Mechanisms of Biosorption of Different Heavy Metals by Brown Marine Macroalgae

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1 Mechanisms of Biosorption of Different Heavy Metals by Brown Marine Macroalgae Ofer Raize, 1 Yerachmiel Argaman, 1 Shmuel Yannai 2 1 Department of Civil Engineering, Environmental and Water Resources Engineering, Technion Israel Institute of Technology, Haifa 32000, Israel 2 Faculty of Food Engineering and Biotechnology, Technion Israel Institute of Technology, Haifa, 32000, Israel; telephone: ; fax: ; s: syannai@tx.technion.ac.il, raize@bezeqint.net Received 4 December 2002; accepted 19 March 2004 Published online 22 July 2004 in Wiley InterScience ( DOI: /bit Abstract: The biosorption mechanisms of different heavy metallic cations (Cd, Ni, Pb) to active chemical groups on the cell wall matrix of the nonliving brown marine macroalga, Sargassum vulgaris in its natural form, were examined by the following instrumental and chemical techniques: Fourier-transform infrared (FTIR) analysis, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and extraction of alginic acid and sulfated polysaccharides, which act as metal-binding moieties present in cell wall. From the different techniques used and the known chemical composition of the algal cell wall, it was observed that biosorption of the metallic cations to the algal cell wall component was a surface process. The binding capacities of the different metal cations were between 1 and 1.2 mmol metal/g on a dry weight basis. The main chemical groups involved in the metallic cation biosorption were apparently carboxyl, amino, sulfhydryl, and sulfonate. These groups were part of the algal cell wall structural polymers, namely, polysaccharides (alginic acid, sulfated polysaccharides), proteins, and peptidoglycans. The main cadmium cation sequestration mechanism by the algal biomass was apparently chelation, while the nickel cation sequestration mechanism was mainly ion exchange. Lead cations exhibit higher affinity to the algal biomass, and their binding mechanism included a combination of ion exchange, chelation, and reduction reactions, accompanied by metallic lead precipitation on the cell wall matrix. During the ion exchange process, calcium, magnesium, hydrogen cations, and probably other cations (sodium and potassium) in the algal cell wall matrix were replaced by the tested heavy metals. B 2004 Wiley Periodicals, Inc. Keywords: biosorption mechanisms; brown marine macroalgae; Sargassum vulgaris; heavy metals; XPS; EDS; FTIR; binding capacities INTRODUCTION Biosorption of heavy metals from aqueous solutions is a relatively new technology for the treatment of wastewater (Schiewer and Volesky, 2000). Absorbent materials (biosorbents), derived from a suitable biomass, can be used for the effective removal and recovery of heavy metallic ions Correspondence to: Ofer Raize and Shmuel Yannai Contract grant sponsor: Ministry of Environment, Israel from wastewater streams. A few species of marine macroalgae, commonly known as brown algae, exhibit high metal binding capacities. These were higher than those of other types of biomass and other sorbents (Zhao and Duncan, 1998; Matheickal and Yu, 1999; Matheickal et al., 1999; Yu and Matheickal, 1999). Biomass of brown marine macroalgae is a renewable biological resource, which is available in large quantities and can form a good base for the development of biosorbent material (Schiewer and Volesky, 2000). Brown algae contain high concentrations of alginic acid and sulfated polysaccharides. It has been postulated that the function of these polysaccharides, which are absent in terrestrial plants, is to enable marine algae to selectively absorb metallic ions in a saline medium through ion exchange (Lewin, 1962; Percival and McDowell, 1967; Stewart, 1974). Biosorption of metals involves several mechanisms that differ qualitatively and quantitatively, according to the species used, the origin of the biomass, and its processing procedure (Holan and Volesky, 1995). There are several chemical groups in biomass that can attract and sequester the metals: acetamido, amino, amido, sulfhydryl, sulfate, and carboxyl (Gardea-Torresdey et al., 1990; Volesky, 1991; Schiewer and Volesky, 2000). Metal sequestration during biosorption follows complex mechanisms that include mainly ionic interactions and formation of complexes between metal cations and ligands contained in the structure of the cell wall biopolymers, as well as precipitation on the cell wall matrix (Crist et al., 1994; Schiewer and Volesky, 1995, 1996, 2000). The binding characteristics of metallic cations during biosorption can partially be explained by Pearson s concept of hard and soft acid and base theory (HSAB) and by Irving Williams series (Forstner and Wittmann, 1981; Schiewer and Volesky, 2000). In the literature are several examples of metal biosorption mechanisms. Kuyucak and Volesky (1989) reported that cobalt biosorption by nonliving biomass of the brown marine macroalgae Ascophyllum nodosum is predominantly an ion-exchange process. They suggested that the carboxyl groups of the cell wall alginates play an important role B 2004 Wiley Periodicals, Inc.

2 in cobalt binding. An ion-exchange biosorption mechanism was also suggested by other investigators (Schiewer and Volesky, 2000). The contribution of other functional groups, such as the strongly acidic sulfate groups (R-OSO 3 ) present in the cell wall polymers (fucoidan, carrageenans) was estimated at 10% of the overall metal-binding sites of these seaweeds (Kratochvil and Volesky, 1998). Gardea-Torresdey et al. (1990) modified carboxyl groups of five different algal species biomasses by using acidic methanol for esterification. All modified biomasses showed major decreases in copper binding. Similar results were obtained for cadmium and lead by Fourest and Volesky (1996), who modified the biomass of the brown seaweed Sargassum by propylene oxide. The researchers also used Fourier transform infrared (FTIR) spectroscopy on cadmium-loaded alginate and Sargassum biomass. The instrumental analysis demonstrated that the cadmium binding mechanism included chelating (bidentate) complex formation with carboxyl groups of the alginate. Figueira et al. (1999) used FTIR and X-ray photoelectron (XPS) spectroscopy for studying the iron binding mechanism of algal biomass. Instrumental analysis revealed the chelating character of the iron complexation to carboxyl and sulfur groups. Ashkenazy et al. (1997) used the same techniques for investigating the mechanism of lead biosorption by acetone-washed biomass of the yeast Saccharomyces uvarum. The FTIR analysis showed a change in the symmetrical stretch of the carboxylate groups, while the XPS analysis showed a shift in the oxygen and a decrease in the nitrogen peaks. These findings indicated that lead uptake occurred mainly through binding to carboxyl groups as well as nitrogen-containing groups. Nakajima et al. (2000) found, by using electron paramagnetic resonance (EPR), that copper cations formed coordinated bonds with nitrogen and oxygen atoms in bacterial cell wall polymers. The metal binding properties of the marine algae were widely investigated, but the mechanisms responsible are still poorly understood. The unknown features of the metal biosorption mechanisms of the brown algae cell wall reduce their chances of being used as competitive products when compared to well-known metal-removal processes, like synthetic ion-exchange resins (Fourest and Volesky, 1996). The aim of the present study was to characterize the nature and binding mechanism of chemical groups occurring in the brown alga Sargassum vulgaris that were responsible for cadmium, nickel, and lead biosorption. In addition, the effects of chemical modification or extractions of some functional groups on these metal cations were also investigated. MATERIALS AND METHODS Biomass Fresh samples of brown marine macroalga, S. vulgaris, were collected from rocky seashores near Haifa, Israel. The macroalga samples were rinsed with distilled water for removal of external salts and sand and then with acetone solution. The algae samples were then roughly chopped and dried to a constant weight at room temperature. Metals Solutions Analytical grade reagents were used in all experiments. Stocks of single and multi-metal solutions (containing 1,000 mg/ L) were prepared by dissolving metallic salts (CdCl 2 6H 2 O, NiCl 2 6H 2 O, PbCl 2 ) in dilute HNO 3. All working solutions were prepared by diluting these stock solutions with distilled water and adjusting the solutions ph to 6 (if needed). The Ni 2+, Cd 2+, Pb 2+, and Mg 2+ concentrations in the multi-metal samples were determined simultaneously by an inductively coupled plasma atomic emission spectrophotometer (ICP-AES, Perkin- Elmer, Optima, 30000V). Single-metal concentrations in the relevant samples were determined by an atomic absorption spectrophotometer (AAS, SpectrAA-300+ Varian ABS). Multi- and Single-Metal Uptake Experiments Dry S. vulgaris biomass portions of approximately 0.1 g were thoroughly mixed with 50 ml of 5 mm multi-metal solution containing 5 mm of each of the following elements: Ni 2+,Cd 2+, and Pb 2+ or, in the case of a single-metal solution, containing 5 mm of just one of these metals. The suspension was stirred with a magnetic stirrer for 1 h at room temperature in a 125-mL Erlenmeyer flask. The algal biomass metallic cation uptake capacities were then calculated from the relevant metallic cation concentration in the algal samples (after total digestion with boiling concentrated nitric acid), according to Eq. (1): q ¼ C f a C ia V; ð1þ S or, from the relevant metallic cation concentration in the solution, according to Eq. (2): q ¼ V ðc i C f Þ : ð2þ S Cell Wall Surface Analyses Scanning Electron Microscopy (SEM)/Electron Dispersive Spectroscopy (EDS) Dry Sargassum samples, before and after metal cation biosorption, were glued onto 10-mm diameter metal mounts and coated with gold under vacuum in an argon atmosphere. The coated samples were put in to a JEOL JSM-5400 SEM/ EDS unit, and different sections in the samples were examined. The voltage used was 10 kev. This technique was 452 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 4, AUGUST 20, 2004

3 Table I. Metal uptake capacities of Sargassum samples after single- and multi-metal uptake capacities. Metal Sargassum metal capacity after single-metal uptake (mmol/g dry) Sargassum metal capacity after multi-metal uptake (mmol/g dry) Pb Cd Ni used to examine the algal cell surface and to evaluate the cell wall composition. X-ray Photoelectron Spectroscopy Analysis (XPS) Dry (dehydrated) Sargassum samples before and after lead, cadmium, and nickel biosorption were examined in high and low resolution with the Perkin-Elmer PHI 5600 Multi- Technique AES/XPS System. The samples were prepared by attaching a very thin layer to an adhesive tape placed on sample holder. An instrument vacuum of at least 10 9 Torr was maintained for all the analysis. The instrument was calibrated for AU4f 7/2 peak binding energy of 84 ev. The thickness of the analyzed layer was Å. This technique was used to evaluate the surface characteristics. A description of this technique and its interpretation were reported by Briggs (1990). Fourier-Transform Infrared Analysis (FTIR) Dry (dehydrated) Sargassum samples, before and after cadmium and nickel biosorption, were examined with the Nicolet IMPACT 400 Bruker FTIR spectrophotometer; the samples were incorporated with KBr Pellets. This technique was used to elucidate the chemical characteristics relevant to metallic ion sorption by the algal biomass. Metal Uptake Before and After Chemical Modification Partial extraction of sulfated polysaccharides (fucoidan) and alginic acid from the Sargassum samples was performed according to the method described by Percival and Figure 1. Sargassum cell wall before (right panel) and after (left panel) Cd adsorption from aquatic solution (original magnification: right panel, 5000; left panel, 10000). McDowell (1967). Two grams of dry algal biomass were boiled in distilled water for 1 h (hereafter referred to as sulfated polysaccharide extraction ) and then soaked for 24 h in a 0.5 M hydrochloric acid solution (referred to as alginic acid extraction ). The medium was replaced 3 times during the extraction periods. The algal samples uptake capacities of the metal cations Ni 2+,Cd 2+,Ca 2+, Mg 2+, and Pb 2+ before and after extraction were evaluated by metal uptake experiments, as described above. RESULTS Metal Uptake Capacities Table I shows the uptake capacities of Cd 2+,Ni 2+, and Pb 2+ in the different metal-laden Sargassum samples after single- and multi-metal uptake tests. The Sargassum uptake capacities for each metal after the single-metal uptake test were 1.05 mmol/g dry for Cd 2+, 0.95 mmol/g dry for Ni 2+, and 1.2 mmol/g dry for Pb 2+. The uptake capacities after the multi-metal uptake test were 0.35, 0.28, and 0.6 mmol/g dry for Cd 2+, Ni 2+, and Pb 2+, respectively. The sum of metal uptake capacities after the multi-metal test was Table II. XPS atomic concentration (in percentage) of relevant chemical elements in Sargassum sample, before and after heavy metal uptake. Atom *No metal uptake 2 Metal 1 Cd Ni Pb Pb+Ni C (+7.2) 64.9 (+36.8) (+8) 57.4 (+8) O ( 34) ( 48.5) ( 35.5) 27 ( 30) N ( 90.7) 2.33 ( 33.8) 1.3 ( 63) 2 ( 43.2) Ca ( 15.3) 0.9 ( 82) ( 100) ( 100) Mg ( 32.2) 1.04 ( 77) 1.6 ( 75) 0.94 ( 79.3) S (+195) 1.06 ( 7) 4.13 (+262) 2.06 (+80) Cd Ni Pb Cu 1 Values in brackets represent changes in XPS atomic concentrations after metal uptake by Sargassum biomass. 2 Raw Sargassum sample. RAIZE ET AL.: HEAVY METAL BIOSORPTION MECHANISMS IN BROWN MARINE MACROALGAE 453

4 Figure 4. High-resolution Pb, 4f XPS spectra of Sargassum and sodium alginate. Figure 2. High-resolution S, 2p XPS spectra of raw Sargassum sample (control) and Sargassum samples after Pb, Cd, and Ni uptake. approximately similar to the uptake capacities of each metal after the single-metal uptake test. Scanning Electron Microscopy (SEM) Scanning electron microscopy was used to examine samples of Sargassum biomass, both before and after metal binding. An electron micrograph of Sargassum biomass, both before and after cadmium binding, is presented in Figure 1. After completion of the metal binding, obvious morphological changes were seen in the cell well matrix, such as shrinking and sticking of layers. The morphological changes in the cell wall electron micrographs after Ni, Cd, and Pb adsorption were very small, and Figure 1 represents the changes before and after the binding of these cations. X-ray Photoelectron Spectroscopy Analysis (XPS) Table II shows the atomic concentrations of Cd 2+,Ni 2+, and Pb 2+ in the different metal-laden Sargassum samples, according to the XPS spectra analysis. Table II also shows changes in the atomic concentrations in the functional groups after the metal binding process (values in brackets). The changes in algal biomass observed after metal uptake included increases in carbon and sulfur atomic concentrations and decreases in nitrogen, oxygen, calcium, and magnesium atomic concentrations. After Ni 2+ uptake, there were significant changes in the atomic concentration of oxygen, carbon, and nitrogen and almost no change in sulfur atomic concentrations. The decrease in nitrogen concentrations was smaller than in the case of Cd 2+ and Pb 2+. The decrease in oxygen concentrations and the increase in carbon concentrations were significantly higher than in the case of Cd 2+ and Pb 2+. After Cd 2+ and Pb 2+ uptake, significant changes in the atomic concentrations of sulfur, nitrogen, and oxygen were noted. In the case of Cd 2+, the decrease in nitrogen concentration was higher, and in the case of Pb 2+, the increase in sulfur concentration was higher. These findings indicate that heavy metal uptake is accompanied by changes in sulfur, nitrogen, oxygen, and carbon binding. High-resolution S, 2p XPS spectra of the raw Sargassum sample and Sargassum samples after Pb 2+,Cd 2+, and Ni 2+ uptake are shown in Figure 2. After Cd 2+ and Pb 2+ uptake, the sulfur peak (165 ev) exhibited a shift of ev, while after Ni 2+ uptake there was almost no shift. High-resolution O, 1s XPS spectra of the raw Sargassum sample and Sargassum samples after Ni uptake are shown Figure 3. High-resolution O, 1s XPS spectra of raw Sargassum sample (control) and Sargassum samples after Ni uptake. Figure 5. FTIR spectra of Sargassum before and after Cd adsorption. 454 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 4, AUGUST 20, 2004

5 in Figure 3. The oxygen peak (533 ev) exhibited a shift of 1.5 ev. Analysis of the oxygen peaks after Pb 2+ and Cd 2+ uptake showed similar shifting. Analysis of high-resolution C, 1s XPS spectra indicated small and nonsignificant shifts in the carbon peak (285 ev) before and after Pb 2+,Cd 2+, and Ni 2+ uptake. High-resolution Pb, 4f XPS spectra of Sargassum and sodium alginate, after lead uptake, are shown in Figure 4. There are two energy peaks for Sargassum, at and ev, which correspond to those reported for 4f 7/2 orbital binding energy in metallic lead and to lead binding to oxygen-containing groups, respectively. For sodium alginate there is just one peak at ev (Moulder et al., 1992; see also the NIST XPS database [http//nist.gov/srd/ nist20.html]). Fourier-Transform Infrared Analysis (FTIR) Infrared spectra of Sargassum biomass samples before and after cadmium binding are shown in Figure 5. The difference between the two spectra was in the absorbance wavenumber and intensities. The cadmium Sargassum displayed new absorbance bands at 876, 779, 693, and 466 cm 1. The spectra of both samples displayed absorbance bands at approximately 1630, 1420, and 1085 cm 1, but the band intensities of the cadmium-loaded Sargassum were significantly higher. These bands indicated stretching of the carbon oxygen bonds in the carboxyl groups and the nitrogen hydrogen bond in amino groups, respectively (Fourest and Volesky, 1996; Ashkenazy et al., 1997; Kokinos et al., 1998). were reduced after the heavy metals binding (calcium and magnesium are present in large amounts in the raw algal biomass cell wall). After the metals binding process, the ph of the solution decreased from 6 to the range of 4 5. The metal binding affinity order, according to the metals binding capacities was: Pb 2+ > Cd 2+ > Ni 2+ > Ca 2+ > Mg 2+. After the extraction, the metals binding capacities decreased by 25%, 55%, and 75% for lead, cadmium, and nickel, respectively. DISCUSSION Metal Uptake Capacities Table I shows the uptake capacities of Cd 2+,Ni 2+, and Pb 2+ in the different metal-laden Sargassum samples after single- and multi-metal uptake tests. Metal cation uptake capacities after the multi-metal uptake test were significantly lower than those of metal cation uptake capacities after the single-metal uptake test, but the sum of metal uptake capacities after the multi-metal test was approximately similar to the uptake capacities of each metal after the single-metal uptake test (Table I). This implies that there exists a certain number of metal cations that can be adsorbed. In the case of the multi-metal adsorption test, this number is divided between the different absorbed metal cations, and each metal uptake capacity is smaller and is affected by competition. The difference between the uptake capacities of the examined metals is discussed in the chemical modification section. Chemical Modification The Sargassum metals binding capacities from multimetal solutions, before and after cell wall soluble component extraction, are shown in Figure 6. In both cases, lead binding capacities were higher than those of the other metals. Calcium and magnesium content in the biomass Figure 6. Metal cation binding capacities to Sargassum biomass from multi-metal solution before and after extraction of cell wall soluble components. Scanning Electron Microscopy (SEM) In Figure 1 obvious morphological changes in the cell wall matrix can be observed, such as shrinking and layer sticking. These changes were probably caused due to strong cross-linking between the cadmium and negatively charged chemical groups in the cell wall polymers. In the raw Sargassum are high concentrations of calcium and magnesium, and these bind to alginic acid monomers (alginic acid is one of the main cell wall components in the brown seaweeds). This binding creates a net of cross-linking (Percival and McDowell, 1967). When the Sargassum samples were exposed to cadmium solutions, the cadmium cations replaced some of the calcium and magnesium in the cell wall matrix and created stronger cross-linking. These observations are supported by the data presented in Table II and Figure 4. In the latter experiments, the calcium and magnesium concentrations in the cell wall, after binding of cadmium and other heavy metals, were smaller than their concentrations in the raw Sargassum. Figueira et al. (1999) and Kuyucak and Volesky (1989) observed the same trends for the brown algae Ulva lactuca and A. nodosum. The replacement of calcium and magnesium, by cadmium in the cell wall matrix, changed the nature of the cross-linking due to RAIZE ET AL.: HEAVY METAL BIOSORPTION MECHANISMS IN BROWN MARINE MACROALGAE 455

6 stronger electrostatic and coordinative bonding between the cadmium and the negatively charged chemical groups in the cell wall polymers (mainly carboxyl groups in the alginic acid monomers). This interpretation is supported by Percival and McDowell (1967), who found that a gel based on sodium alginate, after cross-linking with different divalent metals, changed its volume, depending on the metal being used. They found that gel volume reduction followed the metal order Pb > Cu > Cd > Ba > Ca, where lead formed the densest gel and calcium the least dense gel. X-ray Photoelectron Spectroscopy Analysis (XPS) XPS analysis was used for the characterization of functional groups and chemical bonds involved in the metal binding to Sargassum biomass. From the different XPS spectra, results presented in Table II and Figures 2 4 and from the chemical composition of the brown algae cell wall presented in Table III (Lewin, 1962; Percival and McDowell, 1967; Stewart, 1974; Schiewer and Volesky, 2000), the binding characterization can be estimated. Cadmium cations bind to chemical groups possessing oxygen and carbon (carboxyl groups in the alginic acid), nitrogen (amino/amido groups in the peptidoglycans and proteins), and sulfur (sulfonate, thiol in the sulfated polysaccharides and amino acids), and its binding process brings about smaller changes in calcium and magnesium concentrations, which indicates that ion exchange might not be the main binding mechanism. Fourest and Volesky (1996) reported that cadmium tends to form chelate complexes with carboxyl groups in Sargassum fluitans biomass. The significant change in sulfur and oxygen atomic concentration (Table II) accompanied by the sulfur peak shift (Fig. 1) indicates that cadmium interacts with sulfur and oxygen, providing evidence that sulfur-containing groups like thiol and sulfonate and oxygen-containing groups like carboxyl take part in cadmium biosorption. The same trends were found also for lead, but in this case the binding process also causes a significant decrease in calcium and magnesium concentrations, which indicates the existence and major role of both chelation and ion-exchange binding mechanisms. Lead also precipitates on the cell wall in its metallic form. Nickel cations bind mainly to chemical groups possessing oxygen, and its binding process causes a significant decrease in calcium and magnesium concentrations, which Table III. Main binding groups in brown algae. Binding chemical group Ligand atom Biopolymer Carboxyl Oxygen Alginic acid Thiol Sulfur Amino acids Sulfonate Sulfur Sulfate polysaccharides (fucoidan) Amine Nitrogen Amino acids, peptidoglycan Amide Nitrogen Amino acids indicates that the main binding mechanism is probably ion exchange. The significant change in carbon and oxygen atomic concentration (Table II) accompanied by the oxygen peak shift (Fig. 2) indicates that nickel interacts with oxygen, providing evidence that oxygen-containing groups like carboxyl take major part in nickel biosorption. These results match known theories and approaches concerning metal chemistry, such as hard and soft acid and base theory (HSAB). According to the Pearson s concept of hard and soft acid and base theory (HSAB), cadmium and lead are soft and intermediate-to-soft metals, respectively, nickel is an intermediate-to-hard metal, and calcium and magnesium are hard metals. In the HSAB theory, hard metals involve mainly electrostatic interactions with oxygen-containing ligands, and soft metals involve mainly covalent or coordinative interactions with sulfuror nitrogen-containing ligands (Forstner and Wittmann, 1981). When the current research XPS results were compared to HSAB theory and to other research results, a correlation between the binding characteristics of the examined metals and the principle of HSAB theory could be seen. Lead and cadmium cause significant changes in chemical groups with nitrogen and sulfur, while nickel causes minor changes in these groups. Lead and cadmium also bind, most often, with a higher affinity to negatively charged chemical groups by electrostatic and coordinative interactions (Kuyucak and Volesky, 1989; Figueira et al., 1999). These observations are supported by the metals binding capacities order, presented in Figure 6 (Pb 2+ >Cd 2+ >Ni 2+ >Ca 2+ > Mg 2+ ). Additional support of these observations comes from the simultaneous lead and nickel binding (Table II). XPS scans of Sargassum samples, after simultaneous lead and nickel binding from a solution with equal molar concentrations of these metals, showed that the lead concentration in the scanned area was about 10 times higher than the nickel concentration. This significant difference was due to lead having a higher affinity to the biomass due its ability to bind ligands by coordinative, covalent, and ionic interactions, while nickel binds ligands mostly by ionic interactions. In Figure 4 the presence of an energy peak at ev, which corresponds to the 4f 7/2 orbital energy level in metallic lead (Moulder et al., 1992; NIST XPS database [cited in Results]), indicated a reduction mechanism that occurred in the Sargassum cell wall matrix, in which certain chemical components of the cell wall caused redox reaction with lead cations. Brown algae are known to contain significant amounts of reducing compounds, identified as condensed or hydrolyzable polyphenols and characterized as phloroglucinol (Fourest and Volesky, 1996). Another component with redox activity that can participate in the lead reduction mechanism is probably the carotenoid pigment, fucoxanthin, which is the dominant pigment in brown algae and, due to its hydrophobic nature, must be part of the cell membrane including the cell wall layer (Lewin, 1962). The same reduction step was suggested for gold cations, with a Sargassum biomass, when tannin was the cell wall com- 456 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 87, NO. 4, AUGUST 20, 2004

7 ponent suggested to play a role in the reduction of gold, due to its strong reducing action (Figueira et al., 1999). Fourier-Transform Infrared Analysis (FTIR) FTIR analysis was conducted with raw and cadmiumloaded Sargassum in order to identify groups involved in the biosorption. The IR spectra of the samples were highly complex, reflecting the complex nature of the biomass composition. In spite of this complexity, several features can be observed and compared to results from other studies. The cadmium Sargassum spectra displayed new absorbance bands at 1085, 876, 779, 693, and 466 cm 1, which corresponded, respectively, to nitrogen hydrogen bond stretching in amino groups, carboxyl dimer vibrations (Silverstein et al., 1981; Ashkenazy et al., 1997), metal binding (Mozgawa, 2000), and carboxyl bond bending (Tian et al., 2000). These bands indicate involvement of chemical groups with nitrogen or oxygen. The data shown in Table III suggest that the main chemical groups possessing oxygen and nitrogen atoms are carboxylic groups in alginic acid and amino/amido groups in amino acids and peptidoglycans. These results correlate with the XPS results presented in Table II. The spectra of both samples displayed absorbance bands at approximately 1630 and 1420 cm 1, whereas the cadmium Sargassum absorbance band intensities were significantly higher. These bands correspond to the stretching of the carbon oxygen bonds in the carboxyl groups of the algal cell wall component. The absorbance bands in pure alginic acid, which represent the presence of these bonds, were 1740 and 1240 cm 1, respectively (Fourest and Volesky, 1996; Figueira et al., 1999). The differences in absorbance bands between Sargassum and alginic acid (in the Sargassum sample, the distance between the absorbance bands is smaller) were due to higher symmetry in the Sargassum sample cell wall matrix, which occurred due to complexation with metal cation. In raw Sargassum, these cations are mainly calcium and magnesium, and in cadmium-loaded Sargassum it is cadmium. The presence of absorbance bands, related to carboxyl dimers after cadmium binding, may indicate bridging complexes in which the cadmium cation bonds to two attached carboxyl groups in the cell wall matrix. This description is in line with the former finding of cadmium complexation with carboxylic groups. The cadmium binding is stronger (to form chelation complexes) than calcium and magnesium binding (by ion exchange). The raw alga consist of high concentration of calcium and magnesium which explain the lower absorption intensities of the raw algae samples. Chemical Modification In Sargassum and other brown macro algae, the cell wall contains large amounts of alginic acid (7 14% of the dry weight) and also sulfated polysaccharides (4% of the dry weight) (Lewin, 1962; Percival and McDowell, 1967; Stewart, 1974). In these polysaccharides there are high concentrations of the carboxyl and sulfonate groups, which are supposed to play a key role in the uptake of metal cations (Gardea-Torresdey et al., 1990; Fourest and Volesky, 1996; Figueira et al., 1999). In order to evaluate the contribution of the alginic acid and sulfated polysaccharides to the metal binding by Sargassum, the binding capacities of Pb 2+,Cd 2+,Ni 2+,Ca 2+, and Mg 2+ from multimetal solutions, before and after extraction of cell wall soluble components, were evaluated. In both cases, calcium and magnesium concentrations in the cell wall decreased after binding of heavy metals, which indicates on an ionexchange process when calcium and magnesium cations, which are present in the raw Sargassum, were replaced by the heavy metals in the solution. The same trend was shown in the XPS tests (Tables II and III). Williams et al. (1997) and Figueira et al. (1999) observed the same processes of calcium being replaced by nickel and cadmium during biosorption to brown macroalgae (Ulva lactuca, Ecklomia maxima). The ph decrease also indicated on ion exchange when protons from the cell wall were replaced by the heavy metal cation. The affinity order, according to the metals binding capacities after the multi-metal tests, was Pb 2+ >Cd 2+ >Ni 2+ >Ca 2+ >Mg 2+. After the extraction, the metals binding capacities decreased by 25%, 55%, and 75% for lead, cadmium, and nickel, respectively. After extraction, the total number of binding sites was reduced and most of the remaining sites were occupied by the metal with higher affinity to Sargassum biomass, like lead (which showed smaller decreases). Nickel showed a sharp decrease in its binding capacity. Nickel is mainly involved in electrostatic bonding to oxygen-containing ligands. The decrease in the number of carboxylic groups, due to the extraction of alginic acid, combined with the smaller affinity than the other metals affected severely the binding capacity of nickel. CONCLUSIONS The main cadmium cation sequestration mechanism by the algal biomass was apparently chelation, while the main nickel cation sequestration mechanism was ion exchange. Lead cations exhibit a higher affinity to the algal biomass, and their binding mechanism included a combination of ion exchange, chelation, and reduction reactions, accompanied by metallic lead precipitation on the cell wall matrix. During the ion-exchange process, calcium, magnesium, and also hydrogen cations in the algal cell wall matrix were replaced by the heavy metals investigated herein. Carboxyl groups were the dominant species in the heavy metal biosorption by the Sargassum biomass, especially in the case of nickel. Groups containing nitrogen and sulfur, such as amino/amido and sulfonate/thiol, were also involved in the adsorption of the heavy metals tested, especially lead and cadmium. RAIZE ET AL.: HEAVY METAL BIOSORPTION MECHANISMS IN BROWN MARINE MACROALGAE 457

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