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Influence of extracellular polymeric substances on rheological properties of activated sludge
Biochemical Engineering Journal 77 (2013) 208–213
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Influence of extracellular polymeric substances on rheological properties of activated sludge Dongqin Yuan, Yili Wang ∗ College of Environmental Science and Engineering, Beijing Key Lab for Source Control Technology of Water Pollution, Beijing Forestry University, Beijing 100083, China
a r t i c l e
i n f o
Article history: Received 8 March 2013 Received in revised form 24 May 2013 Accepted 16 June 2013 Available online 24 June 2013 Keywords: Waste-water treatment Activated sludge Extracellular polymeric substances Rheology Non-Newtonian fluids Viscosity
a b s t r a c t Influence of extracellular polymeric substances (EPS) on rheological properties of activated sludge (AS) was conducted by comparing properties of AS before and after EPS extraction at a TSS of 54 g/L. Slime, loosely and tightly bound EPS (LB- and TB-EPS) were stratified by centrifugation and ultrasound method. The results showed that sludge after LB-EPS extraction produced a higher hysteresis loop area (Hla), limiting viscosity (∞ ), yield stress ( y ), energy of cohesion of network sludge (Ec ), and storage modulus (G 0 ), than three other sludge samples, indicating that sludge with TB-EPS exhibited stronger network structure. Strain amplitude sweep (SAS) and frequency sweep (FS) tests revealed that AS before and after EPS extraction produced a gel-like structure in the linear viscoelastic (LVE) domain. TB-EPS appeared to have a positive effect on the gel-like structure of AS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Extracellular polymeric substances (EPS) are large molecular weight components of activated sludge (AS) flocs originating from bacteria secretion, cell lysis and hydrolysis, leakage of exocellular constituents, and absorbed organic matter from wastewater [1,2]. EPS are considered to be of great importance in understanding the structure, function, properties and development of microbial flocs/aggregates [3]. The presence of EPS alters the hydrophobic/hydrophilic characteristics of sludge, charge density and other surface properties [4]. They also entrap water and play an important role in sludge dewatering [4]. In recent years, stratification of EPS is of considerable interest. Yu et al. [5] empolyed centrifugation and ultrasound method to stratify EPS into slime, loosely and tightly bound EPS (LB- and TB-EPS). Wastewater sludge is a non-Newtonian fluid, which possesses both viscous and elastic properties, also called viscoelatic (VE) properties. These VE properties can often be described by rheological models and analyses [6]. Rheology is a powerful tool because it can scientifically describe and predict deformation and flow behaviors in real processes [7]. Understanding rheological properties of sludge is not only important for choosing parameters concerning storage, transportation, and landfill, but also crucial for stabilization
∗ Corresponding author. Tel.: +86 10 62336528; fax: +86 10 62336596. E-mail address: [email protected] (Y. Wang). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.06.011
and dewatering [8]. Rheology can generally characterize the network strength of sludge [9]. The network structure of sludge flocs formed by three-dimensional, gel-like, highly hydrated EPS with multivalent cations and other particulate materials [10], is of significance in maintaining the structural and functional integrity of flocs/aggregates [11] and in restricting water mobility [12]. Hence, to improve the dewatering efficiency, weakening or destroying the network structure of AS might be a possible solution. Effects of EPS concentrations on rheological properties of sludge flocs [1,4,13] and studies on rheological properties of EPS [14–16] have been reported. However, research focusing on the effects of different EPS fractions on rheological properties of AS is scare. This study explores the effects of different EPS fractions on rheological properties of AS by comparing properties of AS before and after EPS extraction. We wish to determine a specific EPS fraction, which mainly affects the network structure of AS. This EPS fraction might have a marked effect on sludge dewatering and drying processes. Hence, these results will provide further information on the influence of EPS on the dewatering properties of AS. 2. Materials and methods 2.1. Characteristics of AS AS was collected from a municipal wastewater treatment plant (WWTP) in Beijing, China. The WWTP treats 6.0 × 105 m3 /day of wastewater using an anaerobic-anoxic-oxic (A2 O) process.
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
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Table 1 Characteristics of AS. pH
Conductivity (mS/cm)
TSS (g/L)
VSS (g/L)
VSS/TSS (%)
COD (mg/L)
SCOD (mg/L)
Zeta potential (mV)
7.13 ± 0.02
1.45 ± 0.01
8.18 ± 0.78
5.64 ± 0.52
69.00 ± 0.30
6630.0 ± 32.5
81.6 ± 7.8
−18.9 ± 1.1
Collected sludge samples were transferred to the laboratory within 2 h and immediately screened through a 1.2 mm sieve. Filtered samples were then stored at 4 ◦ C. Table 1 presents the main characteristics of the AS sample. The pH was measured using a pH meter (PB-10, Sartorius Stedim Biotech Co., Ltd., Beijing, China). Conductivity was measured using a conductivity meter (EC215, Beijing Kanggaote Science and Technology Co., Ltd., China). Total suspended solids (TSS, g/L) and volatile suspended solids (VSS, g/L) were assessed from the weight loss of suspended sludge samples dried at specific temperatures and durations according to the standard method [17]. To obtain a consistent TSS concentration around 54 g/L from sludge samples for rheological testing, sludge samples resuspended after EPS extraction were first centrifuged at 1000 × g for 10 min, after which the bulk solution and solid phase were obtained separately. Collected sediments were successively diluted with supernatant. Chemical oxygen demand (COD) of the filtrate is referred to as soluble COD (SCOD). COD and SCOD analyses were conducted using a COD expedited testing apparatus (HATO CTL-12, Huatong Environmental Protecting Instruments Co., Ltd., Chengde, China). Zeta potential was recorded by a Malvern Zetasizer instrument (Nano Z, Malvern Co., UK). All tests were conducted within a week. All chemical analyses were carried out in triplicate using chemicals of analytical grade. 2.2. Extraction and analytical methods of different EPS fractions Slime, LB- and TB-EPS of AS were extracted by centrifugation and ultrasound method [5]. Slime was obtained from the supernatant after low-speed centrifugation. LB- and TB-EPS were dissolved in a buffer solution (pH 7) containing Na3 PO4 , NaH2 PO4 , NaCl, and KCl at a molar ratio of 2:4:9:1. conductivities of buffers were adjusted with distilled water to match those of the filtrated sludge samples presented in Table 1. Membranes (0.45 m) were used to filter out the particulates present in slime, LB- and TB-EPS solutions after all EPS fractions had been extracted. Proteins (PN) and humic-like substances (HS) of different EPS fractions were measured by the modified Lowry method using bovine serum albumin (Beijing Aoboxing Biotechnology Co., Ltd., China) and humic acid (Sigma, America) as standards, respectively. Polysaccharides (PS) and DNA were determined by the anthrone method, using glucose as the standard, and the diphenylamine colorimetric method, using 2-deoxy-d-ribose (Beijing Ruibo Biotechnology Co., Ltd., China) as the standard, respectively. All chemicals used were of analytical grade, and all tests were performed in triplicate. 2.3. Rheological testing Rheological tests were performed using a rheometer (Physica MCR 300, Anton Paar, Austria) in conjunction with US 200 software which recorded the rheology data. Temperature was maintained at 25 ◦ C by a Peltier control. A PP 50 plate and plate sensor with 49.94 mm diameter and 2.0 mm gap was used. Measurements were initially carried out for the first rheological testing mode in steady flow: (1) increasing shear rate from 0.1 to 1000 s−1 in a logarithm manner; (2) maintaining constant shear rate at 1000 s−1 in 30 s; (3) decreasing shear rate in a logarithm manner from 1000 to 0.1 s−1 . Rheograms of shear stress () as a function of shear rate () ˙ were recorded and analyzed for raw
AS and sludge after EPS extraction. In general, 30 s is sufficient to ensure that the equilibrium point of the stationary state can be reached [18,19]; while during this controlled shear rate (CSR) test, a shorter time span of 5 s for AS response was employed to indicate “network strength” of the sludge at each shear rate [7]. Based on these rheograms, the parameter hysteresis loop area (Hla) can be calculated as follows [9]:
Hla =
t
· d(t) ˙
(1)
0
where (Pa) is the shear stress and ˙ (s−1 ) is the shear rate. The second mode was a type of transient rheological test-strain amplitude sweep (SAS) test, in which strain amplitude varied in a sinusoidal manner over time while the frequency remained fixed at 1 Hz. This test was carried out to exploit the linear viscoelastic (LVE) domain. The rheogram of the moduli (storage modulus G , loss modulus G , complex modulus G*) as a function of strain on the logarithmic coordinates can present that rheological parameters t (including the above moduli) are independent of strain until a critical strain level ( c ), above which the linear domain is reached. Beyond this value, the G* or G values decline as the material loses its structural integrity [7,20,21]. Subsequently, its nonlinear behavior appears, and the transition can be used to determine c and the critical storage modulus (G 0 ). G 0 represents the critical elasticity of the sludge network. The product between G* on the plateau and the critical deformation ( c ) directly provides the value of the yield stress: y = G∗ c . In addition to leaving the sample in a more intact state, the dynamic mechanical test is also advantageous in its simplicity and independence from any model assumptions [7,18,20]. Provided the values of y and c determined from the aforementioned SAS test, the corresponding value of Ec , is also equal to the half product of y and c [19,22]. After determing the LVE range by SAS test, frequency sweep (FS) test was conducted at 0.1% of the strain value. Corresponding G and G values were measured. Rheological tests were performed in duplicate. 3. Results and discussion 3.1. Characteristics of different EPS fractions in AS Characteristics of different EPS fractions in AS are displayed in Table 2. The sum of DNA content from slime, LB- and TB-EPS accounted for only 0.09% of total EPS (the sum of PN, PS, HS, and DNA from all EPS samples), indicating that EPS extraction in this study did not destroy the cell [23]. PN and PS were mainly distributed in TB-EPS (45.5% and 37.4%, respectively) and slime (43.7% and 41.9%, respectively), with the lowest percentage (10.8% and 20.7%, respectively) detected in LB-EPS. HS were mainly found in slime fraction (72.7%), followed by the TB-EPS fraction (21.8%). In comparison to other fractions, LB-EPS had the lowest concentration Table 2 Contents and characteristics of the different EPS fractions of AS. Slime PN (mg/g-VSS) PS (mg/g-VSS) HS (mg/g-VSS) DNA (mg/g-VSS) Zeta potential (mV)
22.53 28.82 0.40 0.24 −13.3
± ± ± ± ±
2.62 0.35 0.05 0.03 2.16
LB-EPS
TB-EPS
5.59 ± 0.31 14.22 ± 0.10 0.03 ± 0.01 0.28 ± 0.01 −10.4 ± 1.24
23.45 25.69 0.12 0.45 −17.0
± ± ± ± ±
1.67 0.24 0.02 0.01 1.08
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
3.2. Rheological characterization of AS before and after EPS extraction Three subsequent rheological tests were conducted at a TSS of 54 g/L and temperature (T) of 303 K.
3.2.1. CSR test Typical rheograms from the CSR test of AS before and after EPS extraction are displayed in Fig. 1. During CSR testing, the imposed shear rate was increased from 0.1 to 1000 s−1 in a ramp manner, then decreased in the same manner. As shown in Fig. 1, the rheograms of AS before and after EPS extraction had almost identical shapes. All samples exhibited non-Newtonian behavior. Shear stress values were higher on the ascending path than on the descending path, which induced a hysteresis loop between the two paths. Hla could be considered to indicate the degree of thixotropy displayed by the sludge [9]. This phenomenon indicates that all sludge samples showed thixotropic properties [18,21]. Thixotropy behaviors could also be demonstrated by apparent viscosity variations, which decreased rapidly initially and then became constant at higher shear rates. The constant viscosity at the infinte shear rate was denoted as the limiting viscosity (∞ ) [19,22,24], wherein the fluid can be assimilated to a continuous fluid with a Newtonian behavior [11].
250
1600
As
1400 1200
150
1000
path ding cen s e D
100
800 600
Shear stress Apparent viscosity
50
400
200
400
600
800
1400 200
1200 th g pa ndin e c s De
150 100
1000
0 200
400
600
800
1000
300
2000
2600
150
Shear stress
100
Apparent viscosity
1300
Shear stress (Pa)
path ding cen s e D
Apparent viscosity (Pa.s)
300
ath
(d)
250 3900
350
Shear stress (Pa)
400
-1
5200
200
600
Apparent viscosity
200 0
h g pat ndin Asce
250
800
Shear stress
Shear rate (s )
500
400
1000
0
-1
(c)
1800 1600
Shear rate (s )
450
2000
As
50
0 0
path
250
200 0
ing cend
(b)
300
Shear stress (Pa)
(a)
path
Apparent viscosity (Pa.s)
ing cend
2200
350
1800
200
Shear stress (Pa)
3.2.2. SAS test and FS test Generally, G represents the energy stored in a sample and characterizes its elastic property during the shearing process, while G represents the viscous dissipation of energy and the corresponding viscous property of the sample. Fig. 2 presents the results of SAS testing for AS before and after EPS extraction. A nearly strain-independent (plateau) G* value was observed with a constant plateau up to a critical strain value ( c ), and the corresponding G*–strain curve interval can be taken as the LVE range. In the LVE range, G was much greater than G , indicating that all sludge samples displayed much stronger elastic behaviors than viscous behaviors. Samples primarily exhibited solid-like behaviors before the critical strain, i.e., G exceeding G , was reached. In all cases, G* and G decreased but G increased beyond the LVE range, which may be explained by the disruption of the EPS-mediated network structure and the release of flowable solution-phase materials at increased strain amplitudes [25]. Fig. 3 presents results from FS test of AS before and after EPS extraction. For raw AS (Fig. 3a), G was slightly higher than G in the range of 0.2–16 Hz, implying a slightly stronger elastic behavior, while an opposite trend was observed for these two moduli in the range of 16–100 Hz. Sludge samples after different EPS fractions extraction displayed similar rheological curves. The critical point f of G exceeding G was around 13 Hz for sludge after slime extraction, 22 Hz for sludge after LB-EPS extraction and 11 Hz for sludge after TB-EPS extraction. In general, if G > G , elastic behavior dominates viscous behavior, which provides evidence of a gel-like structure (or sometimes called paste type behavior) for sludge samples that have low viscosity at high shear rates. If G > G , viscous behavior dominates over elastic behavior [18,20]. Accordingly, rheological curves derived
Apparent viscosity (Pa.s)
of each chemical component (PN, PS, and HS). As listed in Table 2, PN and PS were the major constituents of EPS. In addition, these three EPS fractions all displayed negative charges, as measured by the Zeta potential.
ng p endi
Asc
1600
200
1200 path ding cen s e D
150 100
800
Shear stress Apparent viscosity
50
400
Apparent viscosity (Pa.s)
210
50 0
0 0
200
400
600 -1
Shear rate (s )
800
1000
0
0 0
200
400
600
800
1000
-1
Shear rate (s )
Fig. 1. Typical rheograms at 303 K. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
2.5
3.5
G* G' G''
lgG* or lgG' or lgG'' (Pa)
lgG* or lgG' or lgG'' (Pa)
3.0
(a)
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
0.0
0.5
1.0
1.5
G* G' G''
3.0
(b) 2.5
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
2.0
lgγ
1.0
1.5
2.0
(c) 2.5
2.0
0.5
1.0
1.5
G* G' G''
(d)
3.0
lgG* or lgG' or lgG'' (Pa)
lgG* or lgG' or lgG'' (Pa)
0.5
3.5
G* G' G''
0.0
0.0
lgγ
3.5
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
211
3.0
2.5
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
2.0
lgγ
0.0
0.5
1.0
1.5
2.0
lgγ
Fig. 2. SAS test results. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
from the SAS and FS tests in Figs. 2 and 3 depicted gel-like behaviors of AS before and after EPS extraction in the LVE range.
higher than three other sludge samples, implying that sludge after LB-EPS extraction is more thixotropic. This is likely because a higher shear stress was needed to force the formation of an optimal opening and orientation of sludge after LB-EPS extraction in the flow direction [9] and the sludge sample exhibited higher resistance to shear. This phenomenon also indicated that enhancements in the interactions between flocs/aggregates, in the cohesion energy of the 3D sludge network, and in the elasticity are obtained for the AS after LB-EPS extraction. This finding also implies that the AS with TB-EPS exhibits stronger network structure. Greater values of y , Ec and G 0 from the SAS test for the AS with TB-EPS further suggest that this sludge sample behaved akin to stronger gel-like structure [25]. The network structure of AS could be ascribed to the contributions of slime, LB- and TB-EPS and pellets (the influence of other factors not under consideration). The rheological parameters of slime could be obtained by subtracting those of sludge after slime extraction from raw sludge. Then the corresponding rheological parameters of LB- and TB-EPS could also be determined. Only TBEPS had positive values of these parameters, suggesting that TB-EPS exerts positive effects on thixotropic properties, viscous properties, interactions between flocs/aggregates, the corresponding cohesion
3.2.3. Rheological parameters obtained from CSR and SAS tests As mentioned previously, Hla could be a measure of the degree of thixotropy exhibited by AS [9], the network structure of AS collapses with time and shear rate [9]. The value ∞ could represent the viscosity of sludge matrix corresponding to the maximum dispersion of flocs under the influence of shear rate [24]. It could also be associated with the optimal opening and orientation of sludge in the flow direction [9], where the fluid can be considered Newtonian [22]. The energy required to overcome the cohesion of the sludge network is designated Ec . The sludge network structure and interactions between biosolids are close to disruption under the effect of y . G 0 indicates sludge elasticity. Table 3 provides rheological parameters of AS before and after EPS extraction, including ∞ and Hla derived from the CSR test, y , Ec , and G 0 from the SAS test. Based on the calculation results for the Hla and ∞ in the rheograms in Fig. 1, y , Ec , and G 0 in the curves of Fig. 2, sludge after LB-EPS extraction is about 89.6 ± 11.9 kPa/s of Hla, 112.3 ± 21.5 mPa s of ∞ , 1.2 ± 0.2 Pa of y , 63.0 ± 14.2 × 10−3 J/m3 of Ec , and 11.5 ± 1.1 × 100 Pa of G 0 , which is
Table 3 Rheological parameters of AS before and after EPS extraction derived from CSR and SAS tests. Rheological parameters
∞ (mPa s)
Raw sludge sludge after slime extraction sludge after LB-EPS extraction sludge after TB-EPS extraction
74.8 87.7 112.3 77.5
± ± ± ±
3.9 1.2 21.5 6.8
Hla (kPa/s) 40.7 57.7 89.6 53.2
± ± ± ±
2.5 4.9 11.9 6.9
y (Pa) 0.5 0.7 1.2 0.4
± ± ± ±
0.1 0.1 0.2 0.1
Ec (10−3 J/m3 ) 21.9 30.6 63.0 19.7
± ± ± ±
7.7 13.3 14.2 8.7
G 0 (100 Pa) 4.8 7.8 11.5 4.9
± ± ± ±
1.1 1.2 1.1 0.9
212
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
6
G' G''
4
6
(a)
4
0 -2 -4 -6 -8 -1.0
(b)
2
lgG' or lgG'' (Pa)
lgG' or lgG'' (Pa)
2
G' G''
0 -2 -4 -6
-0.5
0.0
0.5
1.0
1.5
2.0
-8 -1.0
2.5
-0.5
0.0
0.5
lgf 6
G' G''
4
6
(c)
4
1.5
2.0
2.5
G' G''
1.0
1.5
2.0
2.5
(d)
2
lgG' or lgG'' (Pa)
lgG' or lgG'' (Pa)
2 0 -2 -4 -6 -8 -1.0
1.0
lgf
0 -2 -4 -6
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
lgf
-8 -1.0
-0.5
0.0
0.5
lgf
Fig. 3. FS test results. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
energy of the 3D sludge network, and elasticity. This finding indicates that the TB-EPS fraction had a gel-forming property [25]. TB-EPS may be responsible for water retention of AS [26,27] and could highly influence sludge dewatering and drying. 4. Conclusions AS after LB-EPS extraction showed higher rheological parameters including Hla, ∞ , y , Ec , and G 0 , suggesting that sludge obtained after LB-EPS extraction exhibited a stronger network structure. TB-EPS exerted positive effects on the thixotropic properties, limiting viscosity, interactions between flocs/aggregates, cohesion energy of the sludge network, and elasticity of the sludge. Rheological curves derived from SAS and FS tests revealed that AS before and after EPS extraction showed gel-like behaviors in the LVE domain. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 51078035 and 21177010), the Fundamental Research Funds for the Central Universities (Nos. JC2011-1 and TD2010-5), and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20100014110004). References [1] V. Urbain, J.C. Block, J. Manem, Bioflocculation in activated sludge: an analytic approach, Water Res. 27 (1993) 829–838.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Influence of extracellular polymeric substances on rheological properties of activated sludge Dongqin Yuan, Yili Wang ∗ College of Environmental Science and Engineering, Beijing Key Lab for Source Control Technology of Water Pollution, Beijing Forestry University, Beijing 100083, China
a r t i c l e
i n f o
Article history: Received 8 March 2013 Received in revised form 24 May 2013 Accepted 16 June 2013 Available online 24 June 2013 Keywords: Waste-water treatment Activated sludge Extracellular polymeric substances Rheology Non-Newtonian fluids Viscosity
a b s t r a c t Influence of extracellular polymeric substances (EPS) on rheological properties of activated sludge (AS) was conducted by comparing properties of AS before and after EPS extraction at a TSS of 54 g/L. Slime, loosely and tightly bound EPS (LB- and TB-EPS) were stratified by centrifugation and ultrasound method. The results showed that sludge after LB-EPS extraction produced a higher hysteresis loop area (Hla), limiting viscosity (∞ ), yield stress ( y ), energy of cohesion of network sludge (Ec ), and storage modulus (G 0 ), than three other sludge samples, indicating that sludge with TB-EPS exhibited stronger network structure. Strain amplitude sweep (SAS) and frequency sweep (FS) tests revealed that AS before and after EPS extraction produced a gel-like structure in the linear viscoelastic (LVE) domain. TB-EPS appeared to have a positive effect on the gel-like structure of AS. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Extracellular polymeric substances (EPS) are large molecular weight components of activated sludge (AS) flocs originating from bacteria secretion, cell lysis and hydrolysis, leakage of exocellular constituents, and absorbed organic matter from wastewater [1,2]. EPS are considered to be of great importance in understanding the structure, function, properties and development of microbial flocs/aggregates [3]. The presence of EPS alters the hydrophobic/hydrophilic characteristics of sludge, charge density and other surface properties [4]. They also entrap water and play an important role in sludge dewatering [4]. In recent years, stratification of EPS is of considerable interest. Yu et al. [5] empolyed centrifugation and ultrasound method to stratify EPS into slime, loosely and tightly bound EPS (LB- and TB-EPS). Wastewater sludge is a non-Newtonian fluid, which possesses both viscous and elastic properties, also called viscoelatic (VE) properties. These VE properties can often be described by rheological models and analyses [6]. Rheology is a powerful tool because it can scientifically describe and predict deformation and flow behaviors in real processes [7]. Understanding rheological properties of sludge is not only important for choosing parameters concerning storage, transportation, and landfill, but also crucial for stabilization
∗ Corresponding author. Tel.: +86 10 62336528; fax: +86 10 62336596. E-mail address: [email protected] (Y. Wang). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.06.011
and dewatering [8]. Rheology can generally characterize the network strength of sludge [9]. The network structure of sludge flocs formed by three-dimensional, gel-like, highly hydrated EPS with multivalent cations and other particulate materials [10], is of significance in maintaining the structural and functional integrity of flocs/aggregates [11] and in restricting water mobility [12]. Hence, to improve the dewatering efficiency, weakening or destroying the network structure of AS might be a possible solution. Effects of EPS concentrations on rheological properties of sludge flocs [1,4,13] and studies on rheological properties of EPS [14–16] have been reported. However, research focusing on the effects of different EPS fractions on rheological properties of AS is scare. This study explores the effects of different EPS fractions on rheological properties of AS by comparing properties of AS before and after EPS extraction. We wish to determine a specific EPS fraction, which mainly affects the network structure of AS. This EPS fraction might have a marked effect on sludge dewatering and drying processes. Hence, these results will provide further information on the influence of EPS on the dewatering properties of AS. 2. Materials and methods 2.1. Characteristics of AS AS was collected from a municipal wastewater treatment plant (WWTP) in Beijing, China. The WWTP treats 6.0 × 105 m3 /day of wastewater using an anaerobic-anoxic-oxic (A2 O) process.
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
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Table 1 Characteristics of AS. pH
Conductivity (mS/cm)
TSS (g/L)
VSS (g/L)
VSS/TSS (%)
COD (mg/L)
SCOD (mg/L)
Zeta potential (mV)
7.13 ± 0.02
1.45 ± 0.01
8.18 ± 0.78
5.64 ± 0.52
69.00 ± 0.30
6630.0 ± 32.5
81.6 ± 7.8
−18.9 ± 1.1
Collected sludge samples were transferred to the laboratory within 2 h and immediately screened through a 1.2 mm sieve. Filtered samples were then stored at 4 ◦ C. Table 1 presents the main characteristics of the AS sample. The pH was measured using a pH meter (PB-10, Sartorius Stedim Biotech Co., Ltd., Beijing, China). Conductivity was measured using a conductivity meter (EC215, Beijing Kanggaote Science and Technology Co., Ltd., China). Total suspended solids (TSS, g/L) and volatile suspended solids (VSS, g/L) were assessed from the weight loss of suspended sludge samples dried at specific temperatures and durations according to the standard method [17]. To obtain a consistent TSS concentration around 54 g/L from sludge samples for rheological testing, sludge samples resuspended after EPS extraction were first centrifuged at 1000 × g for 10 min, after which the bulk solution and solid phase were obtained separately. Collected sediments were successively diluted with supernatant. Chemical oxygen demand (COD) of the filtrate is referred to as soluble COD (SCOD). COD and SCOD analyses were conducted using a COD expedited testing apparatus (HATO CTL-12, Huatong Environmental Protecting Instruments Co., Ltd., Chengde, China). Zeta potential was recorded by a Malvern Zetasizer instrument (Nano Z, Malvern Co., UK). All tests were conducted within a week. All chemical analyses were carried out in triplicate using chemicals of analytical grade. 2.2. Extraction and analytical methods of different EPS fractions Slime, LB- and TB-EPS of AS were extracted by centrifugation and ultrasound method [5]. Slime was obtained from the supernatant after low-speed centrifugation. LB- and TB-EPS were dissolved in a buffer solution (pH 7) containing Na3 PO4 , NaH2 PO4 , NaCl, and KCl at a molar ratio of 2:4:9:1. conductivities of buffers were adjusted with distilled water to match those of the filtrated sludge samples presented in Table 1. Membranes (0.45 m) were used to filter out the particulates present in slime, LB- and TB-EPS solutions after all EPS fractions had been extracted. Proteins (PN) and humic-like substances (HS) of different EPS fractions were measured by the modified Lowry method using bovine serum albumin (Beijing Aoboxing Biotechnology Co., Ltd., China) and humic acid (Sigma, America) as standards, respectively. Polysaccharides (PS) and DNA were determined by the anthrone method, using glucose as the standard, and the diphenylamine colorimetric method, using 2-deoxy-d-ribose (Beijing Ruibo Biotechnology Co., Ltd., China) as the standard, respectively. All chemicals used were of analytical grade, and all tests were performed in triplicate. 2.3. Rheological testing Rheological tests were performed using a rheometer (Physica MCR 300, Anton Paar, Austria) in conjunction with US 200 software which recorded the rheology data. Temperature was maintained at 25 ◦ C by a Peltier control. A PP 50 plate and plate sensor with 49.94 mm diameter and 2.0 mm gap was used. Measurements were initially carried out for the first rheological testing mode in steady flow: (1) increasing shear rate from 0.1 to 1000 s−1 in a logarithm manner; (2) maintaining constant shear rate at 1000 s−1 in 30 s; (3) decreasing shear rate in a logarithm manner from 1000 to 0.1 s−1 . Rheograms of shear stress () as a function of shear rate () ˙ were recorded and analyzed for raw
AS and sludge after EPS extraction. In general, 30 s is sufficient to ensure that the equilibrium point of the stationary state can be reached [18,19]; while during this controlled shear rate (CSR) test, a shorter time span of 5 s for AS response was employed to indicate “network strength” of the sludge at each shear rate [7]. Based on these rheograms, the parameter hysteresis loop area (Hla) can be calculated as follows [9]:
Hla =
t
· d(t) ˙
(1)
0
where (Pa) is the shear stress and ˙ (s−1 ) is the shear rate. The second mode was a type of transient rheological test-strain amplitude sweep (SAS) test, in which strain amplitude varied in a sinusoidal manner over time while the frequency remained fixed at 1 Hz. This test was carried out to exploit the linear viscoelastic (LVE) domain. The rheogram of the moduli (storage modulus G , loss modulus G , complex modulus G*) as a function of strain on the logarithmic coordinates can present that rheological parameters t (including the above moduli) are independent of strain until a critical strain level ( c ), above which the linear domain is reached. Beyond this value, the G* or G values decline as the material loses its structural integrity [7,20,21]. Subsequently, its nonlinear behavior appears, and the transition can be used to determine c and the critical storage modulus (G 0 ). G 0 represents the critical elasticity of the sludge network. The product between G* on the plateau and the critical deformation ( c ) directly provides the value of the yield stress: y = G∗ c . In addition to leaving the sample in a more intact state, the dynamic mechanical test is also advantageous in its simplicity and independence from any model assumptions [7,18,20]. Provided the values of y and c determined from the aforementioned SAS test, the corresponding value of Ec , is also equal to the half product of y and c [19,22]. After determing the LVE range by SAS test, frequency sweep (FS) test was conducted at 0.1% of the strain value. Corresponding G and G values were measured. Rheological tests were performed in duplicate. 3. Results and discussion 3.1. Characteristics of different EPS fractions in AS Characteristics of different EPS fractions in AS are displayed in Table 2. The sum of DNA content from slime, LB- and TB-EPS accounted for only 0.09% of total EPS (the sum of PN, PS, HS, and DNA from all EPS samples), indicating that EPS extraction in this study did not destroy the cell [23]. PN and PS were mainly distributed in TB-EPS (45.5% and 37.4%, respectively) and slime (43.7% and 41.9%, respectively), with the lowest percentage (10.8% and 20.7%, respectively) detected in LB-EPS. HS were mainly found in slime fraction (72.7%), followed by the TB-EPS fraction (21.8%). In comparison to other fractions, LB-EPS had the lowest concentration Table 2 Contents and characteristics of the different EPS fractions of AS. Slime PN (mg/g-VSS) PS (mg/g-VSS) HS (mg/g-VSS) DNA (mg/g-VSS) Zeta potential (mV)
22.53 28.82 0.40 0.24 −13.3
± ± ± ± ±
2.62 0.35 0.05 0.03 2.16
LB-EPS
TB-EPS
5.59 ± 0.31 14.22 ± 0.10 0.03 ± 0.01 0.28 ± 0.01 −10.4 ± 1.24
23.45 25.69 0.12 0.45 −17.0
± ± ± ± ±
1.67 0.24 0.02 0.01 1.08
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
3.2. Rheological characterization of AS before and after EPS extraction Three subsequent rheological tests were conducted at a TSS of 54 g/L and temperature (T) of 303 K.
3.2.1. CSR test Typical rheograms from the CSR test of AS before and after EPS extraction are displayed in Fig. 1. During CSR testing, the imposed shear rate was increased from 0.1 to 1000 s−1 in a ramp manner, then decreased in the same manner. As shown in Fig. 1, the rheograms of AS before and after EPS extraction had almost identical shapes. All samples exhibited non-Newtonian behavior. Shear stress values were higher on the ascending path than on the descending path, which induced a hysteresis loop between the two paths. Hla could be considered to indicate the degree of thixotropy displayed by the sludge [9]. This phenomenon indicates that all sludge samples showed thixotropic properties [18,21]. Thixotropy behaviors could also be demonstrated by apparent viscosity variations, which decreased rapidly initially and then became constant at higher shear rates. The constant viscosity at the infinte shear rate was denoted as the limiting viscosity (∞ ) [19,22,24], wherein the fluid can be assimilated to a continuous fluid with a Newtonian behavior [11].
250
1600
As
1400 1200
150
1000
path ding cen s e D
100
800 600
Shear stress Apparent viscosity
50
400
200
400
600
800
1400 200
1200 th g pa ndin e c s De
150 100
1000
0 200
400
600
800
1000
300
2000
2600
150
Shear stress
100
Apparent viscosity
1300
Shear stress (Pa)
path ding cen s e D
Apparent viscosity (Pa.s)
300
ath
(d)
250 3900
350
Shear stress (Pa)
400
-1
5200
200
600
Apparent viscosity
200 0
h g pat ndin Asce
250
800
Shear stress
Shear rate (s )
500
400
1000
0
-1
(c)
1800 1600
Shear rate (s )
450
2000
As
50
0 0
path
250
200 0
ing cend
(b)
300
Shear stress (Pa)
(a)
path
Apparent viscosity (Pa.s)
ing cend
2200
350
1800
200
Shear stress (Pa)
3.2.2. SAS test and FS test Generally, G represents the energy stored in a sample and characterizes its elastic property during the shearing process, while G represents the viscous dissipation of energy and the corresponding viscous property of the sample. Fig. 2 presents the results of SAS testing for AS before and after EPS extraction. A nearly strain-independent (plateau) G* value was observed with a constant plateau up to a critical strain value ( c ), and the corresponding G*–strain curve interval can be taken as the LVE range. In the LVE range, G was much greater than G , indicating that all sludge samples displayed much stronger elastic behaviors than viscous behaviors. Samples primarily exhibited solid-like behaviors before the critical strain, i.e., G exceeding G , was reached. In all cases, G* and G decreased but G increased beyond the LVE range, which may be explained by the disruption of the EPS-mediated network structure and the release of flowable solution-phase materials at increased strain amplitudes [25]. Fig. 3 presents results from FS test of AS before and after EPS extraction. For raw AS (Fig. 3a), G was slightly higher than G in the range of 0.2–16 Hz, implying a slightly stronger elastic behavior, while an opposite trend was observed for these two moduli in the range of 16–100 Hz. Sludge samples after different EPS fractions extraction displayed similar rheological curves. The critical point f of G exceeding G was around 13 Hz for sludge after slime extraction, 22 Hz for sludge after LB-EPS extraction and 11 Hz for sludge after TB-EPS extraction. In general, if G > G , elastic behavior dominates viscous behavior, which provides evidence of a gel-like structure (or sometimes called paste type behavior) for sludge samples that have low viscosity at high shear rates. If G > G , viscous behavior dominates over elastic behavior [18,20]. Accordingly, rheological curves derived
Apparent viscosity (Pa.s)
of each chemical component (PN, PS, and HS). As listed in Table 2, PN and PS were the major constituents of EPS. In addition, these three EPS fractions all displayed negative charges, as measured by the Zeta potential.
ng p endi
Asc
1600
200
1200 path ding cen s e D
150 100
800
Shear stress Apparent viscosity
50
400
Apparent viscosity (Pa.s)
210
50 0
0 0
200
400
600 -1
Shear rate (s )
800
1000
0
0 0
200
400
600
800
1000
-1
Shear rate (s )
Fig. 1. Typical rheograms at 303 K. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
2.5
3.5
G* G' G''
lgG* or lgG' or lgG'' (Pa)
lgG* or lgG' or lgG'' (Pa)
3.0
(a)
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
0.0
0.5
1.0
1.5
G* G' G''
3.0
(b) 2.5
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
2.0
lgγ
1.0
1.5
2.0
(c) 2.5
2.0
0.5
1.0
1.5
G* G' G''
(d)
3.0
lgG* or lgG' or lgG'' (Pa)
lgG* or lgG' or lgG'' (Pa)
0.5
3.5
G* G' G''
0.0
0.0
lgγ
3.5
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
211
3.0
2.5
2.0
1.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5
2.0
lgγ
0.0
0.5
1.0
1.5
2.0
lgγ
Fig. 2. SAS test results. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
from the SAS and FS tests in Figs. 2 and 3 depicted gel-like behaviors of AS before and after EPS extraction in the LVE range.
higher than three other sludge samples, implying that sludge after LB-EPS extraction is more thixotropic. This is likely because a higher shear stress was needed to force the formation of an optimal opening and orientation of sludge after LB-EPS extraction in the flow direction [9] and the sludge sample exhibited higher resistance to shear. This phenomenon also indicated that enhancements in the interactions between flocs/aggregates, in the cohesion energy of the 3D sludge network, and in the elasticity are obtained for the AS after LB-EPS extraction. This finding also implies that the AS with TB-EPS exhibits stronger network structure. Greater values of y , Ec and G 0 from the SAS test for the AS with TB-EPS further suggest that this sludge sample behaved akin to stronger gel-like structure [25]. The network structure of AS could be ascribed to the contributions of slime, LB- and TB-EPS and pellets (the influence of other factors not under consideration). The rheological parameters of slime could be obtained by subtracting those of sludge after slime extraction from raw sludge. Then the corresponding rheological parameters of LB- and TB-EPS could also be determined. Only TBEPS had positive values of these parameters, suggesting that TB-EPS exerts positive effects on thixotropic properties, viscous properties, interactions between flocs/aggregates, the corresponding cohesion
3.2.3. Rheological parameters obtained from CSR and SAS tests As mentioned previously, Hla could be a measure of the degree of thixotropy exhibited by AS [9], the network structure of AS collapses with time and shear rate [9]. The value ∞ could represent the viscosity of sludge matrix corresponding to the maximum dispersion of flocs under the influence of shear rate [24]. It could also be associated with the optimal opening and orientation of sludge in the flow direction [9], where the fluid can be considered Newtonian [22]. The energy required to overcome the cohesion of the sludge network is designated Ec . The sludge network structure and interactions between biosolids are close to disruption under the effect of y . G 0 indicates sludge elasticity. Table 3 provides rheological parameters of AS before and after EPS extraction, including ∞ and Hla derived from the CSR test, y , Ec , and G 0 from the SAS test. Based on the calculation results for the Hla and ∞ in the rheograms in Fig. 1, y , Ec , and G 0 in the curves of Fig. 2, sludge after LB-EPS extraction is about 89.6 ± 11.9 kPa/s of Hla, 112.3 ± 21.5 mPa s of ∞ , 1.2 ± 0.2 Pa of y , 63.0 ± 14.2 × 10−3 J/m3 of Ec , and 11.5 ± 1.1 × 100 Pa of G 0 , which is
Table 3 Rheological parameters of AS before and after EPS extraction derived from CSR and SAS tests. Rheological parameters
∞ (mPa s)
Raw sludge sludge after slime extraction sludge after LB-EPS extraction sludge after TB-EPS extraction
74.8 87.7 112.3 77.5
± ± ± ±
3.9 1.2 21.5 6.8
Hla (kPa/s) 40.7 57.7 89.6 53.2
± ± ± ±
2.5 4.9 11.9 6.9
y (Pa) 0.5 0.7 1.2 0.4
± ± ± ±
0.1 0.1 0.2 0.1
Ec (10−3 J/m3 ) 21.9 30.6 63.0 19.7
± ± ± ±
7.7 13.3 14.2 8.7
G 0 (100 Pa) 4.8 7.8 11.5 4.9
± ± ± ±
1.1 1.2 1.1 0.9
212
D. Yuan, Y. Wang / Biochemical Engineering Journal 77 (2013) 208–213
6
G' G''
4
6
(a)
4
0 -2 -4 -6 -8 -1.0
(b)
2
lgG' or lgG'' (Pa)
lgG' or lgG'' (Pa)
2
G' G''
0 -2 -4 -6
-0.5
0.0
0.5
1.0
1.5
2.0
-8 -1.0
2.5
-0.5
0.0
0.5
lgf 6
G' G''
4
6
(c)
4
1.5
2.0
2.5
G' G''
1.0
1.5
2.0
2.5
(d)
2
lgG' or lgG'' (Pa)
lgG' or lgG'' (Pa)
2 0 -2 -4 -6 -8 -1.0
1.0
lgf
0 -2 -4 -6
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
lgf
-8 -1.0
-0.5
0.0
0.5
lgf
Fig. 3. FS test results. (a) Raw sludge; (b) sludge after slime extraction; (c) sludge after TB-EPS extraction; (d) sludge after TB-EPS extraction.
energy of the 3D sludge network, and elasticity. This finding indicates that the TB-EPS fraction had a gel-forming property [25]. TB-EPS may be responsible for water retention of AS [26,27] and could highly influence sludge dewatering and drying. 4. Conclusions AS after LB-EPS extraction showed higher rheological parameters including Hla, ∞ , y , Ec , and G 0 , suggesting that sludge obtained after LB-EPS extraction exhibited a stronger network structure. TB-EPS exerted positive effects on the thixotropic properties, limiting viscosity, interactions between flocs/aggregates, cohesion energy of the sludge network, and elasticity of the sludge. Rheological curves derived from SAS and FS tests revealed that AS before and after EPS extraction showed gel-like behaviors in the LVE domain. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 51078035 and 21177010), the Fundamental Research Funds for the Central Universities (Nos. JC2011-1 and TD2010-5), and the Ph.D. Programs Foundation of the Ministry of Education of China (No. 20100014110004). References [1] V. Urbain, J.C. Block, J. Manem, Bioflocculation in activated sludge: an analytic approach, Water Res. 27 (1993) 829–838.
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