Bioresource Technology 194 (2015) 399–402

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Component analysis and heavy metal adsorption ability of extracellular

polymeric substances (EPS) from sulfate reducing bacteria
Zheng-Bo Yue, Qing Li, Chuan-chuan Li, Tian-hu Chen, Jin Wang ⇑
School of Resources and Environmental Engineering, Hefei University of Technology, Hefei, Anhui 230009, China

h i g h l i g h t s

 SRB EPS included carboxyl, thiol/phosphate, amino/hydroxyl functional groups.

 Heavy metals had no effect on the type of functional groups of the EPS samples.
 Heavy metals increased the concentrations of the surface functional groups.
2+
 EPS extracted from the Zn -dosed system had a higher binding affinity.
2+
 Zn could decrease the toxic effects of Cu2+ and Cd2+ on the SRB.

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

Article history: Extracellular polymeric substances (EPS) play an important role in the treatment of acid mine drainage
Received 3 June 2015 (AMD) by sulfate-reducing bacteria (SRB). In this paper, Desulfovibrio desulfuricans was used as the test
Received in revised form 13 July 2015 strain to explore the effect of heavy metals on the components and adsorption ability of EPS.
Accepted 14 July 2015
Fourier-transform infrared (FTIR) spectroscopy analysis results showed that heavy metals did not influ-
Available online 21 July 2015
ence the type of functional groups of EPS. Potentiometric titration results indicated that the acidic con-
stants (pKa) of the EPS fell into three ranges of 3.5–4.0, 5.9–6.7, and 8.9–9.8. The adsorption site
Keywords:
concentrations of the surface functional groups also increased. Adsorption results suggested that EPS
Sulfate-reducing bacteria
Extracellular polymeric substances
had a specific binding affinity for the dosed heavy metal, and that EPS extracted from the Zn2+-dosed sys-
Heavy metal tem had a higher binding affinity for all heavy metals. Additionally, Zn2+ decreased the inhibitory effects
Adsorption of Cd2+ and Cu2+ on the SRB.
Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Acid mine drainage (AMD) has become a major threat to the
environment because of its low pH and high concentrations of
heavy metals. Previous studies have shown that sulfate-reducing
bacteria (SRB) dominant biological process is an effective means
of treatment AMD (Jalali and Baldwin, 2000). In addition to their
ability to generate S2 , which results in the precipitation of solid
sulfides, high extracellular metal binding capacity of extracellular
polymeric substances (EPS) is another potential advantage of using
SRB to remove heavy metals from AMD (Bridge et al., 1999).
EPS are mainly composed of polysaccharides, proteins, nucleic
acids, and lipids. EPS contain functional groups, such as carboxyl,
phosphoric, amine, and hydroxyl groups, which play a crucial role
⇑ Corresponding author. Tel.: +86 551 62901523; fax: +86 551 62901524.
E-mail address: sophiawj@hfut.edu.cn (J. Wang).
in the biosorption of heavy metals (Comte et al., 2008; Liu and
Fang, 2002). The amount of active functional binding sites in the
EPS has a significant effect on the potential heavy metals removal
efficiency of microbes. EPS has different heavy metal binding
capacities and enhances the heavy metal binding capacities of cell
surfaces (Fang et al., 2011; Li and Yu, 2014). In the SRB-dominated
process for the treatment of AMD or other heavy metal wastewa-
ters, several kinds of heavy metals co-exist and influence microbial
activity, including EPS. However, few reports have examined the
effects of heavy metals on the EPS binding capacities for other
types of heavy metals.
In the current study, a sulfate-reducing strain, Desulfovibrio
desulfuricans, was used. The production of EPS in the presence of
Cu2+, Zn2+, and Cd2+, as well as their binding capacities for heavy
metals, were investigated. The potential functional binding sites
of the EPS were also characterized with Fourier transform infrared
spectroscopy (FTIR) and acid–base titration.

http://dx.doi.org/10.1016/j.biortech.2015.07.042
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
400 Z.-B. Yue et al. / Bioresource Technology 194 (2015) 399–402
2. Methods
2.1. Bacterial cultivation and EPS extraction
The strain of D. desulfuricans (GenBank/HQ022824.1) was
isolated from Wangxiaoying Wastewater Treat Plant and
cultivated at 35 °C using the Starkey medium. The culture medium
consisted of: 0.5 g/L K2HPO4, 1.0 g/L NH4Cl, 1.0 g/L Na2SO4, 0.1 g/L
CaCl2
2H2O, 2.0 g/L MgSO4
7H2O, 2.0 g/L DL-Na-lactate, 1.0 g/L
yeast extract, 0.5 g/L FeSO4
7H2O, and 0.1 g/L ascorbic acid.
EPS were extracted using disodium ethylenediaminetetraacetic
acid (EDTA) as previously described which had limited negative
influence on the cell dialysis (Wan et al., 2012). Copper, cadmium,
zinc standard stock solutions were prepared by dissolving CuCl2,
CdCl2, and ZnCl2 into Milli-Q water (EMD Millipore, Billerica, MA,
USA).
2.2. Experimental design
Solutions (10 mg/L) of Cu2+, Zn2+, Cd2+, in the form of CuCl2,
ZnCl2, and CdCl2, respectively, were added to the medium to the
desired concentrations. A culture without added heavy metals
was used as the control. Cultures were grown in 300 ml
rubber-stopper vials at 35 °C for 40 h to extract the EPS.
Another batch test was performed to investigate whether Zn
could decrease the inhibitory effects of Cu2+ and Cd2+ in the SRB.
Three heavy metal dosages—5, 10, and 20 mg/L—were first used
to determine the effects of the single metals. Mixtures of Cu2+
and Zn2+, as well as Cd2+ and Zn2+, were also used because Zn2+
had no significant influence on SRB activity (p > 0.05). The initial
Cu2+ and Cd2+ concentrations were 20 mg/L. The dosages of Zn2+
were 5 and 10 mg/L. Each batch was examined in triplicate. After
a 40 h cultivation, the sulfate concentration was measured.
A dialysis bag filled with 10 mL of an EPS solution was sus-
pended in 50 mL heavy metal solutions to investigate the binding
capacity of the EPS. All adsorption experiments were conducted
in triplicate at room temperature and a pH of 5.0 at an initial heavy
metal concentration of 50 mg/L. The process was performed as pre-
viously described (Wang et al., 2014).
2.3. Analytical methods
Total organic carbon (TOC) in the EPS solution was analyzed
using a C/N 2100 TOC analyzer (Jena, Germany) and noted as
EPS-C in the following text. Polysaccharides, proteins, and nucleic
acids in the extracted EPS solution were determined as previously
described (Sheng et al., 2005). The concentrations of the EPS con-
stituents were calculated based on the EPS-C. Total EPS were deter-
mined as the sum of the three components. The tests were
repeated three times.
Extracts of EPS were freeze dried to compress them into pellets
(about 1 mg of EPS was mixed with about 180 mg of KBr). The
infrared investigation was performed using a Vertex 70 spectrom-
eter (Bruker, Fällanden, Switzerland). Titrations of EPS solution
were conducted under an N2 atmosphere at 25 °C using an auto-
matic potentiometric titrator (Metrohm, Herisau, Switzerland).
Cu2+, Zn2+, and Cd2+ were measured using atomic adsorption spec-
trometry (WYG 2200, China).
3. Results and discussion
3.1. Effects of heavy metals on SRB activity and EPS components
The results showed that Zn2+ had no significant effect on
the sulfate reducing efficiency (p > 0.05). When the initial
concentration of heavy metals was 20 mg/L, the sulfate reduction
efficiency was reduced by 38.6% and 32.0% for Cu2+ and Cd2+,
respectively. When Zn2+ was added to the Cu-dosed system, the
sulfate reduction efficiency did not change. However, the addition
of Zn2+ significantly increased the sulfate reduction efficiency from
59.6% to 85.1% in Cd-dosed system (p < 0.01). This meant that the
addition of Zn2+ decreased the toxic effect of Cd2+ on the SRB.
The compositions of the EPS extracted from the heavy
metal-dosed cultures are shown in Table 1. Few nucleic acids were
detected, which confirmed that the SRB cells were not disrupted
during the extraction process. The results showed that polysaccha-
rides and proteins were the major components of the EPS. When
Cu2+ and Cd2+ were added, the total production of EPS was not
influenced significantly (p > 0.05). However, the total production
of EPS increased significantly when Zn2+ was added (p < 0.01). In
addition to the difference in the total production of EPS, the com-
position of the EPS, as well as their relative contents, changed sig-
nificantly. The polysaccharide contents of Cu-EPS increased
compared with the control-EPS, while the protein contents
decreased significantly (p < 0.01). Compared with the control-EPS,
Cd-EPS contained lower polysaccharide and higher protein con-
tents (p < 0.05).
The protein content increased and the polysaccharide content
decreased in the presence of Cd2+. Cd2+ has a stronger binding
affinity for the amino groups of proteins, and protein production
increases to protect the bacteria from toxic elements (Yin et al.,
2011). Polysaccharides increased and proteins decreased in the
EPS in the presence of Cu2+ and Zn2+. It was concluded that Cu2+
and Zn2+ had greater binding affinity for the functional groups of
polysaccharides in the EPS. For example, Cu2+ binds the carboxyl
groups of EPS, while carboxyl and phosphoric functional groups
are involved in the binding between Pb2+ and EPS (Li and Yu,
2014; D’Abzac et al., 2013).
3.2. FTIR analysis
A number of absorption peaks that represented various func-
tional groups on the EPS were observed in the FTIR spectra (data
not shown). The FTIR analysis results showed that all EPS gener-
ated from the different heavy metal-dosed cultures did not exhibit
any differences in their functional groups and peak positions.
However, the peak intensity changed in response to dosing with
heavy metals. In the presence of Cu2+, the CAO, C@O, and CAN
(amide I) contents of EPS increased. The presence of Zn2+ increased
the CAH, OAH, and NAH contents. Thus, it is concluded that these
groups play an important role in resisting the toxic effects of heavy
metals. The FTIR analysis results also confirmed that the intensities
of the functional groups in the Cd-EPS decreased, which was in
accordance with the chemical analysis results (Table 1).
3.3. Potentiometric titration
EPS play a crucial role in the biosorption of heavy metals due to
the presence of active functional binding sites (D’Abzac et al., 2010;
Guine et al., 2006; Sheng et al., 2013). The potentiometric titration
using Protofit Software v. 2.1 allows the determination of the acidic
constants (pKa) of the functional groups involved in proton binding
(D’Abzac et al., 2013). The potentiometric titration of EPS showed
three buffering zones. The first zone was located around pH 3.5
and below, which was attributed to carboxyl groups. The second
zone was located at pH 6.4, which was attributed to thiol or phos-
phate groups. The third buffering zone was located at pH 9.6,
which was attributed to amino or hydroxyl groups (D’Abzac
et al., 2013). The peaks indicated a maximum variation in pH cor-
responding to the equivalence points, and the local minima
 Z.-B. Yue et al. / Bioresource Technology 194 (2015) 399–402 401

Table 1
Compositions of EPS extracted from heavy metal dosed systems.

Control-EPS Cu-EPS Zn-EPS Cd-EPS

Polysaccharide (mg/mg EPS-C) 0.63 ± 0.05 0.98 ± 0.08 0.74 ± 0.04 0.51 ± 0.05
Protein (mg/mg EPS-C) 1.44 ± 0.06 1.12 ± 0.08 1.35 ± 0.02 1.54 ± 0.02
Nucleic acid (mg/mg EPS-C) 0.14 ± 0.04 0.14 ± 0.02 0.16 ± 0.01 0.14 ± 0.04
EPS-C (mg/mg EPS) 0.45 ± 0.07 0.44 ± 0.02 0.45 ± 0.04 0.45 ± 0.07
Polysaccharide/protein 0.44 ± 0.03 0.88 ± 0.01 0.55 ± 0.08 0.33 ± 0.05
Total EPS (mg EPS/mg TS) 0.015 ± 0.003 0.016 ± 0.002 0.020 ± 0.005 0.013 ± 0.003

Table 2
pKa values and the site concentrations of EPS.

EPS samples Site 1, 3.5–4.0 carboxyl Site 2, 5.9–6.7 thiol/phosphate Site 3, 8.9–9.8 amino/hydroxyl
pKa Concentrations (mol/kg) pKa Concentrations (mol/kg) pKa Concentrations (mol/kg)
Control EPS 3.5 1.06 6.4 0.93 9.6 1.05
Cu-EPS 3.8 1.3 5.9 0.9 9.8 1.15
Zn-EPS 3.6 1.3 6.0 1.1 9.4 1.35
Cd-EPS 3.8 0.78 6.4 0.99 9.0 1.06

indicated a minimum variation in pH, which is indicative of buffer-

ing (Braissant et al., 2007). 1.8
The pKas of the proton-binding sites, as well as their concentra- Cu
1.6 Zn
tions, were also estimated (Table 2). Moreover, the concentration
1.4 Cd
of carboxyl groups in the Cd-EPS was obviously reduced. The con-

Adsorption density
(mg/mg EPS-C)
1.2
centration of active sites of the Zn-EPS was greater than that of the
control-EPS, indicating that Zn2+ could increase the concentration 1.0
of active sites. Nevertheless, the concentration of active sites on 0.8
the amino/hydroxyl groups of the Zn-EPS was more than those of 0.6
the other EPS, which demonstrated that Zn2+ could better promote 0.4
the production of amino/hydroxyl groups, as well as protecting the 0.2
cells. 0.0
Control-EPS Cu-EPS Zn-EPS Cd-EPS

3.4. Metal binding affinities of EPS EPS

Fig. 1. Adsorption capacity of EPS to heavy metals.

The differences in the active sites on the EPS samples resulted in
competition between different heavy metals for the EPS. Batch ability of EPS to bind other heavy metals. However, the adsorption
adsorption test results showed that EPS extracted from the ability of Cu-EPS to four kinds of metals was weakest. This could be
Cu2+-dosed culture had a higher binding capacity for Cu2+ than attributed to the low production of phosphate/thiol and other
for other heavy metals. Similar results were achieved in the other functional groups in the EPS (Table 2).
batches (Fig. 1). This meant that SRB had the ability to change their
EPS production metabolism to generate more functional groups
4. Conclusions
that had the capacity to bind the dosed heavy metals. This would
be benefit for the application of SRB in the bioremediation process
Chemical and FITR analysis results indicated that the main com-
of AMD. SRB secreted EPS to decrease the toxic effect of heavy
ponents of SRB EPS were proteins and polysaccharides, which
metal and generate S2 to precipitate the heavy metals.
included carboxyl, thiol/phosphate, and amino/hydroxyl functional
The FTIR analysis results showed that the types of functional
groups. The presence of heavy metals had no effect on the type of
groups did not change. There was no significant correlation
functional groups of the EPS samples, while it increased the con-
between the adsorption densities and the concentrations of the
centrations of the surface functional groups. Heavy metals pro-
functional groups (p > 0.05). This meant that the binding capacities
moted the interaction between EPS and metal ions. Zn-EPS had a
of the EPS for the heavy metals were mainly determined by the
higher adsorption density, which partially explained why Zn2+
syntheses of the different functional groups. Regarding adsorption
could decrease the toxic effects of Cu2+ and Cd2+ on the SRB.
to the other metals, the adsorption ability of Zn-EPS to Cu2+ was
the weakest. It was related to the quantity and location of the bind-
Acknowledgements
ing sites on the EPS samples. Just as Cd2+ bound easily to carboxyl,
phosphate, and amino groups, in which the amino group was the
The authors wish to thank the Special Program for National
preferred binding group, CAH, OAH, and NAH groups played an
Natural Science Foundation of China (41130206, 41102214 and
important role in the process of Zn2+ resistance. Cu2+ easily bound
41372347) for the support of this study.
the carboxyl groups of EPS (Li and Yu, 2014). In general, metal
biosorption by EPS involves physicochemical interactions between
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