Bioresource Technology 160 (2014) 119–122
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biosorption of Hg(II) onto goethite with extracellular polymeric substances Wenjuan Song a, Xiangliang Pan a,⇑, Shuyong Mu a, Daoyong Zhang b, Xue Yang a, Duu-Jong Lee b,⇑ a b
Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan
h i g h l i g h t s Interactions between Hg(II), EPS and goethite were investigated. EPS from Chroococcus sp. enhances adsorption capacity of Hg(II) on goethite. EPS binds with Hg(II) to quench the EEM peaks of proteins on EPS. Goethite–EPS soil is a larger Hg(II) sink than goethite alone soil. Biosorption significantly affects the mobility of Hg(II) in goethite soils.
a r t i c l e
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Article history: Available online 21 December 2013 Keywords: Chroococcus sp. Extracellular polymeric substances Mercury Goethite
a b s t r a c t This study characterized the interactions of goethite, EPS from cyanobacterium Chroococcus sp. and Hg(II) using excitation emission matrix (EEM) spectra and adsorption isotherms. Three protein-like fluorescence peaks were noted to quench in the presence of Hg(II). The estimated conditional stability constant (log Ka) and the binding constant (log Kb) of the studied EPS–Hg(II) systems ranged 3.84–4.24 and 6.99– 7.69, respectively. The proteins in EPS formed stable complex with Hg(II). The presence of proteins of Chroococcus sp. enhanced the adsorption capacity of Hg(II) on goethite; therefore, the goethite–EPS soil is a larger Hg(II) sink than goethite alone soil. Biosorption significantly affects the mobility of Hg(II) in goethite soils. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Mercury (Hg) is commonly found in soil and sediment which can induce neurological and renal disturbances and impairment of pulmonary function of human bodies. Iron oxides have strong affinity to Hg(II) so the transport and fate of latter in soil and top sediment layers are significantly affected by the presence of ubiquitous iron oxides. Goethite (a-FeOOH) is the commonly found iron oxide mineral in all types of soils and top-layer freshwater sediments. Goethite has high surface area and surface charge and thus has strong adsorption capacity to numerous heavy metals (Kim et al., 2004; Song et al., 2013). Soil and sediments provide ideal habitats for microorganisms including cyanobacteria. Cyanobacteria are capable of secreting large quantities of extracellular polymeric substance (EPS), which are mainly composed of polysaccharides, proteins, humic substances and nucleic acids (Adav et al., 2010). As one of the
⇑ Corresponding authors. Tel./fax: +86 991 7885446 (X. Pan). Tel.: +886 2 23570516; fax: +886 2 23623040 (D.-J. Lee). E-mail addresses: [email protected] (X. Pan), [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.052
major types of dissolved organic matter (DOM), EPS from cyanobacteria significantly affect mobility and bioavailability of heavy metals (Pan et al., 2012; Zhang et al., 2013). Backstrom (2003) noted that adsorption capacity of Hg(II) by goethite was increased in the presence of fulvic acid. Omoike and Chorover (2006) noted that EPS could be adsorbed to the surface of mineral particles to interefere with film formation on iron oxide surfaces. Organic matters should be able to affect the adsorption process of Hg(II) by goethite, and hence the transport and fate of Hg(II) in soil. However, related studies are lacking. Studies on the adsorption of Hg(II) on goethite in the presence of EPS provide complementary knowledge about biogeochemical processes of mercury in soil and sediments an assess performance of bioremediation of heavy metal contaminated soil and sediment since most bacteria used for remediation persistently excrete EPS. Chroococcus, a typical genus of cyanobacteria, lives luxuriously in various habitats, including soil, the sludge of freshwater lake, river bottoms, and water sources of high salinity. Like other cyanobacteria, Chroococcus produces large quantities of EPS, whose presence may influence the interfacial processes of Hg(II) on the surface of goethite. This study aims at exploring whether proteins, a major
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W. Song et al. / Bioresource Technology 160 (2014) 119–122
part of EPS from Chroococcus sp., would affect adsorption of Hg(II) on goethite. 2. Methods 2.1. Cyanobacterial culture and EPS extraction
When small molecules are bound independently to a set of equivalent sites on a macromolecule, the fluorescence intensity data can be used to obtain the binding constants and the number of binding sites for the complex. The binding constant (log Kb) and the number of binding site (n) can be determined by the following equation (Hill, 1985):
log½ðF 0 FÞ=F ¼ log K b þ n log½HgðIIÞ The cyanobacterium Chroococcus sp., isolated from the soil sample in FuKang, Xinjiang, China, was cultivated in BG-11 medium at 30 °C and 55 lmols photons m2 s1 in a 12 h:12 h light:dark cycle. The collected Chroococcus sp. cells were first centrifugated at 5500 rpm for 10 min at 4 °C. The collected biomass were re-suspended in milli-Q water. The suspension was centrifugated again at 12300 rpm for 20 min at 4 °C. The supernatant was filtered by a 0.45-lm membrane and then purified with dialysis membrane (3500 Da) at 4 °C for 24 h (Adav and Lee, 2008). 2.2. Determination of biochemical composition of EPS The yield of EPS was determined by drying and weighing. The contents of polysaccharides and proteins was measured by phenolsulfuric acid method and Lowry’ method, respectively (Adav and Lee, 2008). 2.3. Preparation of goethite Goethite was synthesized according to the method of Schwertmann and Cornell (2000). Briefly, 1.0 M Fe(NO3)3 solution and 5.0 M KOH solution were mixed fully in a beaker. The mixture was kept at 70 °C for 60 h. The yellow precipitate was collected and washed repeatedly with milli-Q water until the washed water had electrical conductivity close to the milli-Q water. The precipitated iron oxides were then oven-dried at 70 °C. The dried precipitate was ground to pass a 300-mesh sieve. X-ray diffraction and infra-red (IR) spectrometry analysis confirmed the precipitate was a-FeOOH, i.e., goethite. The specific surface area of the goethite prepared using this method was about 20 m2 g1. 2.4. Fluorescence spectroscopy and quenching titration All EEM spectra were measured using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) equipped with 1.0 cm quartz cell and a thermostat bath (Song et al., 2010). The EEM spectra were collected with subsequent scanning emission spectra by varying the excitation wavelength. The width of the excitation/ emission slit was set to 5 nm, and the scanning speed was 1200 nm min1. The fluorometer’s response to a milli-Q water was subtracted from the fluorescence spectra recorded for EPS. EEM data were processed using the software Sigmaplot 10.0 (Systat, USA). The EPS solution was titrated with incremental lL addition of 0.1 M Hg(II) at 25 °C. After each addition of Hg(II), the solution was fully mixed using a magnetic stirrer for 15 min and the fluorescence spectra were recorded. The equilibrium time was set as 15 min since the peak fluorescence intensity varied little after 15 min reaction. The fluorescence quenching data was analyzed by the modified Stern–Volmer equation (Eq. (1)) (Lakowicz, 2006):
F 0 =ðF 0 FÞ ¼ 1=ðfa K a ½HgðIIÞÞ þ 1=fa
ð1Þ
where F0 and F are the fluorescence intensities in absence and presence of Hg(II), respectively. fa is the fraction of the initial fluorescence, which is accessible to quencher, Ka is the conditional stability constant of the accessible fraction and [Hg(II)] is the Hg(II) concentration.
ð2Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Kb is the binding constant and n is the number of binding sites. 2.5. Adsorption experiments Stock Hg(II) solution (0.01 M) was prepared by dissolving HgCl2 of analytical grade in milli-Q water. The pH of EPS solution and goethite suspension was adjusted to 6.0 using 0.01 M HCl/NaOH solution. The adsorption experiments were conducted in batch mode using 50 ml Teflon centrifuge tubes as reaction vessels. The concentration of EPS and goethite suspensions was 10 mg l1 and 4 g l1, respectively. As the electrolyte, the concentration of KCl was 10 mM in the mixed solution. Hg(II) concentrations from 1 to 250 mg l1 were tested at 25 °C to examine the isotherm of Hg(II) to goethite. The pH of mixed solution was adjusted to 6.0. The mixed solutions were centrifuged and the supernatants were collected for measuring Hg(II) concentration. All the adsorption experiments were conducted in duplicate and the experiments in the absence of EPS were the control. The Hg(II) concentration in supernatants were measured using a cold atomic absorption mercury analyzer (F732-VJ, Shanghai, China). The isotherm data were fitted using the Langmuir equation and Freundlich isotherm equation:
Q e ¼ Q max bC e =ð1 þ bC e Þ
ð3Þ
where Qe and Ce correspond to the milligrams of Hg(II) adsorbed per gram of goethite (mg g1) and the equilibrium Hg(II) concentration in the solution (mg l1) (in this case, at 4 h after onset of adsorption experiments), respectively. Qmax is the maximum adsorbed mass (mg g1) and b is an empirical affinity constant; and
Q e ¼ K F C 1=n e
ð4Þ
where Qe is the amount of solute adsorbed per unit weight of adsorbent (mg g1), Ce is the equilibrium concentration of solute in the bulk solution (mg l1), KF is the constant indicative of the relative adsorption capacity of the adsorbent (mg g1), and 1/n is the constant indicative of the intensity of the adsorption. 3. Results and discussion 3.1. EPS–Hg(II) complexation The EPS solution had pH 5.8. The yield of EPS was 264.2 mg1 dry cell g1. The contents of polysaccharides and proteins was 11.5 mg l1 and 9.85 mg l1, respectively. Three fluorescence peaks were observed from the fluorescence spectra of EPS (Fig. 1): peak A at Ex/Em = 230/302 nm, peak B at Ex/Em = 235/350 nm, peak C at Ex/Em = 265/370 nm. These three peaks can be attributed to the contributions of aromatic protein substances. All fluorescence peaks were completely quenched in the presence of 120 lg l1 Hg(II) (Fig. 1b). This observation suggests that >120 lg l1 Hg(II) strongly binds with EPS in solution. The fluorescence intensities of peaks in the spectra of EPS with increasing Hg(II) concentration were illustrated in Fig. S1. All the three peaks fit the modified Stern–Volmer equation (Eq. (1)) at
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W. Song et al. / Bioresource Technology 160 (2014) 119–122
360 EPS
340
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
Ex (nm)
320 300 Peak C
280 260 240
Peak B
220
Peak A
200 200
250
300
(a) 350 400 Em (nm)
450
500
EPS+Hg(II)
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
320
Ex (nm)
300 Peak C
280 260 240 220
Peak B
Peak A
(b)
200 200
250
300
350 Em (nm)
400
450
500
Fig. 1. EEM spectra of EPS from Chroococcus sp. before and after interaction with Hg(II)(a-EPS; b-EPS–Hg(II) system).
r2 > 0.991 and p < 0.001, with the fitting parameters being listed in Table 1. The Hill equation (Eq. (2)) well described all the titration data (r2 > 0.98, p < 0.0001). The binding constants (log Kb) and binding sites (n) of the present EPS–Hg(II) systems were summarized in Table 1. The log Kb values of EPS–Hg(II) system ranged 7.06–7.69, two to three orders of magnitudes greater than those for complexation of bovine serum albumin (BSA) with organic dyes (Song et al., 2009) and a little bit higher than that for EPS-pesticide system (Song et al., 2010). 3.2. Effect of EPS on adsorption of Hg (II) to goethite The Hg(II) absorbed by goethite at pH 6 in the absence and presence of EPS were 58.3% and 73.7%, respectively. The Langmuir equation (Eq. (3)) and Freundlich equation (Eq. (4)) fit the isotherm data (r2 > 0.87, p < 0.05) (Fig. S2) with the best-fit parameters listed in Table 2. The Qm(control) in Langmuir model was lower than than Qm(EPS), suggesting that the maximum absorbed quantity of Hg(II)
Table 1 The conditional stability constants and binding constants of fluorophore–Hg(II) system. Peak
A B C
Conditional stability constant
Langmuir
EPS Control
Freundlich
Qm
b
r2
SD
1/n
KF
r2
SD
40.26 23.71
0.008 0.009
0.878 0.968
0.493 1.139
0.8765 0.8119
0.358 0.273
0.996 0.999
0.063 0.037
to goethite was increased in the presence of EPS. According to the Freundlich equation, KF(EPS) was larger than KF(control), also suggesting that goethite had stronger adsorptive ability to Hg(II)–EPS complexes than Hg(II) ions. 3.3. Discussion
360 340
Table 2 Correlation coefficients and parameters of isothermal adsorption of Hg(II) to goethite in absence and presence of EPS.
Binding constant
log Ka
fa
r2
SD
log Kb
n
r2
SD
4.24 4.09 3.84
1.87 2.05 3.84
0.997 0.991 0.991
0.071 0.075 0.100
7.67 7.46 7.06
1.62 1.60 1.54
0.991 0.989 0.990
0.053 0.060 0.053
The binding constants (log Kb) revealed the interactive intensity between fluorophore and quencher: the greater the binding constant, the stronger of binding capacity of fluorophore and the quencher. The binding constants obtained in Table 1 suggested that EPS from Chroococcus sp. has strong complexation capability to Hg(II). The values of conditional stability constant (log Ka) for EPS–Hg(II) were quite different from those reported for complexation of EPS with the other heavy metals (Guibaud et al. 2006). The relative conditional stability constants of EPS–metal complexes, log K, determined using polarographic methods were 1.54–3.35 for Cd(II), 0.45–1.28 for Pb(II), 3.0–4.4 for Cu(II) and 2.6–3.0 for Ni(II) (Guibaud et al. 2006). Comte et al. (2008) showed that the conditional stability constants for Cu(II), Cd(II) and Pb(II) to EPS from activated sludge were 3.2–4.5, 3.7–5.0 and 3.9–5.7, respectively. Restated, the binding constant data indicated that the EPS may have stronger binding capacity to Hg(II) than to Cd(II), Pb(II) or Cu(II). The constants obtained in this study were close to those for the natural DOM samples (4.2–4.8) (Lu and Jaffe, 2001) and for soil-derived humic substances (Yin et al., 1997). The present constants were also consistent with those for DOM in stream waters (4.3–5.2) (Wu et al., 2004) and those for EPS from cyanobacteria biofilm (3.2–4.5) (Zhang et al., 2010) determined using fluorescence spectroscopy. Effects of organic ligands on the adsorption of heavy metals has received much attention. Zhang et al. (2001) noted that oxalic acid could promote the adsorption of Cd(II) to goethite when the concentration of oxalic acid was 120 lg l1 Hg(II), showing evidence for formation of protein–Hg(II) complex in solution. The EPS–goethite system adsorbed more Hg(II) than the non-EPS system. We proposed that the Hg(II) was bound with proteins first to form proteins–Hg(II) complexes, then the complex was adsorbed onto the goethite. The presence of EPS makes the goethite-soil a larger Hg(II) sink than the non-EPS system, limiting the mobility of Hg(II) in soil. Acknowledgements This work was supported by the West Light Foundation of Chinese Academy of Sciences and National Natural Science Foundation of China (U1120302, 41203088 and 21177127). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 12.052. References Adav, S.S., Lee, D.J., 2008. Extraction of extracellular polymeric substances from aerobic granules with compact interior structure. J. Hazard. Mater. 154, 1120– 1126. Adav, S.S., Lin, J.C.T., Yang, Z., Whiteley, C.G., Lee, D.J., Peng, X.F., Zhang, Z.P., 2010. Stereological assessment of extracellular polymeric substances, exo-enzymes, and specific bacterial strains in bioaggregates using fluorescence experiments. Biotech. Adv. 28, 255–280.
Ali, M.A., Dzombak, D.A., 1996. Competitive sorption of simple organic acids and sulfate on goethite. Environ. Sci. Technol. 30, 1061–1071. Backstrom, M., Dario, M., Karlsson, S., Allard, B., 2003. Effects of a fulvic acid on the adsorption of mercury and cadmium on goethite. Sci. Total Environ. 304, 257– 268. 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, 185–193. Guibaud, G., Hullebusch, V.E., Bordas, F., 2006. Lead and cadmium biosorption by EPS (EPS) extracted from activated sludges: pH-sorption edge tests and mathematical equilibrium modeling. Chemosphere 64, 1955–1962. Hill, T.L., 1985. Cooperativity: Theory in Biochemistry. Springer-Verlag, New York, NY. Kim, C.S., Rytuba, J.J., Brown Jr., G.E., 2004. EXAFS study of mercury (II) sorption to Fe- and Al-(hydr)oxides I. Effects of pH. J. Colloid Interface Sci. 271, 1–15. Lakowicz, J.R., 2006. Principles of Fluorescence Spectroscopy, third ed. SpringerVerlag, New York. Lu, X., Jaffe, R., 2001. Interaction between Hg(II) and natural dissolved organic matter: a fluorescence spectroscopy based study. Water Res. 35, 1793–1803. Omoike, A., Chorover, J., 2006. Adsorption to goethite of extracellular polymeric substances from Bacillus subtilis. Geochim. Cosmochim. Acta 70, 827–838. Pan, X.L., Liu, J., Song, W.J., Zhang, D.Y., 2012. Biosorption of Cu(II) to extracellular polymeric substances (EPS) from Synechoeystis sp.: a fluorescence quenching study. Front. Environ. Sci. Eng. China 6, 493–497. Schwertmann, U., Comell, R.M., 2000. Iron Oxides in the Laboratory, Preparation and Characterization. Wiley-VCH, Weinheim. Song, S.M., Hou, X.L., Wu, Y.B., Shuang, S.M., Yang, C., Inoue, Y., 2009. Study on the interaction between methyl blue and human serum albumin by fluorescence spectrometry. J. Lumin. 129, 169–175. Song, W.J., Mu, G.J., Zhang, D.Y., Pan, X.L., 2010. Interaction of acetamiprid with extracellular polymeric substances (EPS) from activated sludge: a fluorescence study. Afr. J. Biotechnol. 45, 7667–7673. Song, W.J., Zhang, D.Y., Pan, X.L., Lee, D.J., 2013. Complexation of HSA with different forms of antimony (Sb): an application of fluorescence spectroscopy. J. Limin. 136, 80–85. Wu, F.C., Cai, Y.R., Evans, D., Dillon, P., 2004. Complexation between Hg(II) and dissolved organic matter in stream waters: an application of fluorescence spectroscopy. Biogeochemistry 71, 339–351. Yin, Y., Allen, H.E., Huang, C.P., Sanders, P.F., 1997. Adsorption/desorption isotherms of Hg (II) by soil. Soil Sci. 162, 35–45. Zhang, D.Y., Lee, D.J., Pan, X.L., 2013. Desorption of Hg(II) and Sb(V) on extracellular polymeric substances: effects of pH, EDTA, Ca(II) and temperature shocks. Bioresour. Technol. 128, 711–715. Zhang, D.Y., Pan, X.L., Khan, M.G., Chen Mostofa, X., Mu, G.J., Wu, F.C., Liu, J., Yang, J.Y., Liu, Y.L., Fu, Q.L., 2010. Complexation between Hg(II) and biofilm extracellular polymeric substances: an application of fluorescence spectroscopy. J. Hazard. Mater. 175, 359–365. Zhang, G.Y., Dong, Y.Y., Li, X.Y., Wei, Y., Bi, S.Q., 2001. Effects and mechanisms of oxalate on Cd(II) adsorbed on goethite at different pH and electrolyte concentration. Plant Nutr. Fert. Sci. 7, 305–310.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Biosorption of Hg(II) onto goethite with extracellular polymeric substances Wenjuan Song a, Xiangliang Pan a,⇑, Shuyong Mu a, Daoyong Zhang b, Xue Yang a, Duu-Jong Lee b,⇑ a b
Laboratory of Environmental Pollution and Bioremediation, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan
h i g h l i g h t s Interactions between Hg(II), EPS and goethite were investigated. EPS from Chroococcus sp. enhances adsorption capacity of Hg(II) on goethite. EPS binds with Hg(II) to quench the EEM peaks of proteins on EPS. Goethite–EPS soil is a larger Hg(II) sink than goethite alone soil. Biosorption significantly affects the mobility of Hg(II) in goethite soils.
a r t i c l e
i n f o
Article history: Available online 21 December 2013 Keywords: Chroococcus sp. Extracellular polymeric substances Mercury Goethite
a b s t r a c t This study characterized the interactions of goethite, EPS from cyanobacterium Chroococcus sp. and Hg(II) using excitation emission matrix (EEM) spectra and adsorption isotherms. Three protein-like fluorescence peaks were noted to quench in the presence of Hg(II). The estimated conditional stability constant (log Ka) and the binding constant (log Kb) of the studied EPS–Hg(II) systems ranged 3.84–4.24 and 6.99– 7.69, respectively. The proteins in EPS formed stable complex with Hg(II). The presence of proteins of Chroococcus sp. enhanced the adsorption capacity of Hg(II) on goethite; therefore, the goethite–EPS soil is a larger Hg(II) sink than goethite alone soil. Biosorption significantly affects the mobility of Hg(II) in goethite soils. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Mercury (Hg) is commonly found in soil and sediment which can induce neurological and renal disturbances and impairment of pulmonary function of human bodies. Iron oxides have strong affinity to Hg(II) so the transport and fate of latter in soil and top sediment layers are significantly affected by the presence of ubiquitous iron oxides. Goethite (a-FeOOH) is the commonly found iron oxide mineral in all types of soils and top-layer freshwater sediments. Goethite has high surface area and surface charge and thus has strong adsorption capacity to numerous heavy metals (Kim et al., 2004; Song et al., 2013). Soil and sediments provide ideal habitats for microorganisms including cyanobacteria. Cyanobacteria are capable of secreting large quantities of extracellular polymeric substance (EPS), which are mainly composed of polysaccharides, proteins, humic substances and nucleic acids (Adav et al., 2010). As one of the
⇑ Corresponding authors. Tel./fax: +86 991 7885446 (X. Pan). Tel.: +886 2 23570516; fax: +886 2 23623040 (D.-J. Lee). E-mail addresses: [email protected] (X. Pan), [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.052
major types of dissolved organic matter (DOM), EPS from cyanobacteria significantly affect mobility and bioavailability of heavy metals (Pan et al., 2012; Zhang et al., 2013). Backstrom (2003) noted that adsorption capacity of Hg(II) by goethite was increased in the presence of fulvic acid. Omoike and Chorover (2006) noted that EPS could be adsorbed to the surface of mineral particles to interefere with film formation on iron oxide surfaces. Organic matters should be able to affect the adsorption process of Hg(II) by goethite, and hence the transport and fate of Hg(II) in soil. However, related studies are lacking. Studies on the adsorption of Hg(II) on goethite in the presence of EPS provide complementary knowledge about biogeochemical processes of mercury in soil and sediments an assess performance of bioremediation of heavy metal contaminated soil and sediment since most bacteria used for remediation persistently excrete EPS. Chroococcus, a typical genus of cyanobacteria, lives luxuriously in various habitats, including soil, the sludge of freshwater lake, river bottoms, and water sources of high salinity. Like other cyanobacteria, Chroococcus produces large quantities of EPS, whose presence may influence the interfacial processes of Hg(II) on the surface of goethite. This study aims at exploring whether proteins, a major
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W. Song et al. / Bioresource Technology 160 (2014) 119–122
part of EPS from Chroococcus sp., would affect adsorption of Hg(II) on goethite. 2. Methods 2.1. Cyanobacterial culture and EPS extraction
When small molecules are bound independently to a set of equivalent sites on a macromolecule, the fluorescence intensity data can be used to obtain the binding constants and the number of binding sites for the complex. The binding constant (log Kb) and the number of binding site (n) can be determined by the following equation (Hill, 1985):
log½ðF 0 FÞ=F ¼ log K b þ n log½HgðIIÞ The cyanobacterium Chroococcus sp., isolated from the soil sample in FuKang, Xinjiang, China, was cultivated in BG-11 medium at 30 °C and 55 lmols photons m2 s1 in a 12 h:12 h light:dark cycle. The collected Chroococcus sp. cells were first centrifugated at 5500 rpm for 10 min at 4 °C. The collected biomass were re-suspended in milli-Q water. The suspension was centrifugated again at 12300 rpm for 20 min at 4 °C. The supernatant was filtered by a 0.45-lm membrane and then purified with dialysis membrane (3500 Da) at 4 °C for 24 h (Adav and Lee, 2008). 2.2. Determination of biochemical composition of EPS The yield of EPS was determined by drying and weighing. The contents of polysaccharides and proteins was measured by phenolsulfuric acid method and Lowry’ method, respectively (Adav and Lee, 2008). 2.3. Preparation of goethite Goethite was synthesized according to the method of Schwertmann and Cornell (2000). Briefly, 1.0 M Fe(NO3)3 solution and 5.0 M KOH solution were mixed fully in a beaker. The mixture was kept at 70 °C for 60 h. The yellow precipitate was collected and washed repeatedly with milli-Q water until the washed water had electrical conductivity close to the milli-Q water. The precipitated iron oxides were then oven-dried at 70 °C. The dried precipitate was ground to pass a 300-mesh sieve. X-ray diffraction and infra-red (IR) spectrometry analysis confirmed the precipitate was a-FeOOH, i.e., goethite. The specific surface area of the goethite prepared using this method was about 20 m2 g1. 2.4. Fluorescence spectroscopy and quenching titration All EEM spectra were measured using a fluorescence spectrophotometer (F-7000, Hitachi, Japan) equipped with 1.0 cm quartz cell and a thermostat bath (Song et al., 2010). The EEM spectra were collected with subsequent scanning emission spectra by varying the excitation wavelength. The width of the excitation/ emission slit was set to 5 nm, and the scanning speed was 1200 nm min1. The fluorometer’s response to a milli-Q water was subtracted from the fluorescence spectra recorded for EPS. EEM data were processed using the software Sigmaplot 10.0 (Systat, USA). The EPS solution was titrated with incremental lL addition of 0.1 M Hg(II) at 25 °C. After each addition of Hg(II), the solution was fully mixed using a magnetic stirrer for 15 min and the fluorescence spectra were recorded. The equilibrium time was set as 15 min since the peak fluorescence intensity varied little after 15 min reaction. The fluorescence quenching data was analyzed by the modified Stern–Volmer equation (Eq. (1)) (Lakowicz, 2006):
F 0 =ðF 0 FÞ ¼ 1=ðfa K a ½HgðIIÞÞ þ 1=fa
ð1Þ
where F0 and F are the fluorescence intensities in absence and presence of Hg(II), respectively. fa is the fraction of the initial fluorescence, which is accessible to quencher, Ka is the conditional stability constant of the accessible fraction and [Hg(II)] is the Hg(II) concentration.
ð2Þ
where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. Kb is the binding constant and n is the number of binding sites. 2.5. Adsorption experiments Stock Hg(II) solution (0.01 M) was prepared by dissolving HgCl2 of analytical grade in milli-Q water. The pH of EPS solution and goethite suspension was adjusted to 6.0 using 0.01 M HCl/NaOH solution. The adsorption experiments were conducted in batch mode using 50 ml Teflon centrifuge tubes as reaction vessels. The concentration of EPS and goethite suspensions was 10 mg l1 and 4 g l1, respectively. As the electrolyte, the concentration of KCl was 10 mM in the mixed solution. Hg(II) concentrations from 1 to 250 mg l1 were tested at 25 °C to examine the isotherm of Hg(II) to goethite. The pH of mixed solution was adjusted to 6.0. The mixed solutions were centrifuged and the supernatants were collected for measuring Hg(II) concentration. All the adsorption experiments were conducted in duplicate and the experiments in the absence of EPS were the control. The Hg(II) concentration in supernatants were measured using a cold atomic absorption mercury analyzer (F732-VJ, Shanghai, China). The isotherm data were fitted using the Langmuir equation and Freundlich isotherm equation:
Q e ¼ Q max bC e =ð1 þ bC e Þ
ð3Þ
where Qe and Ce correspond to the milligrams of Hg(II) adsorbed per gram of goethite (mg g1) and the equilibrium Hg(II) concentration in the solution (mg l1) (in this case, at 4 h after onset of adsorption experiments), respectively. Qmax is the maximum adsorbed mass (mg g1) and b is an empirical affinity constant; and
Q e ¼ K F C 1=n e
ð4Þ
where Qe is the amount of solute adsorbed per unit weight of adsorbent (mg g1), Ce is the equilibrium concentration of solute in the bulk solution (mg l1), KF is the constant indicative of the relative adsorption capacity of the adsorbent (mg g1), and 1/n is the constant indicative of the intensity of the adsorption. 3. Results and discussion 3.1. EPS–Hg(II) complexation The EPS solution had pH 5.8. The yield of EPS was 264.2 mg1 dry cell g1. The contents of polysaccharides and proteins was 11.5 mg l1 and 9.85 mg l1, respectively. Three fluorescence peaks were observed from the fluorescence spectra of EPS (Fig. 1): peak A at Ex/Em = 230/302 nm, peak B at Ex/Em = 235/350 nm, peak C at Ex/Em = 265/370 nm. These three peaks can be attributed to the contributions of aromatic protein substances. All fluorescence peaks were completely quenched in the presence of 120 lg l1 Hg(II) (Fig. 1b). This observation suggests that >120 lg l1 Hg(II) strongly binds with EPS in solution. The fluorescence intensities of peaks in the spectra of EPS with increasing Hg(II) concentration were illustrated in Fig. S1. All the three peaks fit the modified Stern–Volmer equation (Eq. (1)) at
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W. Song et al. / Bioresource Technology 160 (2014) 119–122
360 EPS
340
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
Ex (nm)
320 300 Peak C
280 260 240
Peak B
220
Peak A
200 200
250
300
(a) 350 400 Em (nm)
450
500
EPS+Hg(II)
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
320
Ex (nm)
300 Peak C
280 260 240 220
Peak B
Peak A
(b)
200 200
250
300
350 Em (nm)
400
450
500
Fig. 1. EEM spectra of EPS from Chroococcus sp. before and after interaction with Hg(II)(a-EPS; b-EPS–Hg(II) system).
r2 > 0.991 and p < 0.001, with the fitting parameters being listed in Table 1. The Hill equation (Eq. (2)) well described all the titration data (r2 > 0.98, p < 0.0001). The binding constants (log Kb) and binding sites (n) of the present EPS–Hg(II) systems were summarized in Table 1. The log Kb values of EPS–Hg(II) system ranged 7.06–7.69, two to three orders of magnitudes greater than those for complexation of bovine serum albumin (BSA) with organic dyes (Song et al., 2009) and a little bit higher than that for EPS-pesticide system (Song et al., 2010). 3.2. Effect of EPS on adsorption of Hg (II) to goethite The Hg(II) absorbed by goethite at pH 6 in the absence and presence of EPS were 58.3% and 73.7%, respectively. The Langmuir equation (Eq. (3)) and Freundlich equation (Eq. (4)) fit the isotherm data (r2 > 0.87, p < 0.05) (Fig. S2) with the best-fit parameters listed in Table 2. The Qm(control) in Langmuir model was lower than than Qm(EPS), suggesting that the maximum absorbed quantity of Hg(II)
Table 1 The conditional stability constants and binding constants of fluorophore–Hg(II) system. Peak
A B C
Conditional stability constant
Langmuir
EPS Control
Freundlich
Qm
b
r2
SD
1/n
KF
r2
SD
40.26 23.71
0.008 0.009
0.878 0.968
0.493 1.139
0.8765 0.8119
0.358 0.273
0.996 0.999
0.063 0.037
to goethite was increased in the presence of EPS. According to the Freundlich equation, KF(EPS) was larger than KF(control), also suggesting that goethite had stronger adsorptive ability to Hg(II)–EPS complexes than Hg(II) ions. 3.3. Discussion
360 340
Table 2 Correlation coefficients and parameters of isothermal adsorption of Hg(II) to goethite in absence and presence of EPS.
Binding constant
log Ka
fa
r2
SD
log Kb
n
r2
SD
4.24 4.09 3.84
1.87 2.05 3.84
0.997 0.991 0.991
0.071 0.075 0.100
7.67 7.46 7.06
1.62 1.60 1.54
0.991 0.989 0.990
0.053 0.060 0.053
The binding constants (log Kb) revealed the interactive intensity between fluorophore and quencher: the greater the binding constant, the stronger of binding capacity of fluorophore and the quencher. The binding constants obtained in Table 1 suggested that EPS from Chroococcus sp. has strong complexation capability to Hg(II). The values of conditional stability constant (log Ka) for EPS–Hg(II) were quite different from those reported for complexation of EPS with the other heavy metals (Guibaud et al. 2006). The relative conditional stability constants of EPS–metal complexes, log K, determined using polarographic methods were 1.54–3.35 for Cd(II), 0.45–1.28 for Pb(II), 3.0–4.4 for Cu(II) and 2.6–3.0 for Ni(II) (Guibaud et al. 2006). Comte et al. (2008) showed that the conditional stability constants for Cu(II), Cd(II) and Pb(II) to EPS from activated sludge were 3.2–4.5, 3.7–5.0 and 3.9–5.7, respectively. Restated, the binding constant data indicated that the EPS may have stronger binding capacity to Hg(II) than to Cd(II), Pb(II) or Cu(II). The constants obtained in this study were close to those for the natural DOM samples (4.2–4.8) (Lu and Jaffe, 2001) and for soil-derived humic substances (Yin et al., 1997). The present constants were also consistent with those for DOM in stream waters (4.3–5.2) (Wu et al., 2004) and those for EPS from cyanobacteria biofilm (3.2–4.5) (Zhang et al., 2010) determined using fluorescence spectroscopy. Effects of organic ligands on the adsorption of heavy metals has received much attention. Zhang et al. (2001) noted that oxalic acid could promote the adsorption of Cd(II) to goethite when the concentration of oxalic acid was 120 lg l1 Hg(II), showing evidence for formation of protein–Hg(II) complex in solution. The EPS–goethite system adsorbed more Hg(II) than the non-EPS system. We proposed that the Hg(II) was bound with proteins first to form proteins–Hg(II) complexes, then the complex was adsorbed onto the goethite. The presence of EPS makes the goethite-soil a larger Hg(II) sink than the non-EPS system, limiting the mobility of Hg(II) in soil. Acknowledgements This work was supported by the West Light Foundation of Chinese Academy of Sciences and National Natural Science Foundation of China (U1120302, 41203088 and 21177127). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 12.052. References Adav, S.S., Lee, D.J., 2008. Extraction of extracellular polymeric substances from aerobic granules with compact interior structure. J. Hazard. Mater. 154, 1120– 1126. Adav, S.S., Lin, J.C.T., Yang, Z., Whiteley, C.G., Lee, D.J., Peng, X.F., Zhang, Z.P., 2010. Stereological assessment of extracellular polymeric substances, exo-enzymes, and specific bacterial strains in bioaggregates using fluorescence experiments. Biotech. Adv. 28, 255–280.
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