In the Laboratory
Zinc Biosorption by Seaweed Illustrated W by the Zincon Colorimetric Method and the Langmuir Isotherm Maria Mar Areco and Maria dos Santos Afonso* DQIAyQF-INQUIMAE, FCEyN, UBA, Cdad Universitaria – Pab. II – (C1428EHA) Buenos Aires, Argentina; *
[email protected] Erika Valdman Center for Mineral Technology, CODS/CETEM/MCT, Rio de Janeiro, Brazil
Enhanced industrial activity during recent decades has led to the discharge of unprecedented volumes of wastewater, which is a serious cause of environmental degradation. Heavy metals are major pollutants in marine, ground, industrial, and even treated waters. Owing to their high toxicity, heavy metals pose a serious threat to biota and the environment (1). Therefore, it is necessary to reduce these metals from industrial effluents, before their discharge into water bodies. Several methods have been suggested for the removal of toxic metals from wastewaters (2). Increasingly stringent regulations are forcing scientists to study new technologies for metal removal from wastewater to determine the best means of attaining today’s toxicity-driven limits (3). Sorption of heavy metals onto live or dead biological materials (biosorption) is a potential method for removing or recovering toxic and precious metals from wastewater (3, 4). Successful metal biosorption has been reported by a variety of biological materials including microalgae and seaweeds,
Figure 1. The green algae Ulva sp. (A) and the red algae Gymnogongrus torulosus (B). The photograph of the Gymnogongrus torulosus algae was kindly provided by Marina Ciancia and J. Estevez from FAUBA-UBA and FCEN-UBA, respectively.
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bacteria, fungi, and crop residues (5, 6). Since the cost of producing biomass specifically for metal removal through cultivation is generally high, we considered the use of marine algae collected directly from the Atlantic coast to study this process. Ulva sp., known as sea lettuce, and Gymnogongrus torulosus, a red algae, are common seaweeds found in the Atlantic coast and easily collected at the high-tide line along the beach. Their physical appearance is quite distinctive and with the aid of an adequate photo (Figure 1) it is possible for students to collect and identify these particular algae. Zinc is a natural element that is essential for man and most living organisms. The average abundance of zinc in the earth’s crust is 76 ppm; in soils it is 25 to 68 ppm; in streams it is 20 µg兾L; and in groundwaters it is lower than 0.1 mg兾L. The solubility of zinc is controlled in natural waters by adsorption on mineral surfaces, precipitation with carbonate ions, and complexation with organic moieties. To be ecotoxic, the metal has to be present in a chemical form that can be taken up by, for example, living organisms, and hence be harmful. The term bioavailability refers to at what rate and to what extent a substance might be taken up by an organism, whereas ecotoxicity refers to a way to assess changes taking place in environmental systems owing to released substances. The heavy metals cause problems by displacing or replacing minerals that are required for essential body functions. Indeed, zinc deficiency is now recognized as a human health problem. Too little zinc can cause health problems such as loss of appetite, decreased sense of taste and smell, slow wound healing and skin sores, or a damaged immune system. On the other hand, too much zinc can also damage human health (7). Also, elevated concentrations of zinc in stream water and sediments have reduced species diversity and abundance of aquatic communities. The atomic absorption spectrometric (AAS) or inductively coupled plasma (ICP) methods are normally preferred for zinc determination in water and wastewater. However, the zincon (2-carboxy-2´-hydroxy-5´-sulfoformazylbenzene; Figure 2) colorimetric method to determine metal ions in solution is suitable for analysis of both potable and polluted waters and may be used if the instrumentation for the preferred methods are not available. The zincon colorimetric method is an easy technique for zinc assessment and adds an interesting motivational aspect to study zinc removal by the green algae Ulva sp. and the red algae Gymnogongrus torulosus. These procedures may be performed as individual or group experiments by undergraduates or beginning graduate students and anyone who wants to verify this interesting
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In the Laboratory
behavior of algae towards metal ions. They are particularly suited for use at locations with biotechnology programs since they combine in a single experiment the need to remove heavy metals from contaminated aqueous solutions and an emerging technology.
O
O
O
O S
S
O
O
NH
N
Experimental Procedure
OH
Biosorbent The green algae Ulva sp. and the red algae Gymnogongrus torulosus were collected at Mar del Plata, Argentina. After collection, algae should be carefully freed of foreign matter, extensively washed, oven dried at 60 ⬚C for 72 hours, and then ground. Algae are stored in a fresh bench and in a stoppered flask. Kinetics Experiment A typical sorption experiment was carried out in 250mL Erlenmeyer flask, applying 0.05 g dry algae to 50 mL of 50 mg L᎑1 zinc solution. The suspension was continuously shaken for 3 h at 28 ⬚C (8). The experiment was conducted in absence of buffer to avoid the effect of buffer components on the reaction course. The pH was adjusted until a constant value was obtained by adding aliquots of NaOH or HCl and was kept constant during the experiment. Aliquots of 1 cm3 were taken from the zinc–algae solution at timed intervals, pH and Zn(II) ions were analyzed to follow the biosorption kinetics. It is important to alert students to take the aliquot carefully to avoid taking algae particles with the sample. The experiment was continued until a constant zinc concentration was observed. Biosorption Isotherms To verify algae performance at different initial metal concentrations, 0.05 g of dry algae were added to 50 mL of ZnSO4 aqueous solutions, ranging from 0–800 mg L᎑1. The experimental procedure was identical to that for the kinetic experiment. Flasks were continuously shaken at room temperature until equilibrium was reached. The heterogeneous suspension was filtered and free Zn(II) ions were quantified. The evaluation of the biosorbent was carried out considering the equilibrium relations (adsorption isotherms). The Langmuir sorption model was chosen for the estimation of maximum sorbate (metal) uptake. The Langmuir isotherm assumes that the adsorption free energy is independent of both the surface coverage and the formation of a monolayer when solid surface reaches saturation (8) and can be expressed as q =
Na
Na
qmax bC f + b Cf )
(1)
(1
where q is the metal ion uptake (mass of zinc ions)兾(mass of algae), qmax is the maximum theoretical uptake upon complete saturation of the surface, b is the Langmuir constant related to the energy of adsorption–desorption (L mg᎑1), and Cf is the final concentration of metal ions in solution (mg L᎑1).
Metal Quantification The concentration of free Zn(II) ions in the aqueous solution was determined spectrophotometrically following the modified protocol of Platte and Marcy (9). The absorbance of the blue zinc–zincon complex in solutions was read at 620 www.JCE.DivCHED.org
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OH
N
N N
N NH
N
O OH
O OH
Figure 2. Structural formula of zincon.
nm. Absorbance measurements were made with a Shimadzu UV–vis spectrophotometer Pharmaspec UV-1700 using a 1cm path-length quartz cuvette at constant temperature. The zinc complex follows Beer’s law in the range of 1 to 8 mg L᎑1 of zinc. Solution Preparation Students prepare different samples from the following solutions: • Buffer solution, pH 9: dissolve 2.4 g of sodium hydroxide in 60 mL of distilled water. Transfer this solution to a 100-mL volumetric flask, add 3.73 g of potassium chloride and 3.1 g of boric acid and complete the volume with distilled water. Check the pH value. • Zincon solution: dissolve 0.065 g of zincon (Figure 2) in 1 mL of 1 M sodium hydroxide solution and dilute to 50 mL. The prepared zincon solution should be refrigerated and kept in the dark to avoid reagent decomposition. This solution is deep red in color and is stable for about one week. • Zinc standard solution: prepare 1 L of 10 mg L᎑1 zinc concentration with distilled water from ZnSO4⭈7H2O. Transfer a 1-mL aliquot of the sample containing no more than 8.0 mg of zinc to a 5-mL volumetric flask. Add reagents to the sample in the following order with mixing between additions: 0.5 mL of buffer, 0.3 mL of zincon, and dilute to 5 mL.
To train students with dilutions, it is recommended to perform zinc concentration higher than the maximum limit of the calibration curve (8.0 mg L᎑1). After students obtain a good linearity with different known zinc solution concentrations, samples with unknown concentrations can be analyzed. Hazards Zincon is harmful by inhalation, ingestion, or skin absorption causing eye and skin irritation. There is insufficient data in the literature to assign complete numerical safety data ratings. For personal protection use safety glasses, handle with caution, and do not breathe dust. Wash thoroughly after handling. ZnSO4⭈7H2O is an eye, skin, and mucous membrane irritant. Boric acid is moderately toxic and may be irritating to eyes. HCl is extremely corrosive. Inhalation of vapor can cause serious injury and liquid can cause severe damage to skin
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Table 1. Values of Experimental Equilibrium Constants Using Langmuir Model for Zinc Biosorption qmax/10−3
b/(L mg−1)
R2
Ulva sp.
26.8
0.019
0.999
Gymnogongrus torulosus
42.8
0.084
0.921
Algae
and eyes. NaOH may cause serious permanent eye damage. It is very harmful by ingestion, skin contact, or inhalation. Results
Figure 3. Calibration curve of standard zinc solution. Absorbance was measured at 620 nm.
Figure 4. Kinetic biosorption of zinc by Ulva sp. ( 䊉 ) and Gymnogongrus torulosus (䊊) (1 g兾L) at pH 5.4. In both cases, the initial zinc concentration (Ci) was 50 mg L᎑1.
50
q / (10−3)
40
30
q = 20
10
0 0
100
200
300
400
500
600
−1
C f / (mg L ) Figure 5. Zinc biosorption by Ulva sp. (䊉) and Gymnogongrus torulosus (䊊) at room temperature and pH 5.4 after 16 h. Data were adjusted using Langmuir model.
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To quantify the unknown zinc concentration in solution, the first step is to obtain a calibration curve with known quantities of the zinc and excess zincon in solution. As shown in Figure 3, the calibration curve is linear (R2 = 0.999) in the range from 1 to 8 mg L᎑1. After students obtained the calibration curve and determined the linear equation, zinc ion solutions of unknown concentrations were analyzed. They used the zincon method to follow zinc uptake from solution by Ulva sp. and Gymnogongrus torulosus. The kinetic biosorption of zinc by both algae with initial concentration of 50 mg L᎑1 are shown in Figure 4. It is important to notice that the biosorption kinetic curve tends to stabilize after 60 minutes, suggesting saturation of the algae binding sites that remove zinc from solution. A fast zinc uptake during the first minutes is also observed. After 2 hours, the zinc ions remaining in solution were approximately 80% and 40% of the initial concentration for the experiment with Ulva sp. and Gymnogongrus torulosus, respectively. The metal uptake mechanism is particularly dependent on the initial heavy-metal concentration (Ci): at low concentrations metals are adsorbed by specific sites, while with increasing metal concentrations the specific sites are saturated and the exchange sites are filled (10). To determine the sorption capacity of a biosorbent, it is necessary to generate the equilibrium sorption data at various metal solution Ci values. These data are necessary for modeling with the Langmuir or Freundlich adsorption isotherms: a fit that is usually used to interpret the efficiency of metal biosorption. Zinc uptake capacity (q) is calculated after stabilization of zinc concentration in solution. It is possible to know the quantity of zinc ions removed by algae by the difference between the initial (Ci) and final (Cf) metal concentration. By dividing this value by the quantity of algae used in the biosorption experiment, the uptake capacity is obtained
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(C i
− Cf ) CA
(2)
where C is the zinc concentration in mg L᎑1 and CA is the algae concentration in g L᎑1. After calculation of q values for each algae and zinc concentration ranging from 0–800 mg L᎑1, the data are fit to the Langmuir isotherm (Figure 5). It is possible to identify a plateau at higher metal concentrations as a monolayer is formed when the solid surface reaches saturation (8). To obtain the constants of Langmuir model (qmax and b), regression features in Statistica 5.0 were used. The values of the constants obtained for each algae are shown in Table 1. It is
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interesting to observe that the maximum zinc biosorption capacity obtained for Gymnogongrus torulosus (42.8 × 10᎑3) was almost 2-fold higher than for Ulva sp. (26.8 × 10᎑3). Results also demonstrated a higher affinity of zinc ions for the red algae (0.084 L mg᎑1) suggesting that this seaweed should be selected to remove zinc ions from wastewater. Finally, the Langmuir model can be verified using the linear transformation of eq 1:
W
Supplemental Material
Additional background information about the experiment is available in this issue of JCE Online. Literature Cited
1 1 1 = + q q max qmax bC f
(3)
By plotting (1兾q) versus 1兾Cf, qmax and b can be determined if a straight line is obtained (11). As the values of R 2 for both algae are close to 1, the data on biosorption of zinc(II) by algae may be concluded to fit the Langmuir isotherms model. The experimental maximum sorption values for the limiting capacities of algae for zinc(II), expressed as qmax, are 26.8 × 10᎑3 and 42.8 × 10᎑3 biosorbent for Ulva sp. and Gymnogongrus torulosus, respectively. The Langmuir affinity constants follow a similar pattern with 0.019 and 0.084 L mg᎑1 for Ulva sp. and Gymnogongrus torulosus, respectively. The relative order of metal sorption affinity of zinc on algae on the basis of qmax and b is Gymnogongrus torulosus > Ulva sp. Conclusion This experiment promotes biotechnology knowledge that is an emerging technology on cleaning treatment showing the potential of seaweed to remove heavy-metal ions from solution. The rapid and accurate determination of zinc in aqueous solution by the zincon colorimetric method gives an
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interesting and simple experiment for any biotechnology program that deals with environmental contamination.
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1. Volesky, B. Hydrometallurgy 2001, 59, 203–216. 2. Volesky, B.; Schiewer, S. In Encyclopedia of Bioprocess Engineering; Flickinger, M. C., Drew, S. W., Eds.; Wiley: New York, 1999; pp 433–453. 3. Esteves, A.; Valdman, E.; Leite, S. G. F. Biotechnol. Lett. 2000, 22, 499–502. 4. Fogarty, R. V.; Dostalek, P.; Patzak, M.; Votruba, J.; Tel-Or, E.; Tobin, J. M. Biotechnol. Tech. 1999, 13, 533–538. 5. Schiewer, S.; Volesky, B. In Environmental Microbe–Metal Interactions; Lovely, D. R., Ed.; ASM Press: Washington, DC, 2000. 6. Saeed, A.; Iqbal, M.; Akhtar, M. W. Pak. J. Sci. Ind. Res. 2002, 45, 206–211. 7. Agency for Toxic Substances and Disease Registry. Toxicological Profile for Zinc. http://www.atsdr.cdc.gov/tfacts60.html (accessed Oct 2006). 8. Valdman, E.; Leite, S. G. F. Bioprocess Eng. 2000, 22, 171–173. 9. Platte, J. A.; Marcy, V. M. Anal. Chem. 1959, 31, 1226–1228. 10. Lehmann, R. G.; Hater, R. D. Soil Sci. Soc. Am. J. 1984, 48, 769–772. 11. Cruz, C. C. V.; da Costa, A. C. A.; Henriques, C. A.; Luna, A. S. Bioresource Technol. 2004, 91, 249–257.
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