobic-uptake of intracellular ortho-P occurred in the 20°C EBPR sludge. An anion-exchange hypothesis that the ortho-P both released from the intracellular ortho-P and came from the hydrolyzed extracellular poly-P could accelerate acetate pass through LB-EPS and TB-EPS was proposed. PAOs were the microbial population basis for the existence and transformation of extracellular poly-P.