Report
PLANT-DERIVED M A T E R I A L S for
METAL
ION-SELECTIVE BINDING and
PRECONCENTRATION
A
s the number of ecological and health problems associated with ^environmental contamination continues to rise, there is considerable interest in the development of biologically derived materials for reclamation and remediation of contaminated sites. These biomass materials can also serve as a means of preventing pollution via treatment of industrial waste streams before their introduction into the environment. Although a great deal of research has been focused on the use of living microorganisms and their inherent ability to degrade organic compounds (1), the potential use of these microorganisms, as well as higher organisms, for metal reclamation and remediation of heavy-metal-containing waste streams and sludges has only recently received attention. Numerous studies indicate that fungi and bacteria are able to adsorb heavy-metal ions (2-5); however, in this Report we will concentrate on the metal sorption ability of algae and higher plant species.
L a w r e n c e R. D r a k e a n d Gary D. Rayson New Mexico State University 22 A
areas or in routine analytBiomass materials inicalenvironmental methods, is determined by screening metal uptake efficiency. The screening can be used to removefor can involve determining the uptake efficiency for a single metal or the ability to metals from selectively adsorb a metal from a mixture several different metal ions or metal ion contaminated water ofspecies. We will discuss the screening
In some instances plant material can be incorporated into routine analytical procedures for use in preconcentration and separation. Commonly used chelating resins often concentrate interfering components in the sample matrix as well as analytes of interest, effectively raising the detection limits. In addition, the binding of components of hard water such as Ca+2 and Mg+2 often limits the utility of ionexchange resins because these ions are frequently present in high concentrations and can compete for heavy-metal-ion binding. One advantage of using plant materials for preconcentration is that Ca+2 or Mg+2 and monovalent ions such as Na+ or K+ do not significantly interfere with the binding of toxic, heavy-metal ions (6). Thus far, the utility of a particular plant biomass, whether it is considered for use
Analytical Chemistry News & Features, January 1, 1996
process, ion-selective binding, and preconcentration, as well as techniques that allow a more fundamental understanding of the metal-binding process. Types of biomass
Two broad categories of binding can occur in biomass-based materials, depending on whether the organism is living or dead (7). Passive binding, which occurs in both living and nonliving cells, involves very rapid physical adsorption and/or ion exchange with the cell surface. Active binding, typical of living cells, is characterized by slower metal uptake as a result of metabolic activity. Complications often arise when living materials are used for remediation because of metal toxicity, which limits the predictability of the metal uptake effectiveness of the biomass and its usefulness in preconcentration and ion-selective bind0003-2700/96/0368-22A/$12.00/0 © 1995 American Chemical Society
ing. Because metal ion uptake of nonliving plant materials occurs only as a result of passive binding, the effects of metal toxicity associated with active binding are not a complication. Metal uptake by nonliving biomaterials is believed to occur through sorption processes involving the functional groups associated with the proteins, polysaccharides, lignin, and other biopolymers found in the cell and cell walls (8,9). Unlike conventional ion-exchange resins, which are designed with a single functionality, biomass materials can contain numerous functionalities, including amino, carboxylate, hydroxide, imidazole, sulfate, and sulfhydryl groups. These polyfunctional biomass materials often exhibit unique metal adsorption abilities and have been successfully used for ionselective binding and preconcentration. In addition, once they are encapsulated in a suitable matrix, these nonliving biomaterials can be regenerated and reused. Biomaterials are capable of selectively sorbing certain metal ions from solutions containing a mixture of several metal ions (6,10-12). The ability to choose the species of plant for the selective recovery of metal ions depends on the generation of
binding data for different biomaterials. Although such screening is often time consuming, it is necessary because different plants can exhibit uniquely different metal binding abilities and efficiencies. Other factors taken into consideration when selecting plants for study include a priori knowledge of chemical components, material availability, cost, and even geographic location. The most convenient means of determining metal uptake abilities is through a batch process in which a quantity of the biomass is placed in contact with a solution containing the ions of interest. After a sufficient amount of time for equilibration, the solution is centrifuged or similarly separated, and the supernatant is removed and analyzed for any remaining metal ions. The concentration of bound ions is then determined by difference. Equilibration times, which depend on the material studied and the analyte concentration, are relatively short; the experiment (from initial contact to final determination) is typically completed in 1-2 h. We have investigated the ability of several biomaterials to bind Cu(II), A1(III), and Au(III) at different pH levels (13). These metals were selected because
they are representative of different classes of metal ions according to the hard and soft acid-base classification scheme. Biomaterials included blue-green algae, Datura innoxia, roots and stems of cattail plants (Typha latifolia), the leaves of young and mature tumbleweeds (Salsola spp.), Spanish moss, and alfalfa sprouts (Medicago sativa). Figure 1 shows the pH-dependent uptake of Cu ions for a representative sample of the biomaterials studied. Although most of these biomaterials exhibit pH-dependent binding profiles, Au+3 adsorption is independent of pH for D. innoxia and is almost 100% efficient under the conditions studied. Thus, there are significant concentrations of soft ligands in the cell wall fragments of D. innoxia (such as sulfur- and nitrogen-containing species). Al(III) binding was rather low for all species studied except alfalfa sprouts, which were 100% efficient at pH 5; thus, alfalfa would be an excellent candidate for preconcentration of AT3. In its native state, biomass often exhibits poor binding abilities because of chemical attack and structural degradation. To alleviate these problems, the biomass has been encapsulated in a supporting matrix using alginate microbeads (14), carra-
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rating the column mode of testing are the necessity of including pumps, fraction collectors, and in-line monitors in the experimental setup, as well as the timeconsuming nature of the experiment. Although a batch experiment can typically be performed in a couple of hours, a single-column experiment may require several days for the evaluation of a material with a relatively high capacity. With the longer time frame of the experiment, experimental complexity is again increased by compensation for influent and effluent instability as a result of temperature fluctuations, C0 2 dissolution, and evaporation effects. Selectivity Figure 1 . C u + 2 uptake efficiency as a function of pH for various biomaterials. Initial Cu concentration is 0.1 mM. (Adapted with permission from Reference 13.)
geenan gel (15), formaldehyde-based polymer (11), polyacrylamide (6,10), or silica-based polymers (6,10,16). Each material exhibits superior mechanical and physical properties with enhanced metalbinding capabilities compared with those of the native material. In addition, improvements in the porosity, chemical resistivity, and structural integrity of the biosorbent allow use of the biomaterial in column or fluidized-bed experiments. Once encapsulated in these matrices, the plant material becomes similar to commercial resins in both metal uptake ability and utility. In fact, the strontiumadsorbing ability of tomato and tobacco roots, immobilized in carrageenan gel, was found to be comparable to or exceed that of several commercial resins (15). These plant materials were tested in both batch andflowingsystems, and the immobilized product was found to be durable and capable of being regenerated by stripping the metal ions from the biomaterial with dilute acid or 1M KC1. In a batch experiment, which is performed in a nonsteady state, the concentration of metal ions continually decreases, the release of protons often increases, and the efficiency of the extraction can be altered because of unfavorable changes in solution parameters. The column experiment is a steady-state separation process in which the material is introduced 24 A
continuously into the biomaterial and the product stream is withdrawn continuously. The solution parameters (concentration and pH) are relatively constant in the column mode because the experiment is performed in a continuous mode. These experiments provide data indicative of flowing systems and should be an integral component in the evaluation of biomaterials for remediation purposes. Column experiments also give an indication of the performance of a specific biomaterial in large-scale applications (such as industrial waste effluent treatment) and are useful in preconcentration studies. Optimum separation parameters (flow rates, particle sizing, and solution conditions) can be evaluated on a bench scale and then successfully transferred to production-scale prototypes. Not only are meaningful capacity data obtained (binding capacity, effective binding capacity, and cation-exchange capacities), but the construction of breakthrough curves or "binding profiles" is also possible (Figure 2). For this hypothetical system, approximately 10 L of solution can pass through the column before the arbitrary breakthrough value is exceeded. Such information enables a more accurate prediction of the amount of influent that can be treated before the material fails to comply with preset standards or regulations. Among the disadvantages of incorpo-
Analytical Chemistry News & Features, January 1, 1996
Metal ions adsorbed by algae have been categorized into three classes (6). Class I includes metal ions (AT3, Cu+2, Cd+2, Cr+3, Co*2, Fe+3, Ni+2, Pb+2, UO^2, and Zn+2) that are bound strongly at pH values near neutrality but are not bound and can be easily stripped from the biomass at pH < 2 because of electrostatic attractions between the metal ion species after the ionization of chemical functionalities (e.g., carboxylates) at these pH values. Class II ions (PtCl^4, CrO;2, and SeOf) exhibit the opposite behavior of those in Class I; they are bound strongly at low pH but do not bind well at pHs above 5. Class III ions (Ag+1, Hg+2, and AuClj1) are the most strongly bound of all metals, and the binding is independent of pH. Some Class II and III metal ions are reduced to elemental metal at the biomass surface; for example, Au+3 is rapidly reduced to Au+1 and eventually to elemental gold. Although it is suspected that sulfur-containing compounds are involved, the exact mechanisms responsible for this reduction are currently unknown (17). Algae are not the only materials for which these three classes of metal-ion uptake are observed; both D. innoxia (13) and organic peat exhibit the binding profiles described above for gold ions. It is interesting that the uptake of silver ions by D. innoxia is pH independent at low concentrations but pH dependent at higher concentrations (18), which suggests that at least two sites are responsible for Ag+ uptake. As the higher affinity but less abundant site, which has been attributed to sulfhydryl functionalities, becomes satu-
rated, the gold ions are adsorbed at the more abundant site and exhibit behavior similar to that of Class I metal ions. Figure 3 shows the pH dependence of metal ion sorption to Chlorella vulagaris from a solution containing equimolar concentrations of Au+3, Cu+2, Pb+2, Zn+2, and AT3 (17). By simply adjusting the solution pH, one metal can be selectively or preferentially bound. For example, Au(III) ions are tightly bound at both high and low pH, but Cu ions do not bind below pH 2. Thus, by binding the metal ions at pH 5, nearly 100% of these two metal ions are removed from the solution. By lowering the pH to 2, Cu ions can be selectively removed from the biomaterial, whereas Au(III) ions remain bound to the biomaterial. The Au(III) ions can then be recovered by adding a complexing agent such as thiourea. Several permutations that might increase the selectivity for metal ion uptake to algae species, including the addition of competing ligands and even the addition of competing metal ions, have been suggested (10).
peat have been used for Pb preconcentration studies, and modified electrodes consisting of different strains of algae have been used in Au and Cu preconcentration studies (12). Preconcentration is simply a method of increasing the concentration of an analyte before quantitative determination. Metal ions from the sample are bound to the biomass and then extracted into a smaller volume, typically by lowering the pH. The analyte concentration is increased by the ratio of the initial volume to that of the extraction volume and the efficiency of metal ion uptake. When the metal adsorp-
M is the metal ion of interest and L is the ligand involved in metal ion adsorption. Equilibrium constants are determined from the slope of Scatchard plots in which [M]/[ML] is plotted against l/[ML] max , where [ML]max is the binding capacity of the biomaterial. Although these plots are useful for determining equilibrium constants near the saturation level, ionexchange equilibria become important at lower concentrations (24). These studies are useful for determining the metal ion uptake efficiency when 100% efficiency is not realized, in the determination of multiple binding sites, and for determining the volume of solution that can be introduced before breakthrough in a column experiment. Before using biomaterials for preconcentration purposes, one must determine both metal ion uptake efficiency of the biomass and optimum solution parameters such as pH. In a common preconcentration method, the batch-and-sluny technique, the biomass makes contact with the sample, the mixture is centrifuged, the supernatant removed, and the biomass resuspended in a smaller volume of acid. The rePreconcentration tion efficiency is less than 100%, the uptake efficiency is determined at several ini- sulting analytical matrix, which consists Algae have been used to preconcentrate of the biomass and the analyte of interest, specific analytes before quantitative deter- tial metal ion concentrations and then interpolated from a plot of these values or is independent of interfering species mination by GFAAS (19, 20), ICP-OES present in the initial sample solution. (21), and flame AAS (22). Biomass materi- from the equilibrium constants obtained Metal ion content is then determined by from these studies. als have also been successfully incorporated into carbon paste electrodes and Metal ion binding to biomass materials GFAAS. Calibration curves can be generated by two methods: by creating stanused for preconcentration (12, 23). Modi- is typically explained with the Langmuir fied electrodes consisting of sphagnum adsorption isotherm, M + L o ML, where dards via serial dilution and accounting for the preconcentration factor and metal uptake efficiency of the biomass or by preparing standards through preconcentration with the biomass.
Immobilized biomass can also be used for preconcentration, usually in a column experiment
Figure 2. Idealized binding profile for a column experiment. The equilibrium binding capacity is reached when the concentration of metal ions in the influent (C,) equals the concentration of metal ions in the effluent (Ce). For this curve, breakthrough is observed after 10 L of solution has passed through the column.
Immobilized biomass can also be used for preconcentration, usually in a column experiment. The volume of solution that can be introduced into the column before breakthrough is estimated from the evaluation of equilibrium constants and the suspected concentration of metal ions in the solution to be treated. As long as the flow through the column is halted before breakthrough, all metal ions from the solution are bound to the column. Dilute acid or a complexing agent is used to strip the metals from the biomaterial, and the ratio of the influent volume to the volume of acid used to strip the metals from the biomass is used as the enrichment or preconcentration factor.
Analytical Chemistry News & Features, January 1, 1996 25 A
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tained in the cell walls of several different algae species resulted in a semiquantitative agreement between the extent of esterification and the reduction in metal ion uptake of Cu (26). These results confirmed that carboxylate groups were the dominant functional groups responsible for adsorption of this metal ion. In a similar experiment, we tested the effects of esterification on D. innoxia and found that, although carboxylate moieties were partially responsible for metal ion uptake, other functionalities also played a significant role in metal adsorption. Interestingly, by saponifying the esterified cell fragments of D. innoxia, we were able to partially reverse the esterification effects and restore a portion of the metal adsorption capacity lost to esterification. Numerous studies using IR (2), X-ray Figure 3. pH-dependent binding profiles for metal uptake by Chlorella (27), luminescence (18, 28), and NMR vulagaris. (29,30) spectroscopies have identified the The metal ions were introduced as equimolar concentrations (0.1 mM). (Adapted from Reference 17.) functionalities responsible for metal ion binding. X-ray absorption near-edge structure (XANES) was used to determine information on the oxidation state of bound The batch-and-slurry method has been the riverine sample; preconcentration of gold in algae-gold samples, and extended used to analyze samples containing partPb, Cd, and Zn was less efficient in soluX-ray absorption fine structure (EXAFS) per-trillion levels of Cd in two seawater tions containing high salt concentrations. standards and ariverinestandard using The immobilized algae were reused as has been used to determine information the algae Stichococcus bacillaris (19). Al- many as 18 times, which attests to the re- about the type and number of atoms though quantitative determinations usability and robustness of these immobi- bound to gold and their distance from the gold atom (27). The ligating atom apwere not successful in seawater because of lized materials. pears to be sulfur for samples derived from the high salt content, an accurate determinaAu(I) complexes; but for samples pretion was obtained for theriverinestanThe basics dard. At the levels studied, Cd uptake was Because biologically derived materials are pared from Au(III), the algae appears to bind Au(I) (after reduction) to a nitrogen 100% efficient and the researchers were a mosaic of chemical functionalities that able to concentrate the analyte in the origi- can potentially bind metal ions, the identifi- moiety. These results indicate that metal speciation plays an important role in the nal solution by 2 orders of magnitude. cation of these chemical moieties is uptake efficiency and in determining the In a similar experiment, a Chlorella sp. rather difficult. Metal adsorption and defunctionalities responsible for metal ion sorption profiles as a function of pH have was used to preconcentrate part-peradsorption. been used to estimate the pKa values of the billion levels of Cu, and the efficiency of functionalities responsible for metal ion metal ion uptake was not affected by iniIn our lab, lanthanide luminescence, uptake (24,25), and from these pKa valtial sample volume, pH (3.5-6.0), or high particularly that of Eu+3, is used to aid in salt content (20). Exposure times for these ues, possible functional groups can be the identification of functionalities as well identified. Although accurate inflection as the number of sites responsible for studies were rather short (20 min for 6 samples in the Cd study), and contact with points in the pH-dependent profiles canmetal binding to biomaterials (18,28). not be determined and a functionality The power of this method lies in the outside contaminants was limited. (e.g., carboxylate groups) can exhibit nuunique singly degenerate 7F0-5D0 transiIn another example, silica-immobilized merous pK values, these studies, com3 tion of Eu(III), which exhibits a single algae were used to preconcentrate trace bined with rates of adsorption and depeak for each unique binding site. In adamounts of Pb, Cu, Zn, and Cd ions that were below the detection limits of flame sorption, are essential when attempting to dition, because the luminescence lifetimes identify the basic chemistry involved in are sensitive to ligation, functionalities AAS in mixed-metal solutions, simulated metal ion uptake. can be identified by comparing the specriverine water, brine, and simulated seatral peak(s) and lifetime (s) to known water solutions (22). Enrichment facChemical modification techniques have +3 Eu -ligand species. We have used this tors ranged from 20- to 200-fold, and Cu also been used to explore the chemistry intechnique to successfully identify the carwas efficiently extracted from all matrices volved in metal ion adsorption. Esterificaand Pb from the mixed-metal solution and tion of carboxylate functionalities con- boxylate and sulfate functionalities re26 A
Analytical Chemistry News & Features, January 1, 1996
sponsible for the high metal ion uptake of anther cells derived from D. innoxia (28). In a related competitive binding experiment, a relative affinity order for metal ion uptake was obtained using the decrease in the sensitivity of the Eu(III) luminescence (18), which may lead to a quick classification scheme for determining a plant's utility for metal ion uptake as well as the functionalities responsible for the metal adsorption capabilities of a certain biomaterial. We are currently using Eu(III) luminescence to classify several plant materials being considered for use as heavy-metal scavengers. With the multitude of plants available, the ability to quickly classify the usefulness of a particu-
NMR spectroscopy can be used to determine the chemical functionalities responsible for metal-ion binding. lar plant for metal ion uptake is of utmost importance. NMR spectroscopy has also proven valuable for determining the chemical functionalities responsible for metal ion binding (29-31). Solution-phase 113Cd NMR is effective in determining the functionalities responsible for Cd uptake by the algae Stichococcus bacillaris (31) and D. innoxia (29). The sensitivity of Cd chemical shifts to variations in the local chemical environment allows this technique to reflect subtle differences in the functional groups involved in Cd ion binding. An extensive chemical shift table was composed for Cd+2 bound to functionalities, including carboxylate, amine, hydroxyl, sulfhydryl, oxalate, sulfate, and sulfonate, that could be present in the cell walls of D. innoxia. The results suggested a preference for Cd uptake by carboxyl functionalities at pH levels < 5 but, as the pH was raised to 6, uptake by diamine-
(9) Greene, B.; Hosea, M.; McPherson, R.; Henzl, M.; Alexander, M. D.; Darnall, D. W. Environ. Sci. Technol. 1986, 20, 627-31. (10) Greene, B.; McPherson, R; Darnall, D. In Metals Speciation, Separation, and Recovery; Patterson, J. W.; Passino, R., Eds.; Lewis Publishers, Inc.: Chelsea, MI, 1987; pp. 315-38. (11) Nakajima, A.; Sakaguchi, T Biomass 1990,21,55-69. (12) Gardea-Torresdey, J.; Darnall, D.; Wang, J. Anal. Chem. 1988,60,72-76. (13) Lujan, J. R; Darnall, D. W.; Stark, P. C; Rayson, G. D.; Gardea-Torresdey, J. L. Solvent Extr. Ion Exch. 1994,12, 803-08. (14) Garnham, G. W.; Codd, G. A; Gadd, G. M. Future directions Environ. Sci. Technol. 1992,26,1764-70. The estimated cost for recovering radio(15) Scott, C. D. Biotechnol. Bioeng. 1992,39, nuclides and heavy metals from waste1064-67. waters and contaminated groundwaters is (16) Mahan, C. A; Holcombe, J. A. Anal. Chem. 1992, 64,1933-39. into the billions of dollars. Biomass tech(17) Darnall, D. W.; Greene, B.; Henzl, M. T; nologies offer an attractive alternative to Hosea, J. M.; McPherson, R. A; Sneddon, conventional reclamation and remediaJ.; Alexander, M. D. Environ. Sci. Technol. 1986,20,206-08. tion methods because of the abundance of (18) Ke, H-Y.D.; Anderson, W. L; Moncrief, materials and their possible selectivity R. M.; Jackson, P. J.; Rayson, G. D. Envifor the desired metal ions. This same naron. Sci. Technol. 1994,28, 586-91. (19) Majidi, V.; Holcombe, J. A / Anal. At. tive selectivity makes these materials Spectrom. 1989,4,439-42. promising preconcentration agents. Un(20) Shengjun, M.; Holcombe, J. A. Anal. fortunately, however, a substantial library Chem. 1990, 62,1994-97. (21) Mahan, C. A; Majidi, V.; Holcombe, J. A of plants that are efficient metal ion scavAnal. Chem. 1989, 61, 624-27. engers and the functional groups responsi(22) Mahan, C. A.; Holcombe, J. A. Spectroble for metal uptake by these plants has chim. Acta 1992,47B, 1483-95. (23) Ramos, J. A; Bermejo, E.; Zapardiel, A; yet to be developed. Armed with informaPerez, J. A; Hernandez, L. Anal. Chim. tion on the fundamental mechanisms by Acta 1993,273,219-27. which metals are bound to biomaterials, (24) Crist, R H; Martin, J. R.; Carr, D.; it will become possible to classify them Watson, J. R; Clarke, H. J.; Crist, D. R. Environ. Sci. Technol. 1994,28,1859-66. based on chemical interactions, which will (25) Crist, R H; Oberholser, K; McGarrity, J.; enable them to be more fully utilized. Crist, D. R; Johnson, J. K; Brittsan, J. M. Environ. Sci. Technol. 1992,26,496-502. (26) Gardea-Torresdey, J. L.; Becker-Hapak, Financial support by the National Science M. K; Hosea, J. M.; Darnall, D. W. EnviFoundation (grant #CHE-9312219) and the New ron. Sci. Technol. 1990,24,1372-78. Mexico Resources Research Institute are (27) Watkins, J. W.; Elder, R. C; Greene, B.; gratefully acknowledged. Darnall, D. W. Inorg. Chem. 1987,26, 1147-51. References (28) Ke, H-Y.D.; Birnbaum, E. R; Darnall, (1) Atlas, R. M. Chem. Eng. News 1995, D. W.; Jackson, P. J.; Rayson, G. D. Envi73(14), 32-42. ron. Sci. Technol. 1992,26,782-88. (2) Tsezos, M.; Volesky, B. Biotechnol. Bio- (29) Ke, H-Y.D.; Rayson, G. D. Environ. Sci. eng. 1982,24,955-69. Technol. 1992,26,1202-05. (3) Nagendra, C. R.; Iyengar, L.; Venkoba(30) Zhang, W.; Majidi, V. Environ. Sci. Techchar, C.J. Environ. Eng. 1993,119,369nol. 1994,28,1577-81. 77. (31) Zhang, W.; Majidi, V. Appl. Spectrosc. (4) Gopalan, R.; Veeramani, H. Biotechnol. 1993,47, 2151-55. Bioeng. 1994,43,471-76. (5) Tan, H.; Champion, J. T.; Artiola, J. F.; Gary D. Rayson, an associate professor of Brusseau, M. L.; Miller, R M. Environ. chemistry at New Mexico State University, Sci. Technol. 1994,25,2402-06. and Lawrence R. Drake, a postdoctoral re(6) Darnall, D. W.; Greene, B.; Hosea, M.; McPherson, R A; Henzl, M.; Alexander, search associate in Rayson's lab, perform reM. D. In Trace Metal Removal from Aque-search on the elucidation of mechanisms of ous Solution; Thompson, R., Ed.; Burlingmetal-ion binding to biomaterials using ton House: London, 1986; pp. 1-24. spectroscopic probes. Address correspon(7) Khummongkol, D.; Canterford, G. S.; Fryer, C. Biotechnol. Bioeng. 1982,24, dence about this article to Rayson at Depart2643-60. ment of Chemistry and Biochemistry, New (8) Crist, R. H.; Oberholser, K.; Shank, N.; Mexico State University, Box 30001, Dept. Nguyen, M. Environ. Sci. Technol. 1981, 3C, Las Cruces, NM 88003. 15,1212-17. type groups was preferred. Unfortunately, these studies are often limited by the same factor that makes them so attractive: their sensitivity. Because solution-phase 113 Cd NMR spectroscopy can determine even minor changes in the chemical environment, it is sensitive to changes not only in the Cd-bound species but also in the surrounding matrix. The use of solid-state NMR is expected to minimize if not eliminate most of these problems.
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