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Applying rheological analysis to better understand the mechanism of acid

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DOI: 10.1016/j.watres.2017.05.002

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 Water Research 122 (2017) 398e406

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journal homepage: www.elsevier.com/locate/watres

Applying rheological analysis to better understand the mechanism of

acid conditioning on activated sludge dewatering
Hou-Feng Wang a, Yun-Jie Ma a, Hua-Jie Wang a, b, Hao Hu a, Hai-Yang Yang c,
Raymond J. Zeng a, *
a
CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
b
School of Environmental and Chemical Engineering, Anhui Vocational and Technical College, Anhui 230011, China
c
Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, China

a r t i c l e i n f o a b s t r a c t

Article history: The pH value is a key parameter and affects sludge dewatering. Comprehensive understanding of the
Received 20 January 2017 effects and mechanism of pH is important for sludge treatment process and sludge dewatering. The goal
Received in revised form of this study was to evaluate the proposed mechanism of acid conditioning on sludge dewatering based
1 May 2017
on rheological analysis. At lower sludge pH, changes in floc structure, surface properties, and flocculation
Accepted 2 May 2017
Available online 13 May 2017
improved the performance of dewatering. Additionally, lower sludge pH caused the hydrolysis of EPS and
intracellular materials, which released greater amounts of bound water. These changes resulted in
altered rheological properties, weakening network strength and shrinking the linear viscoelastic regime,
Keywords:
Acid conditioned sludge
making the sludge system sensitive to shear. Thus, both the sludge dewatering rate and moisture
Dewaterability reduction efficiency were improved by lowering the pH. These factors demonstrate that rheological
Mechanism analysis can understand the mechanism of acid conditioning on activated sludge dewatering better.
Bridge © 2017 Elsevier Ltd. All rights reserved.
Rheological property

1. Introduction Improving sludge compressibility is related to the dewatering rate

A large amount of sewage sludge produced from the activated
sludge process requires further treatment and disposal, for signif-
icant economic and environmental burdens (Mikkelsen and
Keiding, 2002; Mowla et al., 2013). Because the water content of
sewage sludge is about 99%, high-performance dewatering is
beneficial for sludge reduction.
However, the dewatering process is costly and poorly under-
stood as it depends largely on the physical-chemical parameters,
origin of the sludge, and the selection of the dewatering devices (Jin
et al., 2004; Mikkelsen and Keiding, 2002). In the past few decades,
extensive efforts have been made to elucidate the dewatering
mechanism and develop a high-performance dewatering process
(Liu et al., 2012; Mowla et al., 2013; Vesilind and Hsu, 1997). Many
studies suggest that improving sludge filterability and altering the
affinity of extracellular polymeric substances (EPS) for water
molecule capture (bound water) are efficient approaches to
enhance sludge dewatering (Mowla et al., 2013; Qi et al., 2011).
* Corresponding author.
E-mail address: rzeng@ustc.edu.cn (R.J. Zeng).
and the sludge moisture reduction efficiency can usually be
assessed by the bound water content, the moisture content of the
sludge and other parameters. Many treatments including physical
conditioning (pretreatment with non-chemical additives, micro-
wave conditioning, and thermal/freeze pretreatment), chemical
conditioning (salt addition or pH conditioning), and biological or
enzyme conditioning have been used to promote sludge dewater-
ing (Chen et al., 2002; Mowla et al., 2013; Neyens et al., 2004; Ruiz-
Hernando et al., 2013). Overall, the main factors guiding the se-
lection of treatments are cost, environmental impact, and efficiency
(Chu et al., 2005; Mowla et al., 2013).
Many reports have attempted to clarify the mechanism of sludge
dewaterability, and their results mainly focus on the conditioning
process, physicochemical characteristics, and sludge dewaterability
(Chen et al., 2001; Christensen et al., 2015; Kim et al., 2016; Li and
Yang, 2007; Mowla et al., 2013; Neyens et al., 2004). Peeters et al.
(2013) selected polyaluminiumchloride (PACl) as the conditioner
and studied its effect on sludge dewatering-drying operations. Niu
et al. (2013) investigated the effects of inorganic coagulants on
sludge dewatering based on their correlation between physico-
chemical properties and sludge dewaterability. These studies
represent the common approach used to understand the

http://dx.doi.org/10.1016/j.watres.2017.05.002
0043-1354/© 2017 Elsevier Ltd. All rights reserved.
 H.-F. Wang et al. / Water Research 122 (2017) 398e406
mechanism of sludge dewatering focusing on three elements:
conditioning process, physicochemical characteristics, and sludge
dewaterability. However, after sludge conditioning, analysis of the
correlation between physicochemical properties and sludge dew-
aterability has not completely explained the fundamental mecha-
nism of sludge dewatering. It is well-known that dewatering often
involves both filtration (cake formation) and consolidation (cake
compression)(Stickland et al., 2005), and the challenge is to achieve
high performance solid-liquid separation given the challenges of
permeability and compressibility. These factors require us to
consider the hydrodynamics (rheological behavior) during these
processes. Because wastewater sludge is a non-Newtonian fluid and
possesses viscoelastic property, many recent studies point out that
the hydrodynamic properties (rheological behavior) of the sludge
are closely related to the conditioning process (Chaari et al., 2003;
Ormeci, 2007), physicochemistry interaction (Eshtiaghi et al.,
2013), and sludge filterability (Marinetti et al., 2010) during the
dewatering process. However, these studies mainly focused on
relating or assessing dewaterability/filterability with rheological
parameters. Until now, rheological analysis of conditioned sludge
and its correlation with the dewatering processes has been unclear,
and it is unknown if rheological analysis can provide insight into
the mechanism of sludge dewatering. Here we propose a new
approach to characterization of the mechanism of sludge dew-
atering that includes sludge conditioning, physicochemical char-
acteristics, hydrodynamics (rheology behavior) of the sludge
system, and sludge dewaterability. In brief, the process of sludge
conditioning causes changes of sludge's physicochemical charac-
teristics that alter the system's rheological behavior (hydrody-
namics) and change the sludge dewaterability. Of these elements,
the rheological behavior likely acts as a useful indicator for the
activated sludge dewatering process and can reveals mechanistic
insight that can't be determined.
Sludge conditioned with acids prior to dewatering improves the
operating performance of sludge dewatering. Chen et al. (2001)
used sulfuric acid together with surfactant to pretreat activated
sludge and suggested that dewaterability and settleability were
accelerated at pH 2.5 conditioned with surfactant. Zhang et al.
(2016) found that sludge dewaterability could be improved by
lowering the pH level after ferrate treatment. However, information
about the mechanism of acid conditioning action is poorly under-
stood and the systematic application of acid conditioning is still
limited. Furthermore, many synergetic conditions of sludge that are
commonly used often include the variation of pH in the acid-stage
(Chen et al., 2015; Liu et al., 2016a; Niu et al., 2013; Xiao et al., 2016;
Zhai et al., 2012), which underscores the necessity for proper un-
derstanding of the pH effect and its mechanism on sludge
dewatering.
The objective of this study was to evaluate the hypothesis that
rheological analysis can act as a bridge to reveal the mechanism of
sludge dewatering using acid conditioning as an example, and
hopefully to improve our fundamental understanding of sludge
dewatering.
2. Materials and methods
2.1. Raw sludge and chemicals
Raw activated sludge was obtained from the Wangtang munic-
ipal wastewater treatment plant located in Hefei, Anhui Province,
China. This plant has a capacity of 180,000 m3/d and uses the
oxidation ditch process. The initial solid concentration of the ma-
terial was 12.01 g/L and then was gently concentrated to
22.10 ± 0.4 g/L by settling. The sludge samples were stored at 4
C
(for less than 1 week) prior to use (Conrardy et al., 2016). The
399
characteristics of the raw activated sludge are given in Table 1.
Hydrochloric acid (HCl) was purchased from Shanghai Chemical
Reagent Company and was of analytical reagent grade.
2.2. Sludge conditioning and dewatering
For each conditioning test, 2.4 M HCl was added to a 50 mL
portion of sludge to adjust the pH to the designed value. The
mixture was stirred for 1 min at a speed of 300 rpm and then at
150 rpm for 3 min. All the tests were performed in triplicate. The
specific resistance of filtration (SRF, m/kg) and the capillary suction
time (CST, s) are two rapid indicators of the filterability of the
sludge. The SRF was measured based on the method of Wisniewski
and Grasmick (1998) and CST was measured with a CST instrument
(Model 319, Trion, UK). In this study, Buchner-funnel filtration was
used to evaluate the effects of pH on dewatering (Lo et al., 2001) at
0.06 MPa for 2 min, 5min, or 30min. The moisture content of the
filtered sludge cake was calculated according to the study of He
et al. (2015).
2.3. EPS extraction and analysis
First, sludge samples were allowed to settle at 5000 g for 5 min
and the supernatant was collected as the slime (soluble EPS). The
bound EPS of the sludge, including loosely bound EPS (LB-EPS) and
tightly bound EPS (TB-EPS) were obtained using a modified heat
extraction method (Li and Yang, 2007). Both the slime and bound
EPS extractions were analyzed for proteins (PN), polysaccharides
(PS), humic-like substances (HS), and total organic carbon (TOC).
The contents of PN and HS were analyzed by a UV spectropho-
tometer (UV751GD, Analytical Instrument Co., China) following the
modified Lowry method (Frølund et al., 1995) using bovine albumin
(Sigma) and humic acid (Fluka) as standards. The PS content was
determined by the anthrone-sulfuric acid method with glucose as
the standard (Frølund et al., 1996). TOC was measured by a TOC
analyzer (Multi N/C 2100, Analytic Jena Co., Germany) according to
the manufacturer's instructions.
2.4. Physicochemical characteristics of the sludge
The sludge supernatant was obtained by centrifuging the sludge
suspension at 3000 rpm for 5 min, then filtered through the
0.45 mm cellulose acetate membrane. The supernatant was
analyzed for zeta potential using a Nanosizer ZS instrument (Mal-
vern Co., UK) at 25
C based on the previous studies (Li and Yang,
2007; Luo et al., 2015). The particle size distribution of the sludge
samples was measured by a MS2000 laser classifier (Malvern Co.,
UK) and operated as described (Chen et al., 2017; Gunning, 2011).
All measurements were performed at least six independent times
and were conducted at 25.0 ± 0.1
C. Differential scanning calo-
rimetry (DSC, Shimadzu DSC-60, Japan) and a thermos gravimetric
analyzer (TGA, TGA-Q5000, TA Co., USA) were performed to mea-
sure free water and bound water in the sludge cake (Peeters et al.,
2013; Vaxelaire and Cezac, 2004; Wu et al., 1998b). Briefly, the raw
Table 1
Characteristics of the raw activated sludge.
pH 6.80 ± 0.10
Total solids (g L1) 22.1 ± 0.4
Volatile suspended solids (VSS, g/g TS) 0.57 ± 0.02
Specific resistance to filtration (SRF,X1013 m/kg) 8.79 ± 0.61
CST (s) 81.5 ± 0.8
Sludge viscosity (mPa.s) 14.85 ± 0.12
Zeta potential (mV) 11.23 ± 0.72
d(0.5) (mm) 63.48 ± 1.53
400 H.-F. Wang et al. / Water Research 122 (2017) 398e406
and acid-conditioned sludge samples were vacuum filtered at
0.06 MPa for 5 min and then weighed for DSC analysis. About 15 mg
of sludge sample was fast cooled from 20
C to 20
C at a rate of
1
C/min and then increased to 20
C at the same rate (He et al.,
2015). The amount of free water (Wf) was calculated as follows:
Wf ¼ Q/H, where Q is the heat absorbed during the melting process
and H is the water heat of fusion (334 mJ/mg)(Lee and Lee, 1995).
The TGA analyzer recorded the weight loss of the sludge cake when
the temperature increased from 20 to 150
C, and this was defined
as total water (Wt). The bound water (Wb) was then calculated as
the difference between Wt and Wf (Vaxelaire and Ce
zac, 2004; Wu
et al., 1998a).
2.5. Rheological tests
Rheological tests were performed on a rotational AR-2000ex
rheometer (TA instruments, USA) connected to a thermal bath.
The rheometer was equipped with a cup and bob geometry (29-
mm inner diameter, 32-mm outer diameter, 44-mm length). The
temperature was maintained at 25
C in a water bath. Before each
rheological measurement, the sludge sample was pre-sheared for
5 min at a shear rate of 500 s1 then allowed to rest for 10 min
while other tests performed (Baudez et al., 2011; Farno et al., 2014;
Ma et al., 2014).
In dynamic measurement, the angular frequency (5 rad/s) and
temperature (25
C) were held constant as the strain was loga-
rithmically varied from 0.01% to 100% to gain the structural prop-
erty of the sludge and determine the linear viscoelastic region.
Frequency, time, and creep tests were performed at the linear
viscoelastic region and measurements were conducted using an
instrument from Rheometric Scientific. A frequency sweep was
conducted at 0.1% strain and 25
C with the frequency range of
0.1e50 rad s1. A time sweep test (corresponding to a range of time
from 1 to 360 s) was performed under a strain of 0.1% (under LVE
regime) at 25
C. Creep tests were conducted by applying constant
shear stress of 0.02 Pa (<ty ) and the corresponding compliance was
monitored for 10 min.
In the steady shear test, the shear rate was logarithmically
increased from 0.05 to 500 s1 for 10 min to obtain useful infor-
mation about viscous and viscoplastic properties. The Herschel-
Bulkley model (Eq. (1)) was used to fit the rheological data, a
method previously shown to be useful in describing sludge
rheology (Dai et al., 2014).
t ¼ ty þ k$g_ n (1)
where t is the shear stress (Pa), ty is the yield stress (Pa), g_ is the
shear rate (s1), k is the consistency index (Pa,sn ), which can reveal
the average firmness of the sample (Ruiz-Hernando et al., 2014a).
The n is the flow behavior index and a decrease or increase of n
from 1 means that the non-Newtonian flow characteristics are
strengthened (Feng et al., 2014).
2.6. Statistical analysis
Pearson's correlation is the measurement of the linear correla-
tion between two variables. As a statistical analysis tool, it has
widely been used to environment management (Stefanakis and
Tsihrintzis, 2011; Wilen et al., 2003a, b; Xiao et al., 2017). In this
study, correlation analysis on sludge dewatering performance and
the key factors were performed using the software SPSS.20. The
correlations were considered statistically significant at 95% confi-
dence interval (p < 0.05).
3. Results and discussion
3.1. Effects of acid conditioning on the physic-chemical
characteristics of the sludge
The release of EPS and intracellular materials have been proven
to be beneficial for sludge dewatering (Mikkelsen and Keiding,
2002). Fig. 1 shows the variation in organic compounds in the
different fractions of EPS (ie. Soluble EPS and bound EPS) as a
function of pH values. The bound EPS is the sum of LB-EPS and TB-
EPS. Fig. 1a shows that more bound EPS was released as the pH of
the sludge decreased from 6.80 to 2.60, and the biopolymer content
of soluble EPS showed the opposite trend. For example, the
biopolymer content of bound EPS decreased from 37.41 mg/g VSS to
20.94 mg/g VSS as the pH of the sludge decreased from 6.80 to 2.60,
but the opposite pattern was observed for soluble EPS, which
increased from 2.79 mg/g VSS to 28.04 mg/g VSS. Meanwhile, the
changes of TOC contents were similar with the variation of bio-
polymers in the related fractions of EPS (Fig. 1b). The above results
indicated that acid conditioning could release EPS and intracellular
materials as the pH decreased sludge, consistent with Chen et al.
(2001). PN, PS, and HS are likely the major biopolymers of EPS in
activated sludge (Li and Yang, 2007). Fig. 1c expresses the effect of
pH value on the variation of these three major biopolymers within
total EPS. The results presented in Fig. 1c obviously indicate that
with the decrease of pH, the amount of PS and HS in the total EPS
remained almost unchanged, but the amount of PN increased
significantly from 24.84 mg/g VSS to 32.76 mg/g VSS as the pH of
the sludge decreased from 6.80 to 2.60. Frolund et al. (1995) sug-
gested that the predominance of PN in the EPS and intracellular
materials might be due to the presence of a large quantity of exo-
enzymes and cellular enzymes. The above results suggested that
more enzymes were released or degraded from intracellular as the
pH decreased, which gave rise to a high level of PN in the EPS
matrix. To have a deeper understanding of the concentrations of
these biopolymers in different fractions of EPS (soluble EPS and
bound EPS) as a function of pH values, we analyzed the components
of soluble EPS and bound EPS as shown in Fig. 1d. These results
were similar to those presented in Fig. 1a and b, meaning that
concentrations of all of these biopolymers in the soluble EPS
increased as the pH decreased, but the concentrations within the
bound EPS showed the opposite result. Among these biopolymers,
the changes of PN within both types of EPS was the most obviously
affected by pH values. For example, the content of PN within the
soluble EPS increased from 0.67 mg/g VSS to 19.59 mg/g VSS, while
the contrary pattern was observed for PN within the bound EPS, a
decrease from 24.17 mg/g VSS to 13.17 mg/g VSS. Compared with
PN, the contents of PS and HS varied within a smaller range as the
pH decreased, and the related increased values within soluble EPS
were approximately equal to the decreased values within the
bound EPS. The results from Fig. 1c and d showed that PN was the
predominant component in the EPS, constituting 61.50%e66.89% of
the total EPS as the pH of the sludge decreased from 6.80 to 2.60,
which was similar with the results of Yu et al. (2008).
The changes of sludge zeta potentials (ZPs) and particle size
d (0.5) as a function of pH values are shown in Fig. 2. The ZP and
particle size for the raw sludge were 11.23 ± 0.72 mV and
63.48 ± 1.53 mm, respectively. Xu et al. (2006) reported the
disruption of floc would cause more metal cations released
from intracellular materials. So we can see after the pH of the
sludge was decreased from 6.80 to 2.60, the ZPs increased
from 11.23 ± 0.72 mV to 1.87 ± 0.13 mV. These results about low
pH imparting positive charge to sludge were likely due to the
release of EPS and intracellular materials, which were consistent
with previous research on activated sludge and biofilms (Liu et al.,
 H.-F. Wang et al. / Water Research 122 (2017) 398e406 401

Fig. 1. The correlation of sludge between pH and (a) quantities of EPS concentration (sum of PN, PS, and HS); (b) quantities of soluble EPS and Bound EPS in terms of TOC content; (c)
quantities of PN, HS, and PS in the total EPS; (d) PN, PS, and HS contents of the soluble EPS (Sol-EPS) and Bound EPS.

Fig. 2. Impact of pH on sludge zeta potentials and particle size d (0.5).

2016a; Nuhoglu and Oguz, 2003). A similar increase was also

observed for the particle size from 63.48 ± 1.53 mm to
81.69 ± 1.30 mm as the pH of the sludge decreased from 6.8 to 2.6.
These results were similar to those reported by Liu et al. (2016a).
Therefore, lowering pH of the sludge caused the disruption of floc
structure and hydrogen ions competed with metal cations released
from EPS and intracellular materials in the system, which imparted
positive charge to the sludge and resulted in an increase in particle
size.

3.2. Effects of acid conditioning on the rheological profile of the

sludge Fig. 3. Dynamic strain sweep curve (solid squares represent G*, solid circles represent
tan).

3.2.1. Dynamic measurements

The effects of pH on the viscoelastic properties of raw and acid- of curves showed similar evolution of G0 and G00 . G0 and G00 were
conditioned sludge are clearly illustrated in Fig. 3. Five related pairs nearly constant under a low strain, suggesting a linear viscoelastic
402 H.-F. Wang et al. / Water Research 122 (2017) 398e406

regime, and then both moduli became strain dependent. As shown

in Fig. 3a, the G00 of raw sludge peaked before decreasing. The
observed increase in G00 at the peak for acid-conditioned sludge was
significantly smaller than that of raw sludge as the pH decreased.
Based on our knowledge (Farno et al., 2016; Wyss et al., 2005), the
value of the G00 peak can reflect the decrease in structure relaxation
time due to the increase of the shear strain. Thus, the decrease in
the G00 peak for conditioned sludge after lowering its pH might be
related to the solubilisation of intracellular organic matter into the
liquid phase. Additionally, the values of G0 decreased from 26.03 Pa
to 14.30 Pa as the pH of sludge decreased from 6.80 to 2.60 (shown
in Fig. 3a), suggesting that network strength and colloidal forces of
floc were weakened as the pH decreased. Consequently, the linear
viscoelastic region (SI Fig. S1) was decreased (the critical shear
strain: 30.47% (pH ¼ 6.80), 25.01% (pH ¼ 5.02), 23.81% (pH ¼ 4.33),
22.13% (pH ¼ 3.58), and 20.13% (pH ¼ 2.60)). Furthermore, the effect
of pH on G* (the complex modulus) during a strain sweep is shown
in Fig. 3b. High values of G* reflect a strong ability to resist defor-
mation (Feng et al., 2014). The G* decreased from 26.75 Pa to Fig. 4. Frequency sweep in LVE regime (strain ¼ 0.1%).
14.31 Pa as the pH decreased from 6.80 to 2.60. Moreover, the
values of tan(d) increased from 0.10 to 0.13 as the pH decreased
from 6.80 to 2.60, which indicates that the acid-conditioned sludge
showed a trend toward fluidization as the pH decreased. The
related key parameters in the linear regime are shown in Table 2.
In the LVE regime (Fig. 4), a typical frequency sweep was per-
formed at a constant strain of 0.1% for raw and conditioned sludge
with different pH values. All of the sludge presented a solid-like
regime. The viscoelastic properties of the sludge became weaker
as the pH decreased. The values of tan (d) significantly increased
when lowering the sludge pH, suggesting that the sludge that was
subjected to acidification presented weaker solid-like properties.
Next, the time sweep (Fig. 5a) and the creep test (Fig. 5b) were
used to analyze the viscoelastic properties of the raw and condi-
tioned sludge. The storage modulus G0 during time sweep is a good
index of the solid-like character of a material, and fluctuation in-
dicates poor stability. Fig. 5a shows that both the raw and acidified
sludge exhibited good stability, suggesting that they were
homogeneous-like systems. The creep test probes the time-
dependent nature of a sample (Ruiz-Hernando et al., 2014b).
Fig. 5b shows the values of compliance as a function of time. The
creep compliance increased when lowering the pH of the sludge,
suggesting that acidification made the sludge more easily deformed
by a given stress. Taken together with the findings shown in Figs. 2
and 3, these results indicate that acidification can loosen and dis-
rupte the internal structure of the sludge.

3.2.2. Steady shear flow

The steady shear flow in the range of 0.05e500 s1 was used to
analyze the overall rheological behavior from low to high shear. As
shown in Fig. 6a, both the raw sludge and conditioned sludge
presented shear-thinning behavior and the non-Newtonian char-
acteristics gradually decreased with the decrease of pH. To accu-
rately analyze the difference between the treated and untreated
Fig. 5. (a) Time sweep in LVE region (strain ¼ 0.1%); (b) creep tests in LVE region (shear
stress ¼ 0.02 Pa).
Table 2
Fitting results of the Herschel-Bulkley model and the key parameters at various
sludge pHs.
sludge samples and clarify the basic non-Newtonian behavior of
pH Herschele-Bulkley parameters Key parameters in linear regime
these sludge samples, the Herschel-Bulkley equation was adopted.
:
ty (Pa) k n R2 G* tan(delta) gc ð%Þ gc As shown in Fig. 6a, the yield stress (ty) globally decreased when
6.80 1.261 0.375 0.487 0.997 26.749 0.103 26.61 4.961 lowering the pH of sludge. During the shear process, the lower the
5.03 0.959 0.348 0.506 0.998 15.871 0.119 15.63 2.821 pH, the lower the shear stress and the lower the apparent viscosity
4.33 0.878 0.335 0.510 0.998 15.270 0.122 15.24 1.562 at a given shear rate. As pH decreased from 6.8 to 2.6, the value of k
3.58 0.762 0.324 0.523 0.997 15.080 0.124 15.00 0.869 and the yield stress (ty) decreased from 0.375 to 0.300 and
2.60 0.704 0.300 0.540 0.998 14.300 0.127 14.37 0.640
1.26 Pae0.70 Pa, respectively, while the value of n increased from
 H.-F. Wang et al. / Water Research 122 (2017) 398e406
Fig. 6. Flow curves of raw sludge and acid conditioned sludge (solid triangle represent
shear stress, solid circular represent viscosity).
0.487 to 0.540 (Table 2). These results indicated that non-
Newtonian flow characteristics of sludge were weakened and
suggested the internal structure of the sludge was broken at lower
pH value. This is consistent with the release of EPS and intracellular
materials, which can be an indication of the degree of floc rupture
(Liu et al., 2016b). Due to these changes, lowered pH made the
sludge more like fluid. Consequently, Fig. 6b shows that the linear
viscoelastic region was significantly decreased (the critical shear
:
rate (gc ) decreased when lowering the pH of sludge). This is
consistent with the results of the dynamic measurements.
Fig. 7. The correlations of sludge between pH and (a) CST and SRF of sludge; (b) CST
and SRF of sludge the contents of bound water before and after filtration.
403
Hydrolysis of organic matter, especially EPS, can lead to the
destruction of the colloidal properties of macromolecules (Everett,
1974). Among many key organic substances, both proteins and
polysaccharides play important roles in the formation of sludge
flocs (Higgins and Novak, 1997). However, according to the results
presented in Fig. 2, the concentration of proteins of EPS was higher
than that of polysaccharides, and the protein concentration showed
more variation as the pH decreased. Based on this, we can deduce
that the gel-like structure of the sludge flocs after acidification
might be due to the loss of some proteins bonds, as the sludge
structure changed from a highly bound system to a simple colloidal
suspension. These changes were reflected in the rheology behavior
of the sludge system as the acid-conditioned sludge showed a trend
toward fluidization and the treated sludge acted more closely to an
equilibrium material as the pH decreased.
3.3. Effects of acid conditioning on sludge dewatering performance
The effects of acid conditioning on sludge filterability were
assessed in terms of CST and SRF and are illustrated in Fig. 7a. The
filterability of the sludge conditioned with HCl was improved as pH
decreased. As the pH of the sludge decreased from 6.8 to 2.6, the
CST of the raw sludge linearly decreased (R2 ¼ 0.957) from
81.5 ± 0.8 s to 29.3 ± 0.4s and a similar remarkable decrease was
observed for the SRF value (R2 ¼ 0.961), which decreased from
8.79  1013 m/kg to 2.21  1013 m/kg. These results indicated that
acid conditioning led to a significant enhancement of filterability in
terms of CST and SRF as pH decreased. These findings are similar to
previous results (Neyens et al., 2004). Fig. 7b shows the changes of
bound water in the sludge before filtration and after filtration
(dewatered) as a function of pH values. After the pH of the sludge
decreased from 6.80 to 5.02, the bound water in the sludge before
filtration decreased drastically from 3.41 g/g DS to 0.69 g/g DS and
then remained constant near 0.60 g/g DS when the pH reduced to
3.58. However, when the pH decreased further to 2.60, the bound
water increased to 1.29 g/g DS. Overall, low pH resulted in a
remarkable decrease of the bound water. The reduction of bound
water after filtration (dewatered) was quite consistent (R2 ¼ 0.794),
from 0.530 g/g DS to 0.386 g/g DS when the pH of the sludge
decreased from 6.80 to 3.58, and then showed a slight increase to
0.42 g/g DS when the pH continued decreasing to 2.60. Earlier re-
ports suggested that bound water is one of the major limiting
factors affecting sludge dewatering efficiency, and decreasing the
Fig. 8. The correlation of sludge between pH and moisture of filtered cake (%) after
2 min, 5 min, or 30 min filtration under a vacuum pressure of 0.06 MPa.
404 H.-F. Wang et al. / Water Research 122 (2017) 398e406

content of bound water is a key step to enhance dewaterability

0.151

0.021

0.234
0.383
(Robinson and Knocke, 1992). Many earlier reports have focused

BW-2

0.177
0.188
0.020

0.174
0.060
on the changes of bound water within the dewatered sludge cake,

e
without examination of the changes before dewatering. The
bound water within sludge before dewatering (in situ) indicated

0.557

0.766

0.383
BW-1

0.638
0.449
0.731
0.721
0.596

0.868
the potential of moisture reduction efficiency while the bound

e
water within the dewatered sludge (ex situ) might reflect the
degree of moisture reduction. The big changes of bound water

¡0.991**
¡0.995**

0.978*
within the sludge pretreated with HCl indicate that the reduction

0.957
0.967
0.913

0.965

0.766
0.060
of bound water within the dewatered sludge cake was effective

tan

e
(Fig. 7b). Therefore, the water content in the formed filter cake
may decrease for lower pH values. The above results indicated that

0.878

0.978

0.234
the content of bound water was minimized nearly the pH value of

0.899
0.815
0.953
0.959
0.891

0.868
jG*j
3.58.

e
The moisture content of filter-cake with variant pH values is
illustrated in Fig. 8. The moisture content of filter-cake linearly

¡0.995**
0.992**

0.965
decreased from 85.37% to 81.67% for 2-min-filtration time

0.972*
0.974*
0.974*

0.891
0.596
0.171
(R2 ¼ 0.904), 83.32%e79.64% for 5-min-filtration time

h∞

e
(R2 ¼ 0.987) and 75.84%e67.67% for 30-min-filtration time
(R2 ¼ 0.950) when decreasing the pH in the range of 6.80e2.60.

0.974*

0.998**

0.995

0.021
0.980*
0.942

0.974

0.959
0.721
These results illustrated that at low pH, the dewaterability of the

gc
sludge was generally high. Apparently, pH affected dewaterability,

e
suggesting that high pH should be avoided in sludge dewatering.

0.971

0.991
0.987*
0.937

0.998
0.974

0.953
0.731
0.020
3.4. The mechanism of acid conditioning on sludge dewatering

ty

e
In order to accurately study the relationship between sludge

0.942

0.833

0.380
B-TOC

0.944
0.957
0.889
0.875
0.931

0.709

0.414
dewaterability and variations in the key parameters described
above and evaluate the rheological analysis as a bridge to reveal
the mechanism of sludge dewatering, Pearson's correlations were
calculated (Table 3). The results indicated that CST was positively
0.909
0.895
0.945
0.831
0.821
0.886

0.627
0.258
0.440
S-TOC

0.768
and strongly correlated with the value of B-EPS (R ¼ 0.995,
p < 0.01) and h∞(R ¼ 0.992, p < 0.01), the water content of sludge
(WC) was positively and strongly correlated with the value of pH
0.542
0.494
0.647
0.399
0.390
0.485

0.120

0.563

Correlation is significant at the 0.01 level (2-tailed).

(R ¼ 0.990, p < 0.01), but strong negative correlation was found

0.300

0.276
between WC and S-EPS (R ¼ 0.990, p < 0.01). Additionally, strong
PL

negative correlation were found between pH and h∞(R ¼ 0.995,

p < 0.01), B-EPS and h∞(R ¼ 0.989, p < 0.01), and ty and gc
0.910
0.845
0.945
0.786
0.802
0.888

0.634
0.180
0.303
0.770

(R ¼ 0.998, p < 0.01), and moderate correlations were found be-

PN

tween other rheological parameters (Table 3). There are obvious

strong correlations between pH and physicochemical parameters
0.986*
0.995**

0.989**
0.940

and rheological characteristics, rheological behavior, and dew-

B-EPS

0.970
0.967
0.959

0.853
0.380
0.261

atering performance (Table 3). Additionally, it is also important to

note that a lower pH would result in better filterability and
dewaterability as it would tend to release more macromolecular
¡0.990**
0.985*
0.964

0.920
0.920
0.978

0.783
0.258
0.301

components and bound water and improve zeta potential values.

S-EPS

0.895

These changes lead to the sludge showing a trend toward fluid-

ization and closer to an equilibrium system as reflected in the
Pearson's correlation results between different parameters.

rheology behavior. In this study, we confirmed the speculation

0.966
0.944
0.972
0.886
0.886
0.964

0.738
0.366
0.376
**
0.861

that the content of PN was significantly negative to sludge dew-

Zeta

Correlation is significant at the 0.05 level (2-tailed).

atering from the negative R values and p < 0.05 of WC

(R ¼ 0.945, p < 0.05) and CST (R ¼ 0.845, p < 0.07). However Le
et al. (2016) had critical evolution for 00 protein00 measurement, and
0.990**

0.913
0.963

0.937
0.942
0.972

0.815
0.449
0.188

pointed out quantification of these 00 proteins00 using standard

WC

colorimetric assays often shows great variability. So, the influence

of protein within EPS on sludge dewatering performance still need
0.992**
0.967
0.988*

0.963
0.987
0.980

0.899
0.638
0.177

further study. A high content of polysaccharide was reported to

CST

have a negative effect on the net surface charge of sludge, thus

deteriorating sludge dewaterability (Wang et al., 2005). However
0.990**

0.995**
0.957
0.878
0.557
0.151

in this study, we found no obvious correlation of the poly-

0.988

0.971
0.974

saccharide concentration with either CST or WC (Table 3), so the

pH

relationship between PS contents and sludge dewaterability may

be overestimated.
BW-1
BW-2
Table 3

CST

jG*j
WC

Based on our experimental results, we propose that the

tan
pH

h∞
gc
ty

mechanism of acid conditioning on sludge dewatering includes

*
 H.-F. Wang et al. / Water Research 122 (2017) 398e406
Fig. 9. The proposed mechanism of acid conditioning on activated sludge dewatering.
four elements: (1) acid conditioning, (2) physic-chemical behavior,
(3) rheological behavior/analysis, and (4) dewatering performance.
As shown in Fig. 9, lowering the pH of the sludge causes changes in
physicochemical behavior (Figs. 1e2), such as the disruption of floc
structure, the release of bound water caused by the release of EPS
and intracellular materials, and the absorption of sludge flocs due
to neutralization. These changes are reflected in changes in the
rheological properties (Figs. 3e6), including weakening the
network strength, shrinking the linear viscoelastic regime, and
making the sludge system sensitive to shear. As a result, better
filterability and dewaterability were achieved (Figss. 7e8),
including the improvement of filterability and moisture reduction
efficiency. Therefore, we propose that rheological analysis can act
as a bridge to better understand the mechanism of sludge dew-
atering. On this basis, we hope to provide useful guideline to select
right dose of conditioners to reach a good level of dewatering in the
future.
4. Conclusions
This study investigated the effects and mechanism of acid
conditioning on sludge dewaterability by analyzing the changes
and relationships of EPS, physicochemical properties, and rheology
behavior. The main conclusions were:
 The sludge filterability and moisture reduction efficiency were
improved by lowering the pH.
 The floc structure, surface property and flocculation depend
heavily on sludge pH.
 Lowering the pH of the sludge led to a trend towards fluidization
and towards an equilibrium system.
 The mechanism of acid conditioning on sludge dewatering was
elaborated. The data showed that rheological analysis could act
as a bridge between the physicochemical characteristics and the
dewaterability of conditioned sludge to better understand the
fundamental mechanism of sludge dewatering.
Acknowledgments
The authors would like to acknowledge financial support from
the PetroChina Innovation Foundation (2014D-5006-0205), the
Program for Changjiang Scholars and Innovative Research Team in
405
University, the Fundamental Research Funds for the Central Uni-
versities, and the Collaborative Innovation Center of Suzhou Nano
Science and Technology.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.watres.2017.05.002.
References
Baudez, J.C., Markis, F., Eshtiaghi, N., Slatter, P., 2011. The rheological behaviour of
anaerobic digested sludge. Water Res. 45 (17), 5675e5680.
Chaari, F., Racineux, G., Poitou, A., Chaouche, M., 2003. Rheological behavior of
sewage sludge and strain-induced dewatering. Rheol. Acta 42 (3), 273e279.
Chen, C., Bin, L., Tang, B., Huang, S., Fu, F., Chen, Q., Wu, L., Wu, C., 2017. Cultivating
granular sludge directly in a continuous-flow membrane bioreactor with in-
ternal circulation. Chem. Eng. J. 309, 108e117.
Chen, G.H., Yue, P.L., Mujumdar, A.S., 2002. Sludge dewatering and drying. Dry.
Technol. 20 (4e5), 883e916.
Chen, Y.G., Yang, H.Z., Gu, G.W., 2001. Effect of acid and surfactant treatment on
activated sludge dewatering and settling. Water Res. 35 (11), 2615e2620.
Chen, Z., Zhang, W.J., Wang, D.S., Ma, T., Bai, R.Y., 2015. Enhancement of activated
sludge dewatering performance by combined composite enzymatic lysis and
chemical re-flocculation with inorganic coagulants: kinetics of enzymatic re-
action and re-flocculation morphology. Water Res. 83, 367e376.
Christensen, M.L., Keiding, K., Nielsen, P.H., Jorgensen, M.K., 2015. Dewatering in
biological wastewater treatment: a review. Water Res. 82, 14e24.
Chu, C., Lee, D., Chang, C., 2005. Energy demand in sludge dewatering. Water Res. 39
(9), 1858e1868.
Conrardy, J.B., Vaxelaire, J., Olivier, J., 2016. Electro-dewatering of activated sludge:
electrical resistance analysis. Water Res. 100, 194e200.
Dai, X., Gai, X., Dong, B., 2014. Rheology evolution of sludge through high-solid
anaerobic digestion. Bioresour. Technol. 174, 6e10.
Eshtiaghi, N., Markis, F., Yap, S.D., Baudez, J.C., Slatter, P., 2013. Rheological char-
acterisation of municipal sludge: a review. Water Res. 47 (15), 5493e5510.
Everett, J.G., 1974. The effect of pH on the heat treatment of sewage sludges. Water
Res. 8 (11), 899e906.
Farno, E., Baudez, J.C., Parthasarathy, R., Eshtiaghi, N., 2014. Rheological character-
isation of thermally-treated anaerobic digested sludge: impact of temperature
and thermal history. Water Res. 56, 156e161.
Farno, E., Baudez, J.C., Parthasarathy, R., Eshtiaghi, N., 2016. The viscoelastic char-
acterisation of thermally-treated waste activated sludge. Chem. Eng. J. 304,
362e368.
Feng, G.H., Liu, L.Y., Tan, W., 2014. Effect of thermal hydrolysis on rheological
behavior of municipal sludge. Ind. Eng. Chem. Res. 53 (27), 11185e11192.
Frølund, B., Griebe, T., Nielsen, P., 1995. Enzymatic activity in the activated-sludge
floc matrix. Appl. Microbiol. Biotechnol. 43 (4), 755e761.
Frølund, B., Palmgren, R., Keiding, K., Nielsen, P.H., 1996. Extraction of extracellular
polymers from activated sludge using a cation exchange resin. Water Res. 30
(8), 1749e1758.
Frolund, B., Griebe, T., Nielsen, P.H., 1995. Enzymatic-Activity in the activated-sludge
406 H.-F. Wang et al. / Water Research 122 (2017) 398e406
floc matrix. Appl. Microbiol. Biotechnol. 43 (4), 755e761.
Gunning, P.J., 2011. Accelerated Carbonation of Hazardous Wastes. School of Science,
University of Greenwich.
He, D.Q., Wang, L.F., Jiang, H., Yu, H.Q., 2015. A Fenton-like process for the enhanced
activated sludge dewatering. Chem. Eng. J. 272, 128e134.
Higgins, M.J., Novak, J.T., 1997. Characterization of exocellular protein and its role in
bioflocculation. J. Environ. Eng. Asce 123 (5), 479e485.

n, B.-M., Lant, P., 2004. Impacts of morphological, physical and chemical
Jin, B., Wile
properties of sludge flocs on dewaterability of activated sludge. Chem. Eng. J. 98
(1), 115e126.
Kim, M.S., Lee, K.M., Kim, H.E., Lee, H.J., Lee, C., Lee, C., 2016. Disintegration of waste
activated sludge by thermally-activated persulfates for enhanced dewater-
ability. Environ. Sci. Technol. 50 (13), 7106e7115.
Le, C.C., Kunacheva, C., Stuckey, D.C., 2016. “Protein” measurement in biological
wastewater treatment systems: a critical evaluation. Environ. Sci. Technol. 50
(6), 3074e3081.
Lee, D.J., Lee, S.F., 1995. Measurement of bound water content in sludge: the use of
differential scanning calorimetry (DSC). J. Chem. Technol. Biotechnol. 62 (4),
359e365.
Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric sub-
stances (EPS) on the flocculation, sedimentation and dewaterability of activated
sludge. Water Res. 41 (5), 1022e1030.
Liu, F., Zhou, J., Wang, D., Zhou, L., 2012. Enhancing sewage sludge dewaterability by
bioleaching approach with comparison to other physical and chemical condi-
tioning methods. J. Environ. Sci. (China) 24 (8), 1403e1410.
Liu, J.B., Wei, Y.S., Li, K., Tong, J., Wang, Y.W., Jia, R.L., 2016a. Microwave-acid pre-
treatment: a potential process for sludge dewaterability. Water Res. 90,
225e234.
Liu, J.B., Yu, D.W., Zhang, J., Yang, M., Wang, Y.W., Wei, Y.S., Tong, J., 2016b. Rheo-
logical properties of sewage sludge during enhanced anaerobic digestion with
microwave-H2O2 pretreatment. Water Res. 98, 98e108.
Lo, I.M., Lai, K.C., Chen, G., 2001. Salinity effect on mechanical dewatering of sludge
with and without chemical conditioning. Environ. Sci. Technol. 35 (23),
4691e4696.
Luo, K., Yang, Q., Li, X.M., Zhang, S.Y., Pang, Y., Li, X., Liao, X.S., 2015. Effect of calcium
ions on dewaterability of enzymatic-enhanced anaerobic digestion sludge.
Appl. Biochem. Biotechnol. 176 (8), 2346e2357.
Ma, Y.J., Xia, C.W., Yang, H.Y., Zeng, R.J., 2014. A rheological approach to analyze
aerobic granular sludge. Water Res. 50, 171e178.
Marinetti, M., Dentel, S.K., Malpei, F., Bonomo, L., 2010. Assessment of rheological
methods for a correlation to sludge filterability. Water Res. 44 (18), 5398e5406.
Mikkelsen, L.H., Keiding, K., 2002. Physico-chemical characteristics of full scale
sewage sludges with implications to dewatering. Water Res. 36 (10),
2451e2462.
Mowla, D., Tran, H.N., Allen, D.G., 2013. A review of the properties of biosludge and
its relevance to enhanced dewatering processes. Biomass & Bioenergy 58,
365e378.
Neyens, E., Baeyens, J., Dewil, R., De heyder, B., 2004. Advanced sludge treatment
affects extracellular polymeric substances to improve activated sludge dew-
atering. J. Hazard. Mater. 106 (2e3), 83e92.
Niu, M.Q., Zhang, W.J., Wang, D.S., Chen, Y., Chen, R.L., 2013. Correlation of physi-
cochemical properties and sludge dewaterability under chemical conditioning
using inorganic coagulants. Bioresour. Technol. 144, 337e343.
Nuhoglu, Y., Oguz, E., 2003. Removal of copper(II) from aqueous solutions by bio-
sorption on the cone biomass of Thuja orientalis. Process Biochem. 38 (11),
1627e1631.
Ormeci, B., 2007. Optimization of a full-scale dewatering operation based on the
rheological characteristics of wastewater sludge. Water Res. 41 (6), 1243e1252.
Peeters, B., Dewil, R., Vernimmen, L., Van den Bogaert, B., Smets, I.Y., 2013. Addition
of polyaluminiumchloride (PACl) to waste activated sludge to mitigate the
View publication stats
negative effects of its sticky phase in dewatering-drying operations. Water Res.
47 (11), 3600e3609.
Qi, Y., Thapa, K.B., Hoadley, A.F., 2011. Application of filtration aids for improving
sludge dewatering propertiesea review. Chem. Eng. J. 171 (2), 373e384.
Robinson, J., Knocke, W.R., 1992. Use of dilatometric and drying techniques for
assessing sludge dewatering characteristics. Water Environ. Res. 64 (1), 60e68.
Ruiz-Hernando, M., Martinez-Elorza, G., Labanda, J., Llorens, J., 2013. Dewaterability
of sewage sludge by ultrasonic, thermal and chemical treatments. Chem. Eng. J.
230, 102e110.
Ruiz-Hernando, M., Simo
n, F.-X., Labanda, J., Llorens, J., 2014a. Effect of ultrasound,
thermal and alkali treatments on the rheological profile and water distribution
of waste activated sludge. Chem. Eng. J. 255, 14e22.
Ruiz-Hernando, M., Simon, F.X., Labanda, J., Llorens, J., 2014b. Effect of ultrasound,
thermal and alkali treatments on the rheological profile and water distribution
of waste activated sludge. Chem. Eng. J. 255, 14e22.
Stefanakis, A.I., Tsihrintzis, V.A., 2011. Dewatering mechanisms in pilot-scale sludge
drying reed beds: effect of design and operational parameters. Chem. Eng. J. 172
(1), 430e443.
Stickland, A.D., De Kretser, R.G., Scales, P.J., 2005. Nontraditional constant pressure
filtration behavior. Aiche J. 51 (9), 2481e2488.
Vaxelaire, J., Ce
zac, P., 2004. Moisture distribution in activated sludges: a review.
Water Res. 38 (9), 2215e2230.
Vaxelaire, J., Cezac, P., 2004. Moisture distribution in activated sludges: a review.
Water Res. 38 (9), 2215e2230.
Vesilind, P.A., Hsu, C.-C., 1997. Limits of sludge dewaterability. Water Sci. Technol. 36
(11), 87e91.
Wang, Z.W., Liu, Y., Tay, J.H., 2005. Distribution of EPS and cell surface hydropho-
bicity in aerobic granules. Appl. Microbiol. Biotechnol. 69 (4), 469e473.
Wilen, B.M., Jin, B., Lant, P., 2003a. Impacts of structural characteristics on activated
sludge floc stability. Water Res. 37 (15), 3632e3645.
Wilen, B.M., Jin, B., Lant, P., 2003b. The influence of key chemical constituents in
activated sludge on surface and flocculating properties. Water Res. 37 (9),
2127e2139.
Wisniewski, C., Grasmick, A., 1998. Floc size distribution in a membrane bioreactor
and consequences for membrane fouling. Colloids Surf. A Physicochem. Eng.
Aspects 138 (2), 403e411.
Wu, C.C., Huang, C., Lee, D., 1998a. Bound water content and water binding strength
on sludge flocs. Water Res. 32 (3), 900e904.
Wu, C.C., Huang, C.P., Lee, D.J., 1998b. Bound water content and water binding
strength on sludge flocs. Water Res. 32 (3), 900e904.
Wyss, H.M., Tervoort, E.V., Gauckler, L.J., 2005. Mechanics and microstructures of
concentrated particle gels. J. Am. Ceram. Soc. 88 (9), 2337e2348.
Xiao, K., Chen, Y., Jiang, X., Tyagi, V.K., Zhou, Y., 2016. Characterization of key organic
compounds affecting sludge dewaterability during ultrasonication and acidifi-
cation treatments. Water Res. 105, 470e478.
Xiao, K., Chen, Y., Jiang, X., Yang, Q., Seow, W.Y., Zhu, W., Zhou, Y., 2017. Variations in
physical, chemical and biological properties in relation to sludge dewaterability
under Fe (II) e oxone conditioning. Water Res. 109, 13e23.
Xu, H., Liu, Y., Tay, J.H., 2006. Effect of pH on nickel biosorption by aerobic granular
sludge. Bioresour. Technol. 97 (3), 359e363.
Yu, G.H., He, P.J., Shao, L.M., He, P.P., 2008. Stratification structure of sludge flocs
with implications to dewaterability. Environ. Sci. Technol. 42 (21), 7944e7949.
Zhai, L.F., Sun, M., Song, W., Wang, G., 2012. An integrated approach to optimize the
conditioning chemicals for enhanced sludge conditioning in a pilot-scale sludge
dewatering process. Bioresour. Technol. 121, 161e168.
Zhang, W.J., Cao, B.D., Wang, D.S., Ma, T., Yu, D.H., 2016. Variations in distribution
and composition of extracellular polymeric substances (EPS) of biological
sludge under potassium ferrate conditioning: effects of pH and ferrate dosage.
Biochem. Eng. J. 106, 37e47.