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Component analysis and heavy metal adsorption ability of extracellular polymeric substances (EPS) from sulfate reducing bacteria
Bioresource Technology 194 (2015) 399–402
Contents lists available at ScienceDirect
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. Zn
2+
could decrease the toxic effects of Cu2+ and Cd2+ on the SRB.
a r t i c l e
i n f o
Article history: Received 3 June 2015 Received in revised form 13 July 2015 Accepted 14 July 2015 Available online 21 July 2015 Keywords: Sulfate-reducing bacteria Extracellular polymeric substances Heavy metal Adsorption
a b s t r a c t Extracellular polymeric substances (EPS) play an important role in the treatment of acid mine drainage (AMD) by sulfate-reducing bacteria (SRB). In this paper, Desulfovibrio desulfuricans was used as the test strain to explore the effect of heavy metals on the components and adsorption ability of EPS. Fourier-transform infrared (FTIR) spectroscopy analysis results showed that heavy metals did not influence the type of functional groups of EPS. Potentiometric titration results indicated that the acidic constants (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 concentrations of the surface functional groups also increased. Adsorption results suggested that EPS had a specific binding affinity for the dosed heavy metal, and that EPS extracted from the Zn2+-dosed system had a higher binding affinity for all heavy metals. Additionally, Zn2+ decreased the inhibitory effects 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: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.biortech.2015.07.042 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
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 wastewaters, 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.
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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 CaCl22H2O, 2.0 g/L MgSO47H2O, 2.0 g/L DL-Na-lactate, 1.0 g/L yeast extract, 0.5 g/L FeSO47H2O, 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 suspended 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 previously 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 constituents were calculated based on the EPS-C. Total EPS were determined 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 spectrometer (Bruker, Fällanden, Switzerland). Titrations of EPS solution were conducted under an N2 atmosphere at 25 °C using an automatic potentiometric titrator (Metrohm, Herisau, Switzerland). Cu2+, Zn2+, and Cd2+ were measured using atomic adsorption spectrometry (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 polysaccharides 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 composition of the EPS, as well as their relative contents, changed significantly. 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 contents (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 functional groups on the EPS were observed in the FTIR spectra (data not shown). The FTIR analysis results showed that all EPS generated 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 phosphate 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 corresponding to the equivalence points, and the local minima
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Z.-B. Yue et al. / Bioresource Technology 194 (2015) 399–402 Table 1 Compositions of EPS extracted from heavy metal dosed systems.
Polysaccharide (mg/mg EPS-C) Protein (mg/mg EPS-C) Nucleic acid (mg/mg EPS-C) EPS-C (mg/mg EPS) Polysaccharide/protein Total EPS (mg EPS/mg TS)
Control-EPS
Cu-EPS
Zn-EPS
Cd-EPS
0.63 ± 0.05 1.44 ± 0.06 0.14 ± 0.04 0.45 ± 0.07 0.44 ± 0.03 0.015 ± 0.003
0.98 ± 0.08 1.12 ± 0.08 0.14 ± 0.02 0.44 ± 0.02 0.88 ± 0.01 0.016 ± 0.002
0.74 ± 0.04 1.35 ± 0.02 0.16 ± 0.01 0.45 ± 0.04 0.55 ± 0.08 0.020 ± 0.005
0.51 ± 0.05 1.54 ± 0.02 0.14 ± 0.04 0.45 ± 0.07 0.33 ± 0.05 0.013 ± 0.003
Table 2 pKa values and the site concentrations of EPS.
Control EPS Cu-EPS Zn-EPS Cd-EPS
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)
3.5 3.8 3.6 3.8
1.06 1.3 1.3 0.78
6.4 5.9 6.0 6.4
0.93 0.9 1.1 0.99
9.6 9.8 9.4 9.0
1.05 1.15 1.35 1.06
indicated a minimum variation in pH, which is indicative of buffering (Braissant et al., 2007). The pKas of the proton-binding sites, as well as their concentrations, were also estimated (Table 2). Moreover, the concentration of carboxyl groups in the Cd-EPS was obviously reduced. The concentration of active sites of the Zn-EPS was greater than that of the control-EPS, indicating that Zn2+ could increase the concentration of active sites. Nevertheless, the concentration of active sites on the amino/hydroxyl groups of the Zn-EPS was more than those of the other EPS, which demonstrated that Zn2+ could better promote the production of amino/hydroxyl groups, as well as protecting the cells.
1.8
Cu Zn Cd
1.6 1.4
Adsorption density (mg/mg EPS-C)
EPS samples
1.2 1.0 0.8 0.6 0.4 0.2 0.0
Control-EPS
The differences in the active sites on the EPS samples resulted in competition between different heavy metals for the EPS. Batch adsorption test results showed that EPS extracted from the Cu2+-dosed culture had a higher binding capacity for Cu2+ than for other heavy metals. Similar results were achieved in the other batches (Fig. 1). This meant that SRB had the ability to change their EPS production metabolism to generate more functional groups that had the capacity to bind the dosed heavy metals. This would be benefit for the application of SRB in the bioremediation process of AMD. SRB secreted EPS to decrease the toxic effect of heavy metal and generate S2 to precipitate the heavy metals. The FTIR analysis results showed that the types of functional groups did not change. There was no significant correlation between the adsorption densities and the concentrations of the functional groups (p > 0.05). This meant that the binding capacities of the EPS for the heavy metals were mainly determined by the syntheses of the different functional groups. Regarding adsorption 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 binding sites on the EPS samples. Just as Cd2+ bound easily to carboxyl, phosphate, and amino groups, in which the amino group was the preferred binding group, CAH, OAH, and NAH groups played an important role in the process of Zn2+ resistance. Cu2+ easily bound the carboxyl groups of EPS (Li and Yu, 2014). In general, metal biosorption by EPS involves physicochemical interactions between the metal and the functional groups on the cell surface (Yin et al., 2013). It is known that EPS may protect cells by binding toxic metal ions (Li et al., 2011), and that bacteria can increase their EPS production in the presence of toxic metals as a defense mechanism. In addition, more metals were bound by the Zn-EPS than the other EPS, which showed that Zn2+ can greatly improve the
Cu-EPS
Zn-EPS
Cd-EPS
EPS
3.4. Metal binding affinities of EPS
Fig. 1. Adsorption capacity of EPS to heavy metals.
ability of EPS to bind other heavy metals. However, the adsorption ability of Cu-EPS to four kinds of metals was weakest. This could be attributed to the low production of phosphate/thiol and other functional groups in the EPS (Table 2). 4. Conclusions Chemical and FITR analysis results indicated that the main components of SRB EPS were proteins and polysaccharides, which included carboxyl, thiol/phosphate, and amino/hydroxyl functional groups. The presence of heavy metals had no effect on the type of functional groups of the EPS samples, while it increased the concentrations of the surface functional groups. Heavy metals promoted the interaction between EPS and metal ions. Zn-EPS had a higher adsorption density, which partially explained why Zn2+ could decrease the toxic effects of Cu2+ and Cd2+ on the SRB. Acknowledgements The authors wish to thank the Special Program for National Natural Science Foundation of China (41130206, 41102214 and 41372347) for the support of this study. References Braissant, O., Decho, A.W., Dupraz, C., 2007. Exopolymeric substances of sulfatereducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals. Geobiology 5 (4), 401–411. Bridge, T.A.M., White, C., Gadd, G.M., 1999. Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans. Microbiology 145 (10), 2987–2995.
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Comte, S., Guibaud, G., Baudu, M., 2008. Biosorption properties of extracellular polymeric substances (EPS) towards Cd, Cu and Pb for different pH values. J. Hazard. Mater. 151 (1), 185–193. D’Abzac, P., Bordas, F., van Hullebusch, E., Lens, P.N., Guibaud, G., 2010. Effects of extraction procedures on metal binding properties of extracellular polymeric substances (EPS) from anaerobic granular sludge. Colloids Surf. B 80 (2), 161– 168. D’Abzac, P., Bordas, F., Joussein, E., van Hullebusch, E., Lens, P.N., Guibaud, G., 2013. Metal binding properties of extracellular polymeric substances extracted from anaerobic granular sludges. Environ. Sci. Pollut. Res. 20 (7), 4509–4519. Fang, L., Wei, X., Cai, P., 2011. Role of extracellular polymeric substances in Cu(II) adsorption on Bacillus subtilis and Pseudomonas putida. Bioresour. Technol. 102 (2), 1137–1141. Guine, V., Spadini, L., Sarret, G., Muris, M., Delolme, C., Gaudet, J.P., Martins, J.M.F., 2006. Zinc sorption to three gram-negative bacteria: combined titration, modeling, and EXAFS study. Environ. Sci. Technol. 40 (6), 1806–1813. Jalali, K., Baldwin, S.A., 2000. The role of sulfate reducing bacteria in copper removal from aqueous sulfate solutions. Water Res. 34 (3), 797–806. Li, J., Liu, X., Liu, Y., Ramsay, J., Yao, C., Dai, R., 2011. The effect of continuous exposure of copper on the properties and extracellular polymeric substances (EPS) of bulking activated sludge. Environ. Sci. Pollut. Res. 18 (9), 1567–1573. Li, W.W., Yu, H.Q., 2014. Insight into the roles of microbial extracellular polymer substances in metal biosorption. Bioresour. Technol. 160, 15–23.
Liu, H., Fang, H.H.P., 2002. Characterization of electrostatic binding sites of extracellular polymers by linear programming analysis of titration data. Biotechnol. Bioeng. 80 (7), 806–811. Sheng, G.P., Xu, J., Luo, H.W., 2013. Thermodynamic analysis on the binding of heavy metals onto extracellular polymeric substances (EPS) of activated sludge. Water Res. 47 (2), 607–614. Sheng, G.P., Yu, H.Q., Yue, Z.B., 2005. Production of extracellular polymeric substances from Rhodopseudomonas acidophilain the presence of toxic substances. Appl. Microbiol. Biotechnol. 69 (2), 216–222. Wang, J., Li, Q., Li, M.M., Chen, T.H., Zhou, Y.F., Yue, Z.B., 2014. Competitive adsorption of heavy metal by EPS extracted from sulfate reducing bacteria. Bioresour. Technol. 163, 374–376. Wan, Z.Q., Yu, Y.Y., Li, M.M., Wang, J., Peng, S.C., Yue, Z.B., 2012. Proceedings of the 4th International Conference on Environmental Technology and Knowledge Transfer, Hefei, PR China. Yin, Y.R., Hu, Y.Y., Xiong, F., 2011. Sorption of Cu(II) and Cd (II) by extracellular cellular polymeric substances(EPS) from Aspergillus fumigatus. Int. Biodeter. Biodegrad. 65 (7), 1012–1018. Yin, Y.R., Hu, Y.Y., Xiong, F., 2013. Biosorption properties of Cd(II), Pb(II), and Cu(II) of extracellular polymeric substances (EPS) extracted from Aspergillus fumigatu sand determined by polarographic method. Environ. Monit. Assess. 185 (8), 6713–6718.
Contents lists available at ScienceDirect
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. Zn
2+
could decrease the toxic effects of Cu2+ and Cd2+ on the SRB.
a r t i c l e
i n f o
Article history: Received 3 June 2015 Received in revised form 13 July 2015 Accepted 14 July 2015 Available online 21 July 2015 Keywords: Sulfate-reducing bacteria Extracellular polymeric substances Heavy metal Adsorption
a b s t r a c t Extracellular polymeric substances (EPS) play an important role in the treatment of acid mine drainage (AMD) by sulfate-reducing bacteria (SRB). In this paper, Desulfovibrio desulfuricans was used as the test strain to explore the effect of heavy metals on the components and adsorption ability of EPS. Fourier-transform infrared (FTIR) spectroscopy analysis results showed that heavy metals did not influence the type of functional groups of EPS. Potentiometric titration results indicated that the acidic constants (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 concentrations of the surface functional groups also increased. Adsorption results suggested that EPS had a specific binding affinity for the dosed heavy metal, and that EPS extracted from the Zn2+-dosed system had a higher binding affinity for all heavy metals. Additionally, Zn2+ decreased the inhibitory effects 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: [email protected] (J. Wang). http://dx.doi.org/10.1016/j.biortech.2015.07.042 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
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 wastewaters, 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.
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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 CaCl22H2O, 2.0 g/L MgSO47H2O, 2.0 g/L DL-Na-lactate, 1.0 g/L yeast extract, 0.5 g/L FeSO47H2O, 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 suspended 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 previously 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 constituents were calculated based on the EPS-C. Total EPS were determined 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 spectrometer (Bruker, Fällanden, Switzerland). Titrations of EPS solution were conducted under an N2 atmosphere at 25 °C using an automatic potentiometric titrator (Metrohm, Herisau, Switzerland). Cu2+, Zn2+, and Cd2+ were measured using atomic adsorption spectrometry (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 polysaccharides 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 composition of the EPS, as well as their relative contents, changed significantly. 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 contents (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 functional groups on the EPS were observed in the FTIR spectra (data not shown). The FTIR analysis results showed that all EPS generated 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 phosphate 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 corresponding to the equivalence points, and the local minima
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Z.-B. Yue et al. / Bioresource Technology 194 (2015) 399–402 Table 1 Compositions of EPS extracted from heavy metal dosed systems.
Polysaccharide (mg/mg EPS-C) Protein (mg/mg EPS-C) Nucleic acid (mg/mg EPS-C) EPS-C (mg/mg EPS) Polysaccharide/protein Total EPS (mg EPS/mg TS)
Control-EPS
Cu-EPS
Zn-EPS
Cd-EPS
0.63 ± 0.05 1.44 ± 0.06 0.14 ± 0.04 0.45 ± 0.07 0.44 ± 0.03 0.015 ± 0.003
0.98 ± 0.08 1.12 ± 0.08 0.14 ± 0.02 0.44 ± 0.02 0.88 ± 0.01 0.016 ± 0.002
0.74 ± 0.04 1.35 ± 0.02 0.16 ± 0.01 0.45 ± 0.04 0.55 ± 0.08 0.020 ± 0.005
0.51 ± 0.05 1.54 ± 0.02 0.14 ± 0.04 0.45 ± 0.07 0.33 ± 0.05 0.013 ± 0.003
Table 2 pKa values and the site concentrations of EPS.
Control EPS Cu-EPS Zn-EPS Cd-EPS
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)
3.5 3.8 3.6 3.8
1.06 1.3 1.3 0.78
6.4 5.9 6.0 6.4
0.93 0.9 1.1 0.99
9.6 9.8 9.4 9.0
1.05 1.15 1.35 1.06
indicated a minimum variation in pH, which is indicative of buffering (Braissant et al., 2007). The pKas of the proton-binding sites, as well as their concentrations, were also estimated (Table 2). Moreover, the concentration of carboxyl groups in the Cd-EPS was obviously reduced. The concentration of active sites of the Zn-EPS was greater than that of the control-EPS, indicating that Zn2+ could increase the concentration of active sites. Nevertheless, the concentration of active sites on the amino/hydroxyl groups of the Zn-EPS was more than those of the other EPS, which demonstrated that Zn2+ could better promote the production of amino/hydroxyl groups, as well as protecting the cells.
1.8
Cu Zn Cd
1.6 1.4
Adsorption density (mg/mg EPS-C)
EPS samples
1.2 1.0 0.8 0.6 0.4 0.2 0.0
Control-EPS
The differences in the active sites on the EPS samples resulted in competition between different heavy metals for the EPS. Batch adsorption test results showed that EPS extracted from the Cu2+-dosed culture had a higher binding capacity for Cu2+ than for other heavy metals. Similar results were achieved in the other batches (Fig. 1). This meant that SRB had the ability to change their EPS production metabolism to generate more functional groups that had the capacity to bind the dosed heavy metals. This would be benefit for the application of SRB in the bioremediation process of AMD. SRB secreted EPS to decrease the toxic effect of heavy metal and generate S2 to precipitate the heavy metals. The FTIR analysis results showed that the types of functional groups did not change. There was no significant correlation between the adsorption densities and the concentrations of the functional groups (p > 0.05). This meant that the binding capacities of the EPS for the heavy metals were mainly determined by the syntheses of the different functional groups. Regarding adsorption 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 binding sites on the EPS samples. Just as Cd2+ bound easily to carboxyl, phosphate, and amino groups, in which the amino group was the preferred binding group, CAH, OAH, and NAH groups played an important role in the process of Zn2+ resistance. Cu2+ easily bound the carboxyl groups of EPS (Li and Yu, 2014). In general, metal biosorption by EPS involves physicochemical interactions between the metal and the functional groups on the cell surface (Yin et al., 2013). It is known that EPS may protect cells by binding toxic metal ions (Li et al., 2011), and that bacteria can increase their EPS production in the presence of toxic metals as a defense mechanism. In addition, more metals were bound by the Zn-EPS than the other EPS, which showed that Zn2+ can greatly improve the
Cu-EPS
Zn-EPS
Cd-EPS
EPS
3.4. Metal binding affinities of EPS
Fig. 1. Adsorption capacity of EPS to heavy metals.
ability of EPS to bind other heavy metals. However, the adsorption ability of Cu-EPS to four kinds of metals was weakest. This could be attributed to the low production of phosphate/thiol and other functional groups in the EPS (Table 2). 4. Conclusions Chemical and FITR analysis results indicated that the main components of SRB EPS were proteins and polysaccharides, which included carboxyl, thiol/phosphate, and amino/hydroxyl functional groups. The presence of heavy metals had no effect on the type of functional groups of the EPS samples, while it increased the concentrations of the surface functional groups. Heavy metals promoted the interaction between EPS and metal ions. Zn-EPS had a higher adsorption density, which partially explained why Zn2+ could decrease the toxic effects of Cu2+ and Cd2+ on the SRB. Acknowledgements The authors wish to thank the Special Program for National Natural Science Foundation of China (41130206, 41102214 and 41372347) for the support of this study. References Braissant, O., Decho, A.W., Dupraz, C., 2007. Exopolymeric substances of sulfatereducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals. Geobiology 5 (4), 401–411. Bridge, T.A.M., White, C., Gadd, G.M., 1999. Extracellular metal-binding activity of the sulphate-reducing bacterium Desulfococcus multivorans. Microbiology 145 (10), 2987–2995.
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