Effect of ultrasonic power density on extracting loosely bound and tightly bound extracellular polymeric substances

Desalination 329 (2013) 35–40

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Effect of ultrasonic power density on extracting loosely bound and tightly bound extracellular polymeric substances Xiaomeng Han a, Zhiwei Wang a,⁎, Chaowei Zhu b, Zhichao Wu a a b

State Key Laboratory of Pollution Control and Resource Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China Chinese Research Academy of Environmental Sciences, Beijing 100012, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Ultrasonication power density affected LB-EPS extraction significantly. • Power density did not affect TB-EPS extraction until a very high value was used. • DNA evolution could be used to identify the critical power density of EPS extraction. • Critical power density for LB- and TBEPS extraction was 35 and 65 W/10 mL, respectively.

a r t i c l e

i n f o

Article history: Received 26 April 2013 Received in revised form 4 September 2013 Accepted 4 September 2013 Available online 26 September 2013 Keywords: Extracellular polymeric substances (EPS) Membrane fouling Membrane bioreactor Ultrasonic power density Wastewater treatment

a b s t r a c t Ultrasonication has been widely used for bound extracellular polymeric substance (EPS) extraction. However, the used ultrasonic power density is quite different in literature, which makes their results non-comparable. In this study, the effects of different ultrasonic power densities on extracting bound EPS were assessed via analyzing carbohydrates, proteins, humic acids and DNA. Experimental results proved that ultrasonication power density had a significant effect on loosely bound EPS (LB-EPS) extraction, and an appropriate power density termed critical power density was found to be 35 W/10 mL according to the variations of DNA which represented the intactness or damage of cell membrane. Nevertheless, carbohydrates, proteins and humic acids did not change obviously in tightly bound EPS (TB-EPS) until a very high power density was applied. The critical power density was determined as 65 W/10 mL for TB-EPS extraction. The results indicated that a critical power density should be used in order to extract maximal EPS but not to damage cell membrane intactness. © 2013 Elsevier B.V. All rights reserved.

1. Introduction In recent years, membrane bioreactors (MBRs) have gained increasing popularity due to their advantages over conventional activated ⁎ Corresponding author. Tel./fax: +86 21 65980400. E-mail address: [email protected] (Z. Wang). 0011-9164/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.desal.2013.09.002

sludge process, such as reduced footprint, improved effluent quality and decreased sludge production [1]. However, membrane fouling, a major obstacle for further wide-spread applications of MBRs, can lead to reduced flux, increased trans-membrane pressure (TMP) and frequent membrane cleaning, and consequently to high maintenance and operating costs. To date, it has been well accepted that extracellular polymeric substances (EPS) are an important factor causing membrane

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fouling in MBRs [2–4]. A common understanding of EPS is that they are microbially-produced organic materials that contain electrons and carbon, but are not active cells [5]. In other words, EPS are a matrix of large polymeric molecules containing proteins, polysaccharides, nucleic acids, humic acids, lipids and so on, which can induce biofoulant deposition on membranes and alter membrane physicochemical characteristics by blocking membrane pores and changing membrane electrical charge and hydrophobicity [6]. EPS can be classified into soluble EPS and bound EPS including loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS). To date, different extraction methods have been applied to extracting them from biomass in current publications. The common extraction method for soluble EPS is centrifugation, while for bound EPS several kinds of extraction methods have been reported, for example, formaldehyde + NaOH, EDTA, cationic resin, ultrasonication, thermal treatment, etc. [7–9]. Among those extraction methods, ultrasonication has been widely used. However, as shown in Table 1, the ultrasonic power density is quite different or even not clearly stated in literature, which makes their results non-comparable. Although some researchers found the relationship between sludge hydrolysis and exposure time [10], there are few reports focusing on the effect of ultrasonic power density on extracting LB-EPS and TB-EPS. Hence, it is very essential to establish unified ultrasonication parameters for bound EPS extraction. According to the report of Sun et al. [18], an EPS extraction process should ideally extract maximum EPS, minimize cell lysis and cause no damage to EPS structure. Therefore, it is of great significance to discern the effects of extraction parameters on floc disintegration and cell lysis. For ultrasonication extraction, the ultrasonic power density plays an important role in EPS extraction. In this paper, an appropriate ultrasonic power density for extracting bound EPS called as “critical power density” is defined by comparing the concentrations of proteins, polysaccharides, humic acids and DNA, below which the floc disintegration dominates while above which cell lysis occurs to a large extent. Moreover, fluorescence staining and confocal laser scanning microscope (CLSM) imaging, which are usually used to observe granule sludge structure in previous studies [20,21], are also adopted to directly visualize bound EPS distribution in active sludge floc in our study.

2. Materials and methods 2.1. Sludge samples In this study, sludge samples were taken from the oxic zone in a pilot-scale MBR reactor, which was located at the Quyang Municipal Wastewater Treatment Plant of Shanghai. The MBR was fed with real municipal wastewater, including an anoxic zone of effective volume of 21 L and an oxic zone of effective volume of 21 L. The hydraulic retention time of the MBR was 21 h. 2.2. Ultrasonication extraction methods The mixed liquor of sludge was first centrifuged in order to remove bulk solution (6000 ×g, 10 min). After discarding the supernatant, the remaining pellet was resuspended with 0.05% (w/w) NaCl solution and sonicated (the first ultrasonication) at 25 kHz for 2 min according to the report of Ye et al. [16]. The ultrasonication was performed in a probe system (JY90-II, Xinzhi Inc., China) through a tip with a surface area of 0.28 cm2. The liquor was centrifuged at 8000 ×g for 10 min to separate solids and supernatant which was regarded as the LB-EPS. The residual sludge pellet left was re-suspended in 0.05% (w/w) NaCl solution, sonicated for 2 min (the second ultrasonication), then heated at 60 °C for 30 min, and finally centrifuged at 11,000 ×g for 30 min to collect supernatant which was regarded as the TB-EPS in the sludge sample. The temperature of all sludge samples was controlled at 4 °C via ice-water bath to eliminate temperature increase under high power density irradiating. Different ultrasonication power densities (3.2 W/10 mL, 6.5 W/ 10 mL, 13 W/10 mL, 35 W/10 mL, 65 W/10 mL, 100 W/10 mL and 150 W/10 mL) were chosen to investigate the influence of power density on LB-EPS extraction while 3.2 W/10 mL power density ultrasonic treatment for TB-EPS extraction was fixed for the second ultrasonication (Section 3.1). For TB-EPS testing, the ultrasonication power density was varied from 3.2 W/10 mL to 150 W/10 mL in the second ultrasonication while the power density for the first sonication was fixed at 3.2 W/10 mL (Section 3.2). All the experiments were carried out twice under the same condition, and values were given as mean value with standard deviation. 2.3. Analytical methods

Table 1 Comparison of different ultrasonication power densities in literature. No.

Targets

Methods

References

1

EPS

[11]

2

EPS

3 4

EPS EPS

5

LB-EPS and TB-EPS

6

LB-EPS and TB-EPS

7

EPS

8 9

EPS LB-EPS and TB-EPS

Stirring with 36.5% formamide for 1 h and ultrasound treatment at 60 W/10 mL for 2.5 min Ultrasonication at 0.8 W/10 mL for 2 min and stirring with cation exchange resin (CER) for 2 h Ultrasonication at 40 W for 2 min Ultrasonication at 120 W/10 mL for 5 min and stirring with 36.5% formamide for 1 h, 1 mol/L NaOH for 3 h, and at last ultrasound treatment again for 5 min Ultrasonication for 2 min twice to extract LB-EPS; stirring with CER for 12 h to extract TB-EPS Ultrasonication at 3.3 W/10 mL for 2 min to extract LB-EPS; ultrasonication followed by heating at 60 °C for 30 min to extract TB-EPS Ultrasonication at 10 W/10 mL sample for 10 min Ultrasonication at 50 W Oscillating with buffer solution for 1 h to extract LB-EPS; ultrasound treatment for 3 min at 120 W/10 mL sample for TB-EPS extraction

[12]

[13] [14]

[15]

[16]

[17] [18] [19]

The carbohydrate and protein concentrations in bound EPS were determined using the phenol–sulfuric acid method [22] with glucose as a standard and the modified Lowry method [23] with bovine serum albumin (BSA) as the standard reference, respectively. The humic acid was measured according to Frølund's report [24]. The dissolved organic carbon (DOC) of sample was analyzed by a total organic carbon (TOC) analyzer (LiquiTOC trace, Elementar, Germany). The DNA contents in bound EPS were measured by the diphenylamine colorimetric method [25] using salmon semen DNA as the standard. In brief, 0.5 mL of sample was precipitated overnight with 0.5 mL of 25% trichloroacetic acid (TCA), centrifuged at 13,000 ×g for 10 min, and hydrolyzed in Tris–HCl with ethylene diamine tetraacetic acid (EDTA) buffer at 90 °C for 15 min. All samples were incubated with 160 μl of diphenylamine reagent at 37 °C for 4 h, in which 150 mg of diphenylamine was dissolved in 10 mL of glacial acid, and then 150 μl of concentrated sulfuric acid and 50 μl of acetaldehyde solution were added and mixed well. The amount of DNA was determined from its absorbance at 570 nm by a microplate spectrophotometer (Synergy 4, Bio-Tek, America). 2.4. Staining and CLSM analysis According to the procedure reported by Chen et al. [20], 0.1-M sodium bicarbonate buffer was added in the pellet left in the centrifuge tube

X. Han et al. / Desalination 329 (2013) 35–40

after extracting LB-EPS to maintain the amine group in non-protonated form. The mixture was poured in fluorescein-isothiocyanate (FITC) solution (1 mg/mL), and stirred at room temperature for 1 h to stain proteins. Subsequently, the concanavalin A (Con A) solution (0.25 mg/mL) was incubated with the sample for another 30 min to stain αpolysaccharides. After each staining stage, the sample was washed with phosphate-buffered saline (PBS, pH 7.2) to remove the excess dye. The component distribution of floc samples was examined with a CLSM (Leica TCS SP2 confocal spectral microscope imaging system, Germany). The excitation and emission wavelengths for FITC and Con A dye were 488 nm and 500–550 nm, 561 nm and 570–590 nm, respectively. The floc was imaged with a 20× objective and analyzed with the Leica confocal software. 3. Results and discussion 3.1. Effect of ultrasonication power density on LB-EPS extraction In order to figure out the detailed influences of power density on extracting LB-EPS, the following three aspects were investigated: (i) the changes of carbohydrates, proteins, humic acids and TOC, (ii) the variations of DNA concentration, (iii) CLSM observation of sludge after ultrasonication with different power densities. 3.1.1. Carbohydrates, proteins and humic acids in LB-EPS Carbohydrates (EPSc), proteins (EPSp), humic acids (EPSh) and TOC (EPST) were measured to evaluate floc disintegration during ultrasonic pretreatment. The variations of EPSc, EPSp, EPSh and EPST are shown in Fig. 1. It could be observed that with ultrasonication power density changing from 3.2 W/10 mL to 150 W/10 mL, LB-EPSc, LB-EPSp, LBEPSh and LB-EPST all increased steadily from 2.1 mg/g SS to 16.9 mg/g SS, 9.7 mg/g SS to 73.3 mg/g SS, 1.1 mg/g SS to 24.7 mg/g SS and 3.6 mg/g SS to 21.7 mg/g SS, respectively. However, there was no obvious change of TB-EPS concentrations, indicating that the variations of ultrasound power density of the first ultrasonication did not obviously

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influence the extraction of TB-EPS. Statistical analysis by employing SPSS software (SPSS V13.0, USA) demonstrated that ultrasonication power density had a significant positive correlation with LB-EPSc, LBEPSp, LB-EPSh, and LB-EPST with Pearson's correlation coefficient 0.987, 0.975, 0.986 and 0.971 (P b 0.01), respectively, and in turn, influenced LB-EPS extraction and sludge floc disintegration. In addition, the strong linear relationship between the respective LB-EPS components (LB-EPSc, LB-EPSp, LB-EPSh, LB-EPST) and power density is shown in Fig. 1. The findings correspond to Zhang's report [26] that the supernatant soluble chemical oxygen demand (SCOD) increases with the increase of power density. It has been also reported that ultrasonication is an effective method to detach fouling layer from membrane surfaces. Kobayashi [27] investigated the influence of power intensity on polymeric membrane cleaning, and the results showed that the increase of ultrasonic power intensity could enhance permeability recovery due to the formation of more cavitation bubbles and the enlargement of cavitating zone, which is in accordance with our research. However, according to Wen's report [28], scanning electron microscope (SEM) of the membrane surface showed that the polyethylene membrane was somehow damaged when power intensity was increased from 0.122 W/cm2 to 0.203 W/cm2. Therefore, it can be inferred that an increase in power intensity can provide a more effective cleaning of membrane EPS-fouling while extremely high power intensity will affect membrane surface negatively. 3.1.2. DNA in LB-EPS Although some researchers have studied DNA variations in different solution conditions [29] or extraction methods [19], the information on the correlations of DNA concentration in bound EPS with ultrasonication power density, especially high power density, is limited. Hence, in this study, the DNA concentration alteration in LB-EPS under various ultrasonication power density was specified. A cubic regression analysis with acceptable deviation based on the DNA concentration data is shown in Fig. 2. It is obvious that compared to the steady increase of LB-EPSc, LB-EPSp, LB-EPSh and LB-EPST in Fig. 1, no significant changes

Fig. 1. Dependence of LB-EPS component extraction on power density for (a) EPSc, (b) EPSp, (c) EPSh and (d) TOC.

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Fig. 2. Changes in the LB-EPS DNA concentration as a function of the ultrasonication power density.

of DNA concentrations could be observed when ultrasonication power density was below 35 W/10 mL. Nevertheless, the non-linear regression curve in Fig. 2 showed that DNA concentration ascended suddenly when power density was 35 W/10 mL, which might be due to the fact that cell lysis occurred to a large extent at this point. It is believed that floc disintegration and cell lysis take place at the same time and there is dissociated DNA from dead microorganism outside the cells. Therefore, under low power density there was still a little amount of DNA. However, unlike floc disintegration, cell lysis did not obviously occur judging from the evolution of DNA with extraction power density not higher than 35 W/10 mL. It can be inferred that there is a critical power density, below which the floc disintegration dominates while above which cell membrane cannot keep intact and cell lysis occurs to a large extent. The data in Fig. 2 suggests that the critical power density occurs at approximately 35 W/10 mL based upon the departure from the initial values, which is related to cell membrane damaging under high ultrasonication power density. 3.1.3. CLSM results of fluorescence staining sludge In order to directly demonstrate the distribution of components left in sludge flocs, CLSM was applied after fluorescence staining. In previous studies, CLSM was usually employed to investigate structural characteristics of activated sludge floc or granular sludge [30,31], and biofouling layer on the membrane [32]. In our study, the method was used to image the stained pellet after extracting LB-EPS and to calculate the percentage of specific color in the whole area, which represented the amount of different components in bioaggregates. Fig. 3 shows the CLSM images of the stained sludge flocs with a 20× objective, in which green dots represent proteins stained by FITC and red dots represent α-polysaccharides stained by Con A. Green and red areas both decreased with the increase of ultrasonication power density, indicating that both proteins and carbohydrates around cells were extracted with the increase of ultrasonication power density. Based on the fluorescent intensity presented in this figure, a detailed analysis of proteins and carbohydrates is given in Table 2. It is evident that carbohydrates and proteins were gradually reduced based on the variations of stained area percentage in CLSM images. In combination with the figures in Section 3.1.1, proteins and carbohydrates presented in bioaggregates decreased steadily when power density ranged from 3.2 W/10 mL to 150 W/10 mL, suggesting that ultrasonication was efficient for LB-EPS extraction. 3.2. Effect of ultrasonication power density on TB-EPS extraction In current literature, researchers usually focused on the effects of ultrasonication factors on bound EPS that were extracted by one-step ultrasonic treatment [10,26,33], while their specific influences on LB-EPS and TB-EPS have been not well documented. In order to specify the impact of ultrasonication power density on TB-EPS extraction, a two-step ultrasonic treatment was carried out in this study as stated in

Fig. 3. CLSM images of stained sludge flocs with different ultrasonication power densities. (a) without ultrasonication; (b) 3.2 W/10 mL power density; (c) 6.5 W/10 mL power density; (d) 13 W/10 mL power density; (e) 35 W/10 mL power density; (f) 65 W/ 10 mL power density; (g) 100 W/10 mL power density; (h) 150 W/10 mL power density.

Section 2.2. In Li's report [34], a thermal TB-EPS extraction method was employed and found to be sufficient to extract EPS. However, according to our research, ultrasonication followed by heating treatment was more sufficient than merely heating treatment or ultrasonication. For example, protein concentration in TB-EPS by only heating was less than 76% of that by combination of heating and ultrasonication. As illustrated in Fig. 4, there was no significant increase of TB-EPSc, TB-EPSp, TB-EPSh and TB-EPST as power density increased. It is quite

X. Han et al. / Desalination 329 (2013) 35–40

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Table 2 Variations of stained area percentage under different ultrasonication power densities. Power density

Carbohydrates (red area)

Proteins (green area)

0 W/10 mL 3.2 W/10 mL 6.5 W/10 mL 13 W/10 mL 35 W/10 mL 65 W/10 mL 100 W/10 mL 150 W/10 mL

17.6% 7.3% 4.8% 3.5% 1.5% 0.4% 0.8% 0.6%

23.1% 13.6% 8.6% 4.2% 2.4% 1.3% 1.1% 1.0%

different from the change of LB-EPS under different ultrasonication power density in Section 3.1.1, suggesting that power density did not evidently affect TB-EPS extraction. It should be noted that under extremely high power density, namely 100 W/10 mL and 150 W/10 mL, there was a little increase of TB-EPSc, TB-EPSp, TB-EPSh and TB-EPST. That might be attributed to the release of intracellular polymers during cell lysis under those power densities as illustrated in Fig. 5. In Fig. 5, it can be observed that there was no obvious change of DNA when the power density was below 65 W/10 mL. However, cell lysis happened with a dramatic increase of DNA from 4.8 mg/g SS to 19.8 mg/g SS when power density was shifted from 65 W/10 mL to 100 W/10 mL. It can be concluded that 65 W/10 mL was the critical ultrasonic power density for TB-EPS extraction. In combination with Figs. 1(a) and 4(a), it can be found that the total EPSc (the sum of LB-EPSc and TB-EPSc) was both 22 mg/g SS at the power density of 150 W/10 mL and 6.5 W/10 mL to respectively extract LB-EPS and TB-EPS in Fig. 1(a), and 6.5 W/10 mL and 150 W/10 mL to extract LB-EPS and TB-EPS in Fig. 4(a). It has been accepted that LB-EPS and TB-EPS have a dynamic double-layered structure surrounding the cells [15,34]. If low power intensity was applied in the first extraction step, LB-EPS concentration was low and TB-EPS concentration was high, and vice versa. Furthermore,

Fig. 5. Variations of DNA in TB-EPS under different ultrasonication power densities.

Dominguez's research [7] showed that total EPS quantities and EPS constituents were not the same using different extraction methods. The total quantity of LB-EPS and TB-EPS under thermal treatment was only 25% of that using resin extraction method. It indicates that extraction methods may also affect the quality and quantity of bound EPS such as LB-EPS and TB-EPS. Therefore, it is hard to define the strict boundary between them. Nevertheless, it should be noted that the critical power density of 65 W/10 mL is appropriate for TB-EPS extraction of sludge samples in the pilot-scale MBR reactor while it might be changed for other kinds of sludge. Similarly, the critical power density for LB-EPS extraction may be also dependent on the biomass concentration. In this study, the mixed liquor suspended solid (MLSS) concentrations of the samples were in the range of 6.8–8.0 g/L. If the MLSS concentrations significantly deviate from the above-mentioned value, the critical power density for EPS extraction may be varied accordingly, which needs further investigating.

Fig. 4. Variations of (a) EPSc, (b) EPSp, (c) EPSh and (d) TOC with different power densities for TB-EPS extraction.

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4. Conclusions The study revealed that the alteration of ultrasonication power density for bound EPS extraction affected the concentrations of carbohydrates, proteins, humic acids, TOC and DNA in bound EPS, which therefore reflected floc disintegration and cell lysis. Experimental results indicated that ultrasonication power density had a significant effect on LB-EPSc, LB-EPSp, LB-EPSh and LB-EPST and there was a well fitted linear regression relationship between them. CLSM images of stained pellet could well support the variations of the concentrations of carbohydrates and proteins in LB-EPS. According to DNA variations, the critical power density for LB-EPS extraction could be defined as 35 W/10 mL based on the departure from initial values of the cubic regression curve. Compared to the extraction of LB-EPS, concentrations of TB-EPSc, TB-EPSp, TB-EPSh and TB-EPST exhibited no obvious change with the increase of power density until a very high power density was applied. Cell lysis happened with a dramatic increase of DNA when power density was shifted from 65 W/10 mL to 100 W/10 mL, indicating that 65 W/10 mL was the critical power density for TB-EPS extraction. Acknowledgments We thank the National Hi-Technology Development 863 Program of China (2012AA063403), the STCSM research project (11231200400), and the National Science & Technology Pillar Program (2012BAJ21B05) for partial support of this study. References [1] Z.W. Wang, Z.C. Wu, S.J. Tang, Characterization of dissolved organic matter in a submerged membrane bioreactor by using three-dimensional excitation and emission matrix fluorescence spectroscopy, Water Res. 43 (2009) 1533–1540. [2] H. Nagaoka, H. Akoh, Decomposition of EPS on the membrane surface and its influence on the fouling mechanism in MBRs, Desalination 231 (2008) 150–155. [3] S. Lee, M.H. Kim, Fouling characteristics in pure oxygen MBR process according to MLSS concentrations and COD loadings, J. Membr. Sci. 428 (2013) 323–330. [4] Z.W. Wang, Z.C. Wu, S.J. Tang, Extracellular polymeric substances (EPS) properties and their effects on membrane fouling in a submerged membrane bioreactor, Water Res. 43 (2009) 2504–2512. [5] C.S. Laspidou, B.E. Rittmann, A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass, Water Res. 36 (2002) 2711–2720. [6] M.M.T. Khana, S. Takizawab, Z. Lewandowskic, M.H. Rahmand, K. Komatsue, S.E. Nelsonc, F. Kurisub, A.K. Camperc, H. Katayamab, S. Ohgakie, Combined effects of EPS and HRT enhanced biofouling on a submerged and hybrid PAC-MF membrane bioreactor, Water Res. 47 (2013) 747–757. [7] L. Dominguez, M. Rodriguez, D. Prats, Effect of different extraction methods on bound EPS from MBR sludges. Part I: influence of extraction methods over three-dimensional EEM fluorescence spectroscopy fingerprint, Desalination 261 (2010) 19–26. [8] B.M. Lee, H.S. Shin, J. Hur, Comparison of the characteristics of extracellular polymeric substances for two different extraction methods and sludge formation conditions, Chemosphere 90 (2013) 237–244. [9] Y.J. Liu, Z. Liu, A.N. Zhang, Y.P. Chen, X.C. Wang, The role of EPS concentration on membrane fouling control: comparison analysis of hybrid membrane bioreactor and conventional membrane bioreactor, Desalination 305 (2012) 38–43.

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