The Removal Effect of Pollutants and Components in Extracellular Polymeric Substances from Aerobic Granular Sludge in Simultaneous Removal of Organic Matter and Ammonia Nitrogen

Biological processes, such as activated sludge and granular sludge are widely used to remove organic pollutants as well as inorganic pollutants, such as metallic elements1–3, which are known to be potentially toxic, and impact the performance of biological waste treatment processes4. Granular sludge is a dense aggregate of microorganisms, minerals, and microbial produced compounds such as extracellular polymeric substances (EPS)5,6. Compared to traditional activated sludge technology, AGS as a new type of microbe auto-immobilization technology, has many advantages, such as compact structure, fast settling velocity, higher sludge concentration, and ability to withstand high organic loading7,8. Many researches mainly try to study the operating conditions9, microbiological properties10,11, physicochemical characteristics12,13, and extracellular polymeric substances of granular sludge14,15. The AGS technology is used for treating all kinds of wastewater, including organic matter, nitrogen, phosphorus, and toxic substances16–19. EPS is a complex high-molecular-weight mixture of polymers in AGS. The EPS is a network bacteria embedded20, has a significant effect on the physicochemical properties of AGS, including structure, surface charge, flocculation, settling properties, and adsorption ability; plays a crucial role in biological wastewater treatment21–23. It not only results from active bacterial secretion, but also from cell lysis and effluent molecules24. EPS is considered as an organic product with mineral cations bound. Guibaud et al.25 have showed that some mineral particles, such as manganese oxides and iron oxy-hydroxides in the EPS extract can improve the ability of the EPS binding lead or cadmium cations. However, the mineral fraction of the EPS extracts, which is too often neglected in EPS studies, can affect the physicochemical properties of EPS26. The aim of this study is to investigate the removal effect of pollutants and the components, including the organic and mineral fractions, and especially to evaluate the mineral fraction in EPS to improve the stabilization of the AGS.


Introduction
Biological processes, such as activated sludge and granular sludge are widely used to remove organic pollutants as well as inorganic pollutants, such as metallic elements [1][2][3] , which are known to be potentially toxic, and impact the performance of biological waste treatment processes 4 .Granular sludge is a dense aggregate of microorganisms, minerals, and microbial produced compounds such as extracellular polymeric substances (EPS) 5,6 .
Compared to traditional activated sludge technology, AGS as a new type of microbe auto-immobilization technology, has many advantages, such as compact structure, fast settling velocity, higher sludge concentration, and ability to withstand high organic loading 7,8 .Many researches mainly try to study the operating conditions 9 , microbiological properties 10,11 , physicochemical characteristics 12,13 , and extracellular polymeric substances of granular sludge 14,15 .The AGS technology is used for treating all kinds of wastewater, including organic matter, nitrogen, phosphorus, and toxic substances [16][17][18][19] .
EPS is a complex high-molecular-weight mixture of polymers in AGS.The EPS is a network bacteria embedded 20 , has a significant effect on the physicochemical properties of AGS, including structure, surface charge, flocculation, settling properties, and adsorption ability; plays a crucial role in biological wastewater treatment [21][22][23] .It not only results from active bacterial secretion, but also from cell lysis and effluent molecules 24 .EPS is considered as an organic product with mineral cations bound.Guibaud et al. 25 have showed that some mineral particles, such as manganese oxides and iron oxy-hydroxides in the EPS extract can improve the ability of the EPS binding lead or cadmium cations.However, the mineral fraction of the EPS extracts, which is too often neglected in EPS studies, can affect the physicochemical properties of EPS 26 .
The aim of this study is to investigate the removal effect of pollutants and the components, including the organic and mineral fractions, and especially to evaluate the mineral fraction in EPS to improve the stabilization of the AGS.

Experimental sludge and wastewater
The experimental sludge used was the aerobic granular sludge with simultaneous removal of organic matter and ammonia nitrogen taken in a steady lab run for more than one year 27 .A synthetic wastewater was used.Glucose and sodium acetate were used as carbon sources, and ammonium chloride was the nitrogen source.Details of the synthetic wastewater composition are given in Table 1.0.6 mL L -1 trace element was added to the wastewater above to supply the necessary elements for microbial growth 27 .

Reactor description
Plexiglas column SBR reactor was used as experimental set-up.The height of the reactor was 60 cm, with inner diameter of 10 cm, and effective volume of 4.2 L. The volumetric exchange ratio of liquid was 50 %.The aeration system comprised an air compressor and a microporous aerator (sintering sand core) on the bottom.The aeration quantity was controlled by gas flowmeter.A programmable time controller was used to regulate the air compressor to realize the intermittent operation.The reactor was operated for 4 cycles every day.The cycles were distributed as follows: 10 minutes of feeding, 330 minutes of aeration, 5 minutes of settling, and 15 minutes of effluent withdrawal.Control parameters: the operating temperature was room temperature (20 °C), dissolved oxygen (DO) was controlled between 6.0 and 8.0 mg L -1 .The sludge concentration was 4500 mg L -1 .The pH in reactor was controlled by adding 1:14 H 2 SO 4 and 0.03 mol L -1 NaHCO 3 .

Determination of physicochemical properties
The analyses of COD, NH 4 + -N, NO 2 --N, NO 3 --N and mixed liquid suspended solids (MLSS) were performed according to the respective standard method 28 .UV absorbance was measured with a UV-1800 spectrophotometer (Shimadzu Co., Tokyo, Japan).The pH values were measured by a pH meter (pHS-3C, Shanghai Weiye Co., Ltd., China).DO concentration was determined by a DO meter (FG4-FK, Mettler Toledo Co., Ltd, Switzerland).The particle sizes were measured using an image analysis system (Image-pro Express 5.1, Media Cybernetics).The redox potential and the electrical conductivity were determined by pHS-3C precision pH meter (saturated calomel electrode, SCE; platinum electrode) and conductivity meter, respectively.

EPS extraction method
No standard extraction method has been developed for EPS.Chemical extraction protocols have also shown to contaminate the EPS extracts by impurities or carrying over of the extractant itself 26 .According to some studies, the molecular movement is enhanced, which accelerates the EPS dissolution 29 , so heating is the most effective method for EPS extraction due to its minimal cell lysis and more EPS extracted 30,31 .In a typical procedure: 100 mL of sample was centrifuged (4000 rpm, 5 minutes), and the supernatant was discarded, and then diluted with deionized water to its original volume.The diluted sample was heated (80 °C, 10 minutes), centrifuged (8000 × g, 20 minutes), and then filtered through 0.22 μm cellulose membrane filters.The obtained EPS extracts were stored at -20 °C for further analysis.

Analysis of organic matter
Protein measurement was performed using a modified Lowry method 32 .Bovine Serum Albumin (BSA) was used as standard.Polysaccharide concentration was determined using the Anthrone method 33 .Glucose was used as standard.Each measurement was undertaken on duplicate samples.

Analysis of mineral fractions
The mineral fractions in EPS were determined according to literature 26 .The details are as followed: A total of 3 mL of EPS were digested through the addition of 2 mL of hydrogen peroxide for 24 hours.Then, 3 mL of nitric acid and 1 mL of perchloric acid were added to the solution.Digestion was again performed after another 24 hours.After the second digestion, the samples were diluted with ultrapure water to 50 mL and then filtered through 0.22 μm cellulose nitrate membrane filters.Inductively coupled plasma optical emission spectrometry (ICP-OES, Perkin Elmer Optima 8300, America) determined the contents of mineral fractions.

Fourier-transformed IR (FTIR) spectroscopy
The FT-IR spectra of the EPS samples were recorded using an FTIR spectrometer (Avatar 370, Thermo Nicolet, America).The freeze-dried EPS samples were initially mixed with KBr (1 mg of EPS per 100 mg of KBr).The KBr pellets were heated until dried, and the dried samples were kept under vacuum in desiccators prior to use.The blank was corrected using a clean KBr pellet.The spectra were obtained with a resolution of 4 cm -1 and a scan range of 400-4000 cm -1 .

Results and discussion
Particle size distribution characteristics of granules Fig. 1 is particle size distribution of granules in stable operation reactor.It was observed that the main particle size of the AGS was more than 1,400 μm, of which the masses of particle sizes that were less than 500 μm, 500-830 μm, 830-1,000 μm, 1000-1400 μm, more than 1400 μm accounted for 15.73 %, 3.32 %, 0.49 %, 19.86 %, 60.60 %, respectively, of the total mass of AGS.The mature granular sludge was tawny, its outline was spheroidic or round, its boundary was clear (Fig. 2a).There are many cocci and a small number of bacillus distributing on the surface of granular sludge (Fig. 2b).The mature granular sludge having bigger particles, and dense structure has an excellent settling property 34 .Compared with traditional sludge (particles of traditional activated sludge are smaller than 150 μm, loose sludge flocs, settling rate no more than 10 m h -1 ), the average settling rate of the mature granular is 3.30 cm s -1 .The particle size of the granules was influenced by many factors, such as the COD/N, short operating time (60 days) and the configuration of reactors 10 .COD/N ratio plays a significant role in nitrifying sludge granulation.To some extent, there was a trend towards larger average granule diameter under higher COD/N ratio and load 35 .In contrast, Tsuneda et al. 36 obtained smaller granule diameter of granular sludge which took longer because the influent did not contain organic substrate, whereas Gao et al. 37 also obtained a smaller granule diameter because of lower substrate load.Moreover, granule diameter of granular sludge was related to the operating time and configuration of reactors, granule diameter of granular sludge increased with the extension of operating time generally 38 .However, the effect of reactor configuration on granule diameter of granular sludge is more complicated, which needs further research 10 .

Removal ratio of AGS on pollutants
Removal ratio of AGS on NH  -N removal efficiency was 99 % running 8 h with the use of granular sludge investigated by Cydzik-Kwiatkowska et al. 42 However, from days 1 to 17 in the experiment, the NH 4 + -N removal efficiencies were only between 69.30 and 89.22 %, the average removal efficiency was 92.66 %.Compared to the literature, lower removal efficiency resulted from the small specific area of particles due to their larger sizes in this experiment, NH 4 + -N incompletely exposed to the aerobic bacteria grown outside.Otherwise, because of the dense structure of the granules and the sticky EPS accumulated on the surface of bacterial cells for adhesion during the granulation process, NH 4 + -N cannot be oxidized by the ammonium-oxidizing bacteria in the middle.

Removal ratio of AGS on COD
Fig. 4 shows the influent and effluent COD concentrations and COD removal efficiency of AGS.Unstable behavior of practical wastewater with a wide fluctuation of influent organics was observed.From days 1 to 35, the influent COD concentrations increased from 106.35 to 303.14 mg L -1 .From days 23 to 49, the effluent COD concentrations decreased stepwise from 91.71 to 1.31 mg L -1 .Afterwards, the effluent COD concentrations were between 2.93 and 28.85 mg L -1 .From days 31 to 63, the removal efficiency of COD was between 80.59 and 99.37 %.The average removal efficiency was 90.22 %.The result of this study indicated good performance in organics removal in accordance with those performed in other researches.Ni et al. 43 reported that the average COD removal effi-ciency of aerobic granulation kept at 90 % for a long time in the bioprocess.The organics removal efficiency of AGS cultivated by Schwarzenbeck et al. 44 reached 90 % after complete granulation.The researches above have well documented the perfect removal efficiency of AGS on organics in the treatment of various organic wastewater.

Effluent nitrite and nitrate of AGS
As shown in Fig. 5, the effluent concentration of nitrite nitrogen was between 12.52 and 57.59 mg L -1 , and nitrate nitrogen concentration maintained below 8.56 mg L -1 .Nitrite accumulation rates (NO 2 -N/NO x -N) were between 78.81 and 96.83 % in the effluent, so that partial nitrification occurred in the reactor.Nitrite bacteria could be confirmed as the dominant nitrifying bacteria in the system.Controlling the DO concentration, temperature, SRT, pH, free ammonia (FA), and free nitrous acid (FNA) in the reactor could achieve nitrite accumulation.In this experiment, DO controlled between 6.0 and 8.0 mg L -1 , the inflow concentration of nitrite nitrogen controlled below 4 mg L -1 , at room temperature (25 °C), and SRT controlled at 15 d, were adverse to nitrite accumulation.Therefore, according to analysis, high pH (between 7.5 and 8.0) and the FA concentration (1.20-1.71mg L -1 ) might be the main reasons for nitrite accumulation.This result was in accordance with the literature [45][46][47] .Besides, delamination of AGS might also be a reason for the nitrite accumulation.Shi et al. 39 reported that the amount of nitrosation bacteria was more than that of nitrifying bacteria in AGS, and most of the nitrosation bacteria that adhered to the surface of granules could obtain sufficient DO to oxidate ammonia nitrogen to nitrite nitrogen.While nitrifying bacteria had distributed in the inner portion of the granules lacking in DO, the oxidizing ability was inhibited.This corresponded to the experimental results.

Analysis of EPS
The organic fraction and physicochemical properties of EPS The organic fraction and physicochemical properties of EPS are summarized in Table 2.The results show that the electrical conductivity was 2.25• 10 2 μm cm -1 , and redox potential was 367.2 mV.Recently, a few studies on the electrical conductivity of EPS have been performed, whose theory and the influence factors need to be further studied.The organic fraction of EPS is mainly composed of protein and polysaccharide.The results show that each gram of MLSS, the contents of polysaccharide and protein in the EPS extracted by the heating method were 18.67 and 38.36 mg, respectively, and the ratio of protein/polysaccharide 2.05.The data for protein and polysaccharide contents obtained for EPS are in accordance with the concentrations observed by Sheng et al. 48Moreover, the large yields of both are due to the fact that, during the extraction procedure by heating, the proteins and polysaccharides could have hydrolysed 49 , and the bound EPS, strongly attached to activated sludge, was allowed to be extracted, since both kinds of EPS exist in the sludge in this extraction procedure.In addition, the result of the ratio of protein/polysaccharide is in accordance with the one reported by Liu and Fang 50 , and confirms that the EPS extracted in this study were not contaminated by intracellular materials from bacteria of AGS.These authors have shown that the ratio of protein/ polysaccharide from activated sludge varies from 0.5 to 21.2.When the ratio is high, the EPS are predominantly composed of protein, thus the protein originated mainly from cell lysis or exocellular enzymes.

Mineral fractions
Fig. 6 shows the chemical composition of the mineral fractions in the EPS.Many different inorganic cations were present in EPS samples.Phosphorus was the main mineral compound (data not presented), whereas Ca, K, Fe, Mg, Zn and Cu were the main metallic elements.The presence of Ca and Fe confirms their role in the maintenance of the granular structure, and they are necessary for the granulation of sludge, and integrated in the granule in important quantities 51 .Ca and Fe act as the bridging ions 52 , therefore, the binding of divalent cations with EPS stabilizes the structure of the EPS matrix.Moreover, in the EPS, the ionic nature of cell surfaces and the molecules allows interactions with cations, Zn and Cu should be preferentially present inside the bacterial cell due to their biological activity.Due to the presence of various types of binding sites for cations in the organic compounds of the EPS 24,26 , a part of the multivalent inorganic cations, such as Ca 2+ , Mg 2+ , and Fe 2+ , measured in the EPS extracts, are very likely bound to the organic molecules in the EPS in order to provide structural stability in AGS 53,54 .Mn and Ni are the trace elements in the EPS, the contents are 0.008 and 0.017 mg L -1 respectively.F i g .6 -Main elements content of the mineral fractions in the EPS (mg g -1 , MLSS) IR spectra examples of EPS extracted from AGS are represented.The IR spectra show the contribution, as well as the superposition of absorption bands from organic and inorganic compounds, except for absorption bands below 1000 cm -1 which may be essentially attributed to inorganic compounds 55 , such as phosphate or sulfur functional groups.Several intense characteristic bands can be attributed to protein and polysaccharide functional groups.Carboxylic groups, under acid or basic salt form, are responsible for some less intense bands.The stretching vibration of OH can be observed 55 at 3392.19 cm -1 as well as 1654.49cm -1 , and a weak symmetric stretching vibration band of CH 2 at 2927.57cm -1 and 1454.26cm -1 .The stretching vibration of C-N observed at 1544.15 cm -1 of proteins.The bands at 1400.51 cm -1 and 1245.86 cm -1 may respectively be assigned to the C=O in the carboxylates and carboxylic acids.The absorption peak observed at 1083.46 cm -1 in the range from 1000 cm −1 to 1200 cm −1 is known to be typical of all sugar derivatives 56 .The different functional groups observed in the EPS samples agree with Comte et al. 57 .Divalent cations are able to link to the phosphate and carboxyl groups.Therefore, FTIR was used to study the functional groups of microbial aggregates and their associated EPS.

Conclusion
The removal ratio of COD and NH 4 + -N were analyzed.The AGS demonstrated good performance in simultaneous organic carbon and nitrogen removal, and the removal rates were 90.22 and 92.66 %.The particle size above 0.5 mm was 84.27 %, and the main particle size above 1.4 mm was 60.60 %.The electrical conductivity of EPS was 2.25• 10 2 μm cm -1 , and the mineral fractions of the EPS extract were determined to exist.Ca, K, Fe, Mg, Zn, and Cu were the main mineral elements found in EPS extracts, Mn and Ni were the trace elements in EPS extracts.In this study, the organic and mineral fractions in EPS were studied; however, the relationship between them is unclear.Meanwhile, in the forming process of granular sludge, the function of EPS need further research.
efficiency of AGS is shown in Fig. 3.The effluent NH 4 + -N concentrations were stable between 0.08 and 8.78 mg L -1 at a volumetric exchange ratio of 50 %.From days 19 to 63, the NH 4 + -N removal efficiencies improved between 92.33 and 99.92 %.The NH 4 + -N removal capacity indicated a good enrichment of nitrifiers, which could have resulted in the formation of AGS.According to the literature, which have well demonstrated good performance in nitrogen removal due to the composition of granular microbial communi-F i g . 1 -Particle size distribution of granules

F i g . 2 -
Shape (a) and SEM observation (b) of AGS ties, including heterotrophic, aerobic bacteria grown on the outside, ammonium-oxidizing bacteria in the middle, and facultative and anaerobic bacteria in the core of the granules 39,40 .Wei et al. 41 reported that the average NH 4 + -N removal efficiency kept at 96 % for a long time in the aerobic granule-based bioreactor.The NH 4 +

F i g . 3 -+
Influent and effluent NH 4 efficiency of AGS F i g .4 -Influent and effluent COD concentrations and COD removal efficiency of AGS F i g .5 -Effluent NO 2 --N and NO 3 --N concentrations of AGS