Environ Sci Pollut Res (2011) 18:1567–1573
DOI 10.1007/s11356-011-0492-6
RESEARCH ARTICLE
The effect of continuous exposure of copper on the properties
and extracellular polymeric substances (EPS) of bulking
activated sludge
Jia Li & Xiang Liu & Yan Liu & Juliana Ramsay &
Chenchao Yao & Ruihua Dai
Received: 12 December 2010 / Accepted: 11 March 2011 / Published online: 17 May 2011
# Springer-Verlag 2011
Abstract
Introduction This study investigated the effect of copper on
chemical oxygen demand (COD) removal efficiency and on
the properties (mainly settling and dewatering) and the
composition of extracellular polymeric substances (EPS)
when 20 mg/L Cu(II) was continuously dosed to a sequencing
batch reactor (SBR) inoculated with activated sludge.
Materials and methods The results showed that the
continuous addition of 20 mg/L Cu(II) seriously inhibited
the removal of sodium benzoate (provided as a model
organic pollutant) by activated sludge in a SBR.
Results and discussion After 40 days of acclimation, the
removal efficiency presented a slight but unsteady recovery
and the settling and dewatering properties improved,
indicating that sludge bulking had been inhibited. Addi-
tionally, the proportion of loosely bound EPS in the total
EPS increased with time and the relative composition of the
total organics was polysaccharide > humic substances >
protein > DNA.
Responsible editor: Zhihong Xu
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-011-0492-6) contains supplementary material,
which is available to authorized users.
J. Li : X. Liu (*) : Y. Liu (*) : C. Yao : R. Dai
Department of Environmental Science and Engineering,
Fudan University,
220 Handan Road,
Shanghai 200433, China
e-mail: liuxiang@fudan.edu.cn
Y. Liu
e-mail: liuyan@fudan.edu.cn
J. Ramsay
Department of Chemical Engineering,
Queen’s University, Kingston, Ontario, Canada K7L 3N6
Conclusion The effects of copper on the composition of
EPS and the settling and dewatering properties of bulking
activated sludge were also discussed for the first time in this
paper.
Keywords Copper . Bulking sludge . Settling property .
Dewatering property . Extracellular polymeric substances
(EPS)
1 Introduction
Heavy metals are usually present in wastewaters from the
automobile, metal finishing, leather tanning, electroplating,
petroleum, and textile dyeing industries. Copper, zinc, lead,
mercury, chromium, cadmium, iron, nickel, and cobalt are
commonly encountered (Fergusson 1990; Aksu 2005).
Direct disposal of these wastewaters can have a detrimental
impact on the environment. Heavy metals can adversely
affect biological wastewater treatment since they would
accumulate in soil when the treated sludge is recycled
creating a hazard for plants, animals, and humans.
Copper is widespread in the wastewater and sludge in
Shanghai, China. Previous studies have focused on its toxic
effect to activated sludge systems (Cabrero et al. 1998; Sin
et al. 2000; Wong 1999; Principi et al. 2006; Hu et al.
2004). Low Cu(II) concentrations stimulated microbial
growth in sludge while high concentrations were inhibitory
or may cause serious instability of the system. The effects
of copper on the growth kinetics of activated sludge
(Cabrero et al. 1998; Dilek and Yetis 1992), on activity of
the sludge (Oviedo et al. 2002), on the microbial commu-
nity (Madoni et al. 1996), and on the minimum inhibitory
concentration for filamentous microorganism (Nicolau et al.
2005) have been examined. The minimum inhibitory
1568
copper concentration ranged from 2 to 10 mg/L (Cabrero et
al. 1998; Dilek and Yetis 1992; Oviedo et al. 2002). A few
studies which examined the recovery of an activated sludge
system after a single exposure to copper (Benetti 2000)
mainly emphasized the recovery of the chemical oxygen
demand (COD) removal efficiency. However, it remains
unknown whether microbial adaptation and recovery can
occur after continuous exposure. This is where the novelty
lies in this study.
Extracellular polymeric substances (EPS) make up
50~90% percent of the total organic component of activated
sludge (Benetti 2000) and play an important role in floc
aggregation, the sludge settling ability (Wingender et al.
1999; Liao et al. 2001), and the sorption of heavy metals
(Guibaud et al. 1999; Fukushi et al. 2001). The quantity
and composition of EPS are related to the nutrients and
substrates in the influent (Kiff 1978). Microorganisms
release large amounts of soluble microbial products
(SMP) which increased the COD and BOD5 in the effluent
(Kuo and Parkin 1996). Recent studies have mainly focused
on the adsorption of heavy metal to EPS and SMP
components of activated sludge, or on whether heavy
metals affected the ability to remove pollutants. However,
few studies have examined whether copper affects the
composition of EPS and/or the sludge properties.
This study has investigated the effect of continuously
dosing 20 mg/L copper (1) on the removal of an organic
pollutant using sodium benzoate (C6H5COONa) as the
model compound and (2) on the sludge properties (includ-
ing its settling and dewatering abilities, and the quantity
and organic composition of EPS and SMP) over a period of
160 days. This paper also discusses how the change of EPS
composition under the influence of copper affected the
settling and dewatering abilities of activated sludge.
2 Materials and methods
2.1 Experimental methods
Two sequencing batch reactors (SBR), one dosed with
copper (experimental reactor, ER) and the control which
was not exposed to copper (control reactor, CR), were
inoculated with a seed sludge obtained from a local sewage
treatment plant in Shanghai. A synthetic wastewater
influent contained C6H5COONa as the carbon source, in
the range of 650~820 mg COD/L. The COD:N:P mass ratio
of the wastewater was kept at 100:5:1, and mineral
nutrients were added to each liter as follows: 0.0894 mg
FeCl3·6H2O, 0.0894 mg H3BO3, 0.0179 mg CuSO4·5H2O,
0.1074 mg KI, 0.0716 mg MnCl2·4H2O, 0.0716 mg
ZnSO4·7H2O, and 0.0894 mg CoCl2·6H2O. After steady
state, 20 mg/L Cu(II) (refers to the concentration of Cu(II)
Environ Sci Pollut Res (2011) 18:1567–1573
in the synthetic wastewater) was added in the influent of the
ER. In a previous batch experiment to examine the effect of
copper loading at 0, 10, 20, and 40 mg/L Cu(II), the relative
toxic effect of Cu(II) was 10 mg/L<20 mg/L≤40 mg/L.
However, since there was no significant difference between
20 and 40 mg/L Cu(II) and the COD removal efficiency
recovered after 24 h at both concentrations, 20 mg/L Cu(II)
was selected for the continuous experiment. The reactors
were run over 160 days, and all parameters were measured
repeatedly.
The reactors had a 4.0-L working volume and were
operated for 160 days at 20–30°C with a 12-h cycle of four
stages: fill (0.5 h), aeration (6.0 h), settling (2.0 h), decant
(0.5 h), and idle (3 h). At the end of each cycle, 2 L of
supernatant was replaced with an equal volume of fresh
synthetic wastewater. The dissolved oxygen level in the
aeration stage was maintained at 2.0~2.5 mg/L. The pH in
the reactors maintained at 6.5–7.0 in ER and 7.0–8.0 in CR.
The SBR schematic diagram is shown in Fig. 1.
2.2 Analytical methods
The analyses of COD, sludge settling ratio (SV30), sludge
volume index (SVI), mixed liquor suspended solids
(MLSS), and specific resistance to filtration (SRF) were
done according to the standard methods of APHA (1995).
To extract EPS (Comte 2006), sludge was collected
30 min before the end of the aeration period, 10 mL was
filtered through a 0.22-μm filter membrane, and the
composition of the SMP in the filtrate was measured.
Sludge (5 mL) was centrifuged at 1,063×g for 10 min in a
7-mL centrifuge tube, and the supernatant filtered through a
0.22-μm filter membrane was considered to be the low-
speed centrifugation products (LSCP). Filtered samples
were washed and resuspended in 5 mL of distilled H2O,
and recentrifuged at 9,564×g for 20 min. This supernatant
was filtered through a 0.22-μm membrane and the
composition of EPSs measured. The retentate after EPSs
extraction was mixed with 5 mL of distilled H2O, heated in
an 80°C water bath for 30 min, and then centrifuged at
Fig. 1 The SBR schematic diagram
Environ Sci Pollut Res (2011) 18:1567–1573 1569

9,564×g for 20 min. The supernatant was filtered through a
0.22 μm membrane, and the composition of EPSb in the
filtered solution was measured. Loosely bound EPS (LB-
EPS) was defined as the sum of LSCP and EPSs, tightly
bound EPS (TB-EPS) was EPSb, and total EPS (EPSt) was
the sum of LB-EPS and TB-EPS.
The main components of EPS were analyzed: polysac-
charide by an anthrone colorimetric assay (Gaudy 1962),
protein and humic substances by a modified Lowry method
(Frϕlund et al. 1995), and DNA by a diphenylamine
method (Frϕlund et al. 1996).
3 Results and discussion
3.2 The settling properties of activated sludge
Parameters which applied to evaluate settling ability of
activated sludge, such as SV30, MLSS, and SVI, are shown
in Fig. 3. Before Cu(II) was dosed (day 1~day 42), SV30,
MLSS, and SVI values in both reactors were similar
(Fig.3a–c), where SVI fluctuated between 290~400 mL/g
(Fig. 3c). This indicated that the sludge in both reactors was
seriously bulking (generally, the SVI value of a sludge with
a good settling property is 60~150, a bulking sludge
150~300, and >300 is thought to be a severely bulking
sludge) (Jenkins et al. 1993). The sludge was bulking due
to several potential reasons, such as temperature, types of

3.1 COD removal efficiency

To investigate the effect of copper on the removal of organic

matter (C6H5COONa) from activated sludge, the COD
removal efficiency was measured, as is shown in Fig. 2.
The COD of the influent of both the reactor continuously
dosed with Cu(II) (ER) and the control without Cu(II) (CR)
was maintained at 650~820 mg/L. Initially, the performance
of both reactors was similar since neither had been exposed
to Cu(II) with a steady COD removal efficiency above 95%
(Fig. 2). This decreased to 42% the day after 20 mg/L copper
was added, with a poor COD removal in the following
40 days (day 43~day 80) of only 2~15%.
Although COD removal increased to 78% after Cu(II)
acclimatization, it was unstable and decreased to zero by
day 162. The recovery at day 80~day 100 might indicate a
short-term adaptation of the microorganisms to Cu(II), but
long-term, continuous dosing absolutely inhibited sludge
activity by day 162. Cu(II) toxicity may have impeded the
formation of flocs and led to sludge disintegration.
However, with no Cu(II) exposure, the COD removal
efficiency remained stable above 90% throughout its
162 days of operation.

Fig. 3 Effect of Cu(II) on the settling properties of bulking activated

Fig. 2 The effect of Cu(II) on the COD removal efficiency of bulking sludge. a SV30, b MLSS, and c SVI in CR (without Cu(II)) and ER
sludge with (ER) and without (CR) continuous exposure to 20 mg/L Cu(II) (with Cu(II))
1570
substrate, temperature, and carbon/nitrogen ratio. This was
similar with previous study on nickel (Li et al. 2011). Ten
days after dosing with Cu(II) (about day 50), the SVI value
began to decrease until day 70, from 578 to 126 mL/g.
After which, it remained at 100~150 mL/g. In contrast, the
bulking property of the unexposed sludge was always high,
achieving as much as 1,974 mL/g on day 133.
The significant improvement in the settling property after
Cu(II) exposure (Fig. 3a) may be due to inhibition of the
growth of the filamentous microorganisms which may be more
vulnerable to Cu(II) toxicity. According to Shuttleworth and
Unz (1998), Cu(II) concentrations above 1 μM (63.5 μg/L)
could totally inhibit the growth of filamentous bacteria and
the SBR was dosed with 20 mg/L Cu(II). As will be
discussed later, the settling ability of activated sludge was
also correlated to the EPS composition.
3.3 Dewatering of activated sludge
To evaluate the dewatering ability of the sludge, the specific
resistance to filtration (SRF) was measured for three sludge
samples taken on days 83, 96, and 110 (Table 1).
Continuous dosing of 20 mg/L Cu(II) improved the
dewatering ability of the bulking sludge as the SRF values
decreased from 9.72×1011 to 5.02×1010 s2/g. However,
without Cu(II) exposure (CR), the dewatering ability was
poorer and deteriorated as the SRF increased from 3.72×1012
to 1.63×1013 s2/g from day 83 to day 110.
3.4 EPS of bulking sludge
3.4.1 EPS quantity and composition
The EPS composition of the activated sludge from both
reactors is shown in Fig. 4. The data for day 42 was
obtained before copper was dosed. EPS consists of loosely
(LB-EPS) and tightly (TB-EPS) bound EPS with LB-EPS
being only a small part of the total EPS (EPSt) (Wingender
et al. 1999).
Although the total EPS was generally much lower, the
proportion of LB-EPS increased after dosing with Cu(II)
(ER in Fig. 4) when compared to no Cu(II) exposure (CR).
Additionally, TB-EPS achieved its highest level of 134 mg/g
of volatile suspended solid (VSS) on day 61 and fell to a
Table 1 Dewatering ability of sludge samples taken from SBRs with
(ER) and without dosing of 20 mg/L Cu(II) (CR)
Average specific resistance filtration (s2/g)
Sample Day 83 Day 96 Day 110
CR (3.72±0.17)×1012 (1.63±0.08)×1013 (1.00±0.05)×1013
ER (9.72±0.49)×1011 (1.76±0.09)×1011 (5.02±0.26)×1010
Environ Sci Pollut Res (2011) 18:1567–1573
Fig. 4 The effect of Cu(II) on EPS content of the activated sludge
with (ER) and without (CR) continuous exposure to 20 mg/L. Sample
at day 42 in ER was taken prior to Cu(II) exposure
minimum on day 105, with only 39 mg/g-VSS. From Fig. 2,
the minimum of COD removal efficiency occurred on day 61
but recovered to a maximum around day 100 in the Cu(II)-
dosed reactor. Without Cu(II) dosing, TB-EPS content was
obviously higher than when dosed with Cu(II), from 103 to
439 mg/g-VSS (Fig. 4). From these data, it would appear that
there is an inverse relationship between COD removal and the
TB-EPS content such that high COD removal occurred when
the TB-EPS content was low and that COD removal was
decreased as the TB-EPS content increased. TB-EPS also
always made up a large proportion of EPSt, ranging from 70%
to 91%. The corresponding COD removal was fairly steady
and high in CR, with an average value of 95.3%.
3.4.2 Comparison of SMP and EPS content
Soluble microbial products (SMP) are usually the major
component of the soluble organic matter in effluents from
biological treatment processes and consist of the utilization-
associated products and the biomass-associated products
(Rittmann et al. 1987). According to Huang et al. (2009),
EPS and SMP have excellent buffering action towards
external environmental changes because the microorganisms
can survive by degrading EPS and SMP under poor nutrient
conditions. The content of total EPS and SMP in CR (without
Cu(II)) and ER (with Cu(II)) reactors is shown in Fig. 5.
Prior to Cu(II) exposure, the EPSt and SMP content were
the same in two reactors, which were 130 and 35 mg/g-
VSS, respectively (Fig. 5). After copper was dosed in ER,
the trend for both EPSt and SMP production was similar.
Both increased and achieved a maximum of 271 and
134 mg/g-VSS on day 61, respectively, while the COD
removal efficiency fell to its minimum (3~5%) with a high
effluent COD around that time. This increase indicated a
microbial response by producing EPSt and SMP to relieve
the sudden exposure to Cu(II). Later decrease
(day 62~day 77) of these values indicated that the micro-
organisms in the sludge might have utilized the biodegrad-
able components in EPSt and SMP to sustain vital
processes. After which, the EPSt content was relatively
Environ Sci Pollut Res (2011) 18:1567–1573
Fig. 5 EPSt and SMP with (ER) and without (CR) continuous dosing
of 20 mg/L Cu(II)
steady till the end of the experiment (day 78~day 105),
while SMP decreased from 104 to 64 mg/g-VSS. Interest-
ingly, recovery of COD removal occurred around day 100
(about 78%). However, the EPSt and SMP trends without
Cu(II) dosing were not regular, with EPSt and SMP widely
varying at 115~522 mg/g-VSS and at 32~220 mg/g-VSS,
respectively. This large fluctuation was possibly related to
severely bulking sludge in CR (without Cu(II)).
Fig. 6 The effect of Cu(II) on the organic composition of EPS from
activated sludge with (ER) and without (CR) exposure to 20 mg/L Cu
(II). a Polysaccharide, b protein, c humic substances, and d DNA
1571
Fig. 7 The effect of Cu(II) on EPS and SVI value corresponding with
time
3.5 Organic composition of bulking sludge
EPS is complex and contains mainly polysaccharide,
protein, humic substances, and DNA, among which,
polysaccharide and protein make up 75~89% of the total
EPS by mass (Zinkevich et al. 1996). The change of these
four major organic components in the activated sludge from
both reactors is shown in Fig. 6. Without exposure to Cu(II)
content in both reactors. Sample at day 42 in ER was taken prior
to Cu(II) exposure
1572
or prior to its exposure, the composition of the activated
sludge was the same. In the control reactor, there was a
general increase in DNA with the achievement of a steady
state by day 67 indicative of microbial growth even though
there was a fluctuation in the protein, polysaccharide, and
humic substances content.
However, after continuous exposure to Cu(II), DNA
content was much higher even though the protein and
polysaccharide levels were significantly lower than in the
control reactor. Different from CR, the increased DNA in
ER was more contributed by intracellular DNA released
through cell lysis after exposure to Cu(II) than the DNA in
EPS, which indicated microbial growth. Average content
for DNA, polysaccharide, and protein in ER were 31.1,
48.5, and 47.9 mg/g-VSS.
3.6 Relationship between EPS and sludge properties
3.6.1 Relationship between EPS and sludge settling
Consistent with previous studies (Liu and Fang 2003), SVI
increased linearly with the EPS content in the sludge
(Fig.7). However as LB-EPS increased, the SVI decreased,
conflicting with the general conclusion of Zinkevich et al.
(1996) who found that LB-EPS played an essential role
in sludge settling since SVI increased with increasing
LB-EPS resulting in decreasing sludge settling. This
inconsistency might be caused by a serious toxic reaction
on the sludge.
Although copper did improve the settling property of
bulking activated sludge, it may not have been due to a
lower quantity of total EPS. Three additional explanations
may account for the improvement. First, even though a
higher proportion of LB-EPS led to poorer settling, the
absorbed copper ions might increase the density of the
activated sludge, which in turn, improved its settling
property. Secondly, negatively charged groups on the EPS
might interact with positively charged Cu(II) ions and
counteract repulsive forces in the sludge flocs making them
easier to settle. Finally, filamentous bacteria, a potential
cause for sludge bulking in this experiment, are more
sensitive to Cu(II) toxicity. The elimination of filamentous
bacteria curbed sludge bulking efficiently.
3.6.2 Relationship between EPS and sludge dewatering
Previous studies found that increasing the EPS content
reduces the dewatering ability of activated sludge and is
reflected as an increase in SRF (Higgins and Novak 1997).
A majority of researchers think that hydrophilic polysac-
charide negatively affects dewatering (Wu et al. 1982).
Activated sludge containing high concentrations of Ca2+
and Mg2+ generally had better dewatering ability (Higgins
Environ Sci Pollut Res (2011) 18:1567–1573
and Novak 1997). Bruus et al. (1993) showed that sludge
containing Cu(II) had the best dewatering ability among
three sludges containing Cu2+, Mg2+, and Ca2+. Total EPS
(Fig.4), polysaccharide (Fig. 6a), and SRF (Table 1)
decreased in the later stages of Cu(II) exposure. This
demonstrated that changes in the dewatering property of
sludge might be caused by copper and affected the
quantity and composition of EPS: copper toxicity
reduced the quantity of EPS and then reduced the water
trapped by it (Vogt et al. 2000), improving the dewatering
property.
4 Conclusion
Continuous exposure in 20 mg/L Cu(II) (1) strongly
inhibited organic matter (i.e., C6H5COONa) removal
even though there was slight but unsteady recovery after
acclimatization to the Cu(II), (2) improved settling and
dewatering properties, and (3) affected EPSt composition
and was eventually at a steady level after day 78. The
settling properties as evaluated by SVI were directly
related to the total EPS while dewatering properties as
measured by SRF were related to both total EPS and the
polysaccharide content.
Acknowledgments The authors wish to express their gratitude to
the Chinese Natural Science Foundation (no. 20377010) and the State
Key Laboratory of Pollution Control and Resources Reuse Foundation
(no. PCRRF10003) for the financial support of this study.
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