Environ. Sci. Technol. 2010, 44, 4355–4360
Contribution of Extracellular Polymeric Substances (EPS) to the Sludge Aggregation X I A O - M E N G L I U , † G U O - P I N G S H E N G , * ,† HONG-WEI LUO,† FENG ZHANG,† SHI-JIE YUAN,† JUAN XU,† RAYMOND J. ZENG,† JIAN-GUANG WU,‡ A N D H A N - Q I N G Y U * ,† Department of Chemistry, University of Science & Technology of China, Hefei, 230026 China, and Anhui Academy of Environmental Science Research, Hefei, 230061 China
Received June 8, 2009. Revised manuscript received April 16, 2010. Accepted April 24, 2010.
The contribution of extracellular polymeric substances (EPS), including loosely bound EPS (LB-EPS) and tightly bound EPS (TBEPS), to the aggregation of both aerobic and anaerobic sludge is explored using the extended DLVO theory. It is observed that the aggregation abilities of both sludge samples decrease with the extraction of LB-EPS and TB-EPS, implying the crucial roles of EPS in sludge aggregation. Furthermore, through analyzing the interaction energy curves of sludge before and after the EPS extraction using the extended DLVO theory, it is found that both LB-EPS and TB-EPS have a substantial contribution to the sludge aggregation. The interaction energy of LB-EPS is always negative, suggesting that the LB-EPS always display a positive effect on the sludge aggregation. On the other hand, the interaction energy of TB-EPS is not always negative, depending on the separation distance between sludge cells. These results imply that the LB-EPS and TBEPS have different contributions to the sludge aggregation.
Introduction In biological wastewater treatment systems, the sludge aggregation is essential for the solid/liquid separation. Poor sludge aggregation leads to an increase in the effluent turbidity. The sludge aggregation depends mainly on the sludge structure. Sludge usually has loose structure and the cells are glued together by a matrix of extracellular polymeric substances (EPS) (1, 2). Sludge cells in the floc matrix have a double layered EPS structure: loosely bound EPS (LB-EPS) diffused from the tightly bound EPS (TB-EPS) surrounding the cells (1, 3). EPS play an important role in maintaining the sludge floc structure and functions (4, 5). The sludge cells in the outer region of flocs are entangled by weak interactions through LB-EPS. The TB-EPS are reported to be responsible for cell adhesion and attachment in the inner floc structure through strong interactions (1). The quantity and properties of EPS are therefore recognized to be crucial to the sludge aggregation (6, 7). Removal of EPS from sludge would lead to worse aggregation (6). * Address correspondence to either author. Fax: +86 551 3601592 (G.-P.S.); +86 551 3601592 (H.-Q.Y.). E-mail:
[email protected] (G.-P.S.);
[email protected] (H.-Q.Y.). † University of Science & Technology of China. ‡ Anhui Academy of Environmental Science Research. 10.1021/es9016766
2010 American Chemical Society
Published on Web 05/06/2010
To access the chemical compositions and functions of EPS extracted from various microorganisms, new techniques have been developed to qualitatively and quantitatively analyze the EPS. The chromatography-mass spectrometry combination technique has been used to determine the monosaccharide and amide acid components (8). The X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, and nuclear magnetic resonance have also been utilized to elucidate the functional groups and element compositions in EPS (9, 10). With these analytical methods, more structure information about EPS has become available. However, the precise roles of EPS in sludge aggregation are not clear yet, and the results are inconsistent (11, 12). Some studies have demonstrated that the proportion of LBEPS and TB-EPS, rather than their quantities, is more important in sludge aggregation (13). The excessive LB-EPS in the outer region might deteriorate sludge aggregation, while it was not true for the case of the TB-EPS (14). The complex composition of EPS makes it difficult to explore their roles in the sludge aggregation, and thus, the contribution of EPS has not been quantified. The aggregation of sludge is primarily governed by the interactions between sludge cells, which could be described by the DLVO theories (15). The classical DLVO theory was proposed to describe the stability of colloidal suspensions. It involves the estimation of the energy due to the overlap of electric double layers (usually repulsion) and the van der Waals energy (usually attraction) in terms of interparticle distance. In addition to the van der Waals attractive and the electrostatic repulsive interaction, the Lewis acid-base interactions are also taken into account in the extended DLVO theory (16-18). According to this theory, sludge cells adhere to each other as they approach sufficiently close to be attracted and to be pulled into a deep energy minimum. Although both LB-EPS and TB-EPS have a significant impact on the interaction energy (19), the contribution of the EPS to the sludge aggregation has not been well elucidated by the extended DLVO theory (20, 21). Here, a new approach based on the extended DLVO theory for quantifying the contribution of EPS and their subfractions in sludge aggregation is proposed. The change of sludge aggregation abilities after the LB-EPS and TB-EPS extractions were evaluated. In addition, the surface thermodynamic approach was used to calculate the interaction energy terms in the extended DLVO theory (22), and then the LB-EPS and TB-EPS interactions are quantified. In this way, the crucial roles of LB-EPS and TB-EPS in the sludge aggregation are elucidated. To the best of our knowledge, this might be the first attempt to qualitatively evaluate the contribution EPS and their subfractions to the sludge aggregation.
Materials and Methods Sludge. The activated sludge was collected from the aeration tank at the Wangxiaoying municipal wastewater treatment plant in Hefei, China, whereas the anaerobic methanogenic sludge was sampled from a pilot-scale upflow anaerobic sludge blanket reactor treating soybean-processing wastewater in Bengbu, China. Before use, the sludge samples were passed through 0.4-mm sieves and then washed with tap water twice to remove the residual components and dispersed small particles. EPS Extraction and Chemical Analysis. The LB-EPS and TB-EPS of both aerobic and anaerobic sludge samples were extracted using a mild method and a harsh method in sequence, that is, the oscillation-ultrasound method followed VOL. 44, NO. 11, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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by the cation exchange resin (CER) technique (14, 23). For sludge samples, the flocs were harvested by centrifugation at 550g for 15 min, and then the pellet was washed twice with 0.1 mol L-1 NaCl solution. After that, the sludge pellet was resuspended to a predetermined volume and the solution was transferred to an extraction beaker with some glass beads. An ultrasound was performed first on the suspension for 2 min, followed by the horizontal oscillation for 10 min, and then rthe ultrasound was repeated a second time for 2 min. Thereafter, the LB-EPS were harvested by centrifugation at 9000g and 4 °C for 15 min. The residual sludge pellets were resuspended to a predetermined volume and the solution was transferred to an extraction beaker to extract the TBEPS. Afterward, CER was added and the suspensions were stirred for 12 h at 4 °C. After the CER was removed by settlement, the TB-EPS were harvested by centrifugation at 9000g and 4 °C for 30 min to remove remaining sludge components. The supernatants were used as the TB-EPS fraction. The LB-EPS and TB-EPS solutions were then filtrated through 0.45-µm acetate cellulose membranes and used as the EPS fraction for chemical analysis. The Zeta potential and zero point of charge of each sample were recorded using a Zetasizer Nano ZS Instrument (Malvern Co., U.K.) at 25 °C. The surface thermodynamic properties of sludge, such as the surface free energy, were estimated using a contact angle approach, as described in the Supporting Information. All chemicals used in this work were of analytical grade. The total EPS content was defined as the sum of carbohydrates, proteins, and humic substances. Their contents were determined as described previously (23). The suspended solid (SS) of the sludge was measured following the Standard Methods (24). The detailed method of FTIR and XPS measurements for EPS structure analysis were provided in the Supporting Information. The Extended DLVO Theory. The total energy of the interaction (Wtot) in the extended DLVO theory can be obtained through the summation of electric double layer (WR), van der Waals energies (WA), and the acid-base interaction (WAB): Wtot ) WR + WA + WAB. The total interaction energies were expressed as a function of the separation distance H. The calculation of the terms of WR, WA, WAB in the extended DLVO theory was based on the surface thermodynamic calculation approach (15). The detailed calculation for the terms in the extended DLVO theory could be found in the Supporting Information. Aggregation Tests. The aggregation kinetics was used to characterize the bacterial aggregation. The aggregation rate constant k was used to evaluate the sludge aggregation (18, 25, 26). A greater value indicates a better aggregation ability of sludge. Sludge samples were harvested by centrifugation at 9000g for 10 min and washed twice with 0.1 mol L-1 NaCl solution. The pellets were then resuspended in NaCl solution with various concentrations for the aggregation kinetic test, which was conducted with a 2-h temporal optical density measurement. The value of k can be determined from the initial slop of the 2-h temporal optical density variation curve of suspension at 650 nm. Calculation of the Contributions of LB-EPS and TBEPS. After the LB-EPS and TB-EPS extraction, the contributions of LB-EPS and TB-EPS to the interaction between sludge cells disappeared step by step, which caused a significant change in the sludge surface characteristics. Thus, the total interaction energies between sludge cells were assumed to be attributed to the contributions of the sludge cells, the LB-EPS, and the TB-EPS at the corresponding stages. Thus, it was assumed that Wtot could be expressed as the sum of the interaction energy of the sludge after the TB-EPS aTB LB-EPS extraction W tot , and the contributions of LB-EPS W pi TB-EPS and TB-EPS W pi : 4356
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aTB LB-EPS TB-EPS + W pi + W pi Wtot ) W tot
(3)
Thus, the contributions of LB-EPS and TB-EPS to the interaction energy between sludge flocs can be estimated from the extended DLVO theory and expressed as follows: LB-EPS aLB W pi ) Wtot - W tot
(4)
LB-EPS aLB aTB W pi ) W tot - W tot
(5)
aLB is the interaction energy of the sludge after the where W tot LB-EPS extraction, and can be calculated from the extended DLVO theory based on the surface thermodynamic approach. In this way, the contributions of LB-EPS and TB-EPS onto the interaction energy between sludge flocs could be addressed as a function of the separation distance H.
Results Characteristics of LB-EPS and TB-EPS Extracted from Aerobic and Anaerobic Sludge. The LB-EPS and TB-EPS contents extracted from both aerobic and anaerobic sludge are summarized in Table 1. For the two sludge samples, carbohydrates and proteins were found to be the main components in both LB-EPS and TB-EPS. Compared with the LB-EPS, the carbohydrate and protein concentrations were higher in the TB-EPS solutions. Hence, the total amount of the TB-EPS was greater than that of the LB-EPS for both sludge samples. The humic substance concentration was lower in the EPS extracted from the aerobic sludge, only 1.0 mg g-1 SS for LB-EPS and 0.4 mg g-1 SS for the TB-EPS, respectively. However, the humic substances were the main components in the LB-EPS and the TB-EPS extracted from the anaerobic sludge, with corresponding contents of 3.6 and 8.4 mg g-1 SS, respectively. The zero point values of charge of each EPS sample were in a range of 2.0-2.6 (Table 1). This indicated that all the EPS samples were negatively charged under neutral conditions. To elucidate the chemical structures of the two EPS fractions, FTIR and XPS of the LBand TB-EPS extracted from aerobic and anaerobic sludge were measured. The detailed results were presented in the Supporting Information. Changes of Surface Characteristics and Aggregation of Sludge before and after the EPS Extraction. After EPS extraction, the sludge surface characteristics, either in the aerobic sludge or in the anaerobic sludge, changed significantly. The changes in the contact angles of sludge before and after the EPS extraction are shown in Table 2. The contact angles of both sludge measured before and after the LB-EPS and TB-EPS extractions also changed significantly. The zeta potentials of sludge before and after EPS at various pH and ionic strengths are shown in Figure 1. The sludge zeta potential changed slightly after the EPS extraction. With the increase in pH, the zeta potentials of both sludge became more negative. However, it became less negative with the increase of ionic strength. Subsequently, to identify the alteration of sludge aggregation, the sludge samples at different extraction stages, including initial sludge, sludge after the LB-EPS extraction, and finial residual sludge, were resuspended in NaCl solutions with various concentrations, and then the aggregation tests were conducted. Results show that the initial sludge before the EPS extraction had the best aggregation ability. The dispersed sludge cells reflocculated rapidly. After the LBEPS extraction, the aggregation of sludge decreased slightly. However, after the TB-EPS extraction, the dispersed sludge became relatively stable and was not able to reflocculate. As shown in Table 3, the aggregation rate constant of sludge decreased subsequently after LB- and TB-EPS extraction, implying that the sludge before and after the EPS extraction
TABLE 1. Contents and Zero Point of Charges of LB-EPS and TB-EPS Extracted from Aerobic and Anaerobic Sludge aerobic sludge -1
carbohydrates (mg g SS) proteins (mg g-1 SS) humic substances (mg g-1 SS) total EPS (mg g-1 SS) zero point of charge
anaerobic sludge
LB-EPS
TB-EPS
LB-EPS
TB-EPS
3.3 ( 0.4 2.6 ( 0.4 1.0 ( 0.1 6.9 ( 0.9 2.55
10.6 ( 0.3 11.4 ( 0.5 0.4 ( 0.1 22.4 ( 0.9 2.20
4.6 ( 0.7 6.0 ( 0.4 3.6 ( 0.4 14.2 ( 1.5 2.02
10.7 ( 0.7 16.6 ( 0.7 8.4 ( 0.8 35.7 ( 1.2 2.00
TABLE 2. Change of the Contact Angles of Sludge before and after EPS Extraction aerobic sludge
water 1-bromonaphthalene formamide
anaerobic sludge
initial
after LB-EPS extraction
after TB-EPS extraction
initial
after LB-EPS extraction
after TB-EPS extraction
65.0 ( 2.3 39.0 ( 3.4 63.5 ( 2.5
61.0 ( 3.3 41.0 ( 2.6 66 ( 3.7
46.0 ( 2.1 51.0 ( 1.9 72.0 ( 2.8
58.0 ( 1.6 41.0 ( 2.3 64.0 ( 1.5
54.0 ( 1.5 46.0 ( 1.4 57.0 ( 2.2
30.0 ( 0.9 55.0 ( 1.8 35.0 ( 1.3
could be reflocculated after dispersed, but that the reflocculated rate decreased significantly after the EPS extraction. The solution ionic strength had significant effects on the aggregate kinetics of sludge both before and after EPS extraction. With the increase of the ionic strength of sludge solution, the aggregation rate constant of sludge increased. This might be due to fact that the repulsive force between sludge cells was reduced by the increasing ionic strength. Dose of LB- or TB-EPS to the sludge solution after the EPS extraction could induce the aggregation of sludge. The sludge aggregation ability was recovered by above 80%, indicating
that the EPS had the same properties as bioflocculants and could play a key role in sludge aggregation. Total Interaction Energy of Sludge before and after the EPS Extraction. The reduced aggregation of sludge after the extraction of LB-EPS and TB-EPS could be also predicted from the interaction energy between sludge cells before and after the EPS extraction. The interaction energies calculated from the extended DLVO theory are shown in Figure 2 for the aerobic and anaerobic sludge. The calculation was performed at 0.1 mol L-1 ionic strength for example. The interaction energy profiles of both sludge samples before
FIGURE 1. The zeta potentials of sludge before and after EPS extraction versus pH and ionic strength: (a and b) aerobic sludge; (c and d) anaerobic sludge.
TABLE 3. The Aggregation Rate Constants (s-1) of Aerobic and Anaerobic Sludge aerobic sludge
anaerobic sludge
ionic strength (mol L-1)
Initial
after LB-EPS extraction
after TB-EPS extraction
initial
after LB-EPS extraction
after TB-EPS extraction
0.001 0.01 0.1
0.008 0.018 0.027
0.005 0.011 0.021
0.0003 0.0019 0.005
0.001 0.0025 0.006
0.00076 0.002 0.002
0.00011 0.00019 0.00059
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FIGURE 2. Total interaction energy curves of (a) aerobic and (b) anaerobic sludge before and after EPS extraction. and after the EPS extraction match the results from the aggregation dynamic tests. For the aerobic sludge, the energy barrier increased subsequently after the LB-EPS extraction and the TB-EPS extraction (Figure 2). A higher energy barrier means a more stable sludge suspension. The dispersed sludge cells in suspension should have sufficient kinetic energy to overcome this barrier to reflocculate. In addition, the secondary energy minimum in the interaction energy profiles represents the dispersible ability of sludge cells, that is, the ability of sludge cell desorption from sludge surface (27). Loose and unstable flocs could be formed when the separation distance was in this range, as shown in the inset of Figure 2, which could be deflocculated by the external force readily. Therefore, a higher potential well of the secondary energy minimum value indicates that more energy was needed to disperse the sludge cells, and thus, the sludge structure was more stable. As shown in Figure 2a, the secondary energy minimum value corresponding to the initial aerobic sludge was approximately -50 KT, but changed into -40 KT after the LB-EPS extraction. Finally, the sludge after the TB-EPS extraction had a potential well of -20 KT only. The increasing value of the secondary energy minimum in the interaction energy profiles of sludge after the EPS extraction also suggests that the aggregation of sludge decreased after the EPS extraction.
Discussion The experimental and calculation results above show that the interaction energies of sludge before and after the EPS extraction changed significantly. These results also demonstrate the crucial roles of EPS in the aggregation of both aerobic and anaerobic sludge. Contribution of LB-EPS. As shown in Figure 3, the LBLB-EPS ) for both aerobic and EPS interaction energy values (W pi anaerobic sludge were always negative when the separation distance was less than 20 nm, indicating that the role of LB-EPS was attractive. The attractive interaction energy decreased from about -60 KT to nearly -5 KT with an increase in the separation distances H. Therefore, the total interaction energies (Wtot) and the potential well of the secondary energy minimum value decreased with the removal of the LB-EPS 4358
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FIGURE 3. LB-EPS interaction energy curves as a function of separation distance H for (a) aerobic sludge and (b) anaerobic sludge. from the sludge surface. This was also confirmed by the aggregation dynamic test results (Table 3). The attractive LB-EPS interaction energy values for the aerobic sludge decreased more substantially than for the anaerobic sludge (Figure 3). The contribution of the LB-EPS to the total interaction energy depended on the separation distance H. For the anaerobic sludge, at a separation distance H of about 3.4 aLB LB-EPS , and W tot ) nm, the total interaction energy Wtot ) W pi 0, suggesting that the interaction between sludge came entirely from the LB-EPS contribution (Figure 3b). At a LB-EPS aLB ) 2W tot , separation distance H of 4.3 nm, Wtot ) 2W pi in which the LB-EPS and their inner cells in sludge had the same levels of contribution to the total interaction energy. Contribution of TB-EPS. Different from the LB-EPS, the TB-EPS were located in the inner region surrounding the sludge cells. The contribution of the TB-EPS to the interaction energy was different from that of the LB-EPS (Figure 4). When the sludge cells approached sufficiently closely, the TB-EPS had a repulsive contribution to the total interaction energy for both aerobic and anaerobic sludge. A higher rigidity of the polymers in the short-distance might result in a steric effect, which would be responsible for the TB-EPS repulsion (28). There was an energy barrier in the TB-EPS interaction energy curves, indicating a relative high repulsive contribution of TB-EPS (Figure 4). This suggests that the TB-EPS had a negative effect on the total interaction energy when the sludge cell separation distance fell in the range above. With a gradual increase in the separation distance H, the TB-EPS displayed an attractive contribution to the total interaction energy (Figure 4). The TB-EPS attraction increased and the potential well peaked at -27.8 and -22.5 KT for the aerobic and anaerobic sludge, respectively. Then, the attractive contribution of the TB-EPS decreased with a further increase in H. Thereafter, with a further decrease in H, the TB-EPS redisplayed an attractive interaction.
FIGURE 4. TB-EPS interaction energy curves as a function of separation distance H for (a) aerobic sludge and (b) anaerobic sludge.
FIGURE 5. Interaction energy curves of EPS extracted from (a) aerobic sludge and (b) anaerobic sludge.
Comparison of the Roles of EPS in the Aggregation of Aerobic and Anaerobic Sludge. In this work, it was assumed that the total EPS contribution was the sum of the LB-EPS and the TB-EPS interactions, as shown in Figure 5. It was found that either for the aerobic or anaerobic sludge, the total interaction energy curves were similar to their corresponding TB-EPS interaction curves. For the aerobic sludge, with an increase in distance H, the total EPS interaction was initially repulsive and reached a maximum value. With a further increase in H, the total interaction decreased and changed to be attraction for both sludge samples. However, for the anaerobic sludge, the total contribution of EPS was attractive. These results indicate that the roles of EPS in sludge aggregation also depended on the sludge characteristics. Significance of This Work. EPS are found to crucially affect the properties of sludge in biological wastewater treatment reactors, such as mass transfer, surface characteristics, adsorption ability, shear stability, and the formation of sludge flocs (1). However, the roles of EPS in these sludge characteristics are not clear and the reports about such roles are not consistent. In the present work, based on the determination of the aggregation of sludge before and after the EPS extraction, and the subsequent calculation of the interaction energy of EPS using the extended DLVO theory, the roles of EPS in sludge aggregation could be effectively evaluated. Although some idealized assumptions were made and simplified considerations were given, it is still helpful to elucidate the crucial roles of the LB-EPS and the TB-EPS in sludge aggregation, and is thus useful to clarify the inconsistent results of roles of EPS in sludge aggregation reported in previous studies. The EPS subfractions have different effects on sludge aggregation attributed to their characteristics. Furthermore, understanding of such roles of EPS and their subfractions may provide a new way to improve the sludge aggregation through manipulating the contents of EPS under various operating conditions. Since the sludge characteristics are heavily dependent on the interaction between EPS and
sludge cells, the approach proposed in this work might also be used to evaluate the effects of EPS on the sludge physicochemical characteristics, in addition aggregation.
Acknowledgments Authors wish to thank the Natural Science Foundation of China (50625825, 50708106 and 50978243) and Foundation for the Author of the Outstanding PhD Thesis of China for the partial support of this study.
Supporting Information Available Zeta potential measurement; surface thermodynamics analysis; extended DLVO theory; FTIR; the FTIR spectra of the LBand TB-EPS extracted from aerobic and anaerobic sludge; XPS; XPS wide survey scans of the LB- and TB-EPS extracted from aerobic and anaerobic sludge; high resolution C, O, and N 1 s XPS spectra of the LB-EPS and the TB-EPS extracted from aerobic and sludge; the O, N and C atomic ratio and the relative areas of various functional groups. This material is available free of charge via the Internet at http:// pubs.acs.org.
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