Environ. Sci. Technol. 1996, 30, 110-114
Chemical Modification and Metal Binding Studies of Datura innoxia
information obtained from these determinations would be important in any future attempts at chemically or biosynthetically altering the material to enhance the binding capacity or to selectively bind specific metals. The identity of the functional groups would also aid in determining the mechanisms responsible for the binding of targeted metals.
LAWRENCE R. DRAKE, SHAN LIN, AND GARY D. RAYSON*
A nonviable biomaterial of interest in our laboratory is derived from Datura innoxia. This plant exhibited a high affinity for heavy metals in a survey of biologically derived materials (11). D. innoxia belongs to the plant family Solanaceae (e.g., potatoes and tobacco) and is indigenous to Mexico and the southwestern United States. This plant is commonly found in the tailings of abandoned mines, which also demonstrates D. innoxia’s tolerance for heavy metals. Metal ion binding to D. innoxia at various pH values has been determined for Cu2+, Ag+, Ni2+, Cd2+, Eu3+, Sr2+, Ba2+, and UO22+ (12-18). D. innoxia exhibits a pHdependent metal binding profile with metal adsorption increasing from pH 1 to pH 6. For most metals, maximum adsorption occurs at pH 5 or 6.
Department of Chemistry and Biochemistry, P.O. Box 30001, Department 3C, New Mexico State University, Las Cruces, New Mexico 88003
PAUL J. JACKSON Genomics and Structural Biology Group, Life Sciences Division, LS-2, MS M880, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
The esterification of carboxylate functionalities present in the cell walls of Datura innoxia results in a decrease in metal uptake by as much as 40%, depending on the metal studied. These findings suggest that carboxylate groups are important in metal ion adsorption to this biomaterial. Base hydrolysis of the native plant material resulted in a slight increase in metal ion uptake for Cu2+ and Sr2+ and a decrease in uptake for Cd2+. These results are attributed to the hydrolysis of esters native to the plant material, which increases the carboxylate content but also results in conformational changes in the macromolecules that comprise the cell fragments. Both the esterified product and the hydrolyzed material were examined via infrared spectroscopy. A peak occuring at 1735 cm-1 (attributed to the carbonyl stretch) confirmed the esterification process. The infrared spectra of the hydrolyzed samples indicate further ionization of carboxylate groups or hydrolysis of esters native to D. innoxia.
Introduction Nonliving biomass materials have been shown to rapidly bind metals in high capacities. These materials have been used in the recovery of precious metals, preconcentration of metals, and trace metal detection via biomass-based electrode sensors (1-7). Commercial development of these materials for purposes of waste remediation has shown promise due to their low cost and selectivity against more common metal ions such as Mg2+ and Ca2+ (8, 9). Metal ion uptake by these biomaterials is believed to occur through interactions with functional groups that are native to the proteins, lipids, and carbohydrates that make up the cell walls (3, 10). To maximize the efficiency of these materials, the identity of the functional groups responsible for metal binding is vastly important. The * Corresponding author telephone: 505-646-2505; fax: 505-6462649; e-mail address:
[email protected].
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Spectroscopic techniques have been a valuable aid in determining functional groups that are responsible for metal binding to D. innoxia. Both Eu3+ and UO22+ luminescence have been utilized in determining the functional groups responsible for metal binding (13-18). Previous investigations indicated that carboxyl and dicarboxyl groups are responsible for Eu3+ metal binding to D. innoxia above pH 3 (16). Additionally, Eu3+ luminescence was used to assign a relative affinity order for various metal ions to the carboxyl sites that are responsible for Eu3+ metal ion adsorption at pH 5 (13). UO22+ luminescence studies indicated that carboxylate and phosphate functionalities were responsible for UO22+ adsorption (17, 18), and 113Cd NMR studies indicated that amine and carboxyl groups are responsible for the binding of Cd2+ to D. innoxia (19). Chemicial modification techniques are also of value in characterizing the functional groups responsible for metal binding. Gardea-Torresdey et al. (20) recently reported on the esterification of carboxyl groups on algae cells. This “capping” technique was utilized in metal binding studies involving five algae species. A decrease in metal uptake suggested that carboxyl groups were partially responsible for Cu(II) and Al(III) binding, but an increase in Au(III) uptake suggested that ionized carboxyl groups could only play a minor role in the binding of Au(III) to algae. The increased uptake of Au(III) was attributed to a decrease in the net negative charge of the algae cells upon esterification, which would favor the attraction of the anionic gold complex. Chemical modification of the biomaterial could also result in a material that is more efficient in the adsorption of metal ions. Crist et al. (21) recently reported on the sorption of amines to algae. They suggested that addition of amines to algae could result in a biomaterial that more efficiently complexes toxic metals. In this paper, we describe the esterification of carboxylate functionalities and the base hydrolysis of D. innoxia. The effects of these modifications on the metal binding capabilities of this biomaterial are determined for metal ions that have been demonstrated to have some affinity for the carboxyl groups on D. innoxia. In addition, structural
0013-936X/96/0930-0110$12.00/0
1995 American Chemical Society
effects of the modifications are confirmed and examined with infrared spectroscopy.
Experimental Section Materials. The conditions under which D. innoxia cells were cultivated have been described previously (16, 22). Cell wall fragments of cultured cells, isolated from the anther of the plant, were used in these studies. Complexities due to possible binding differences in cells originating from different parts of the plant were eliminated in this way. D. innoxia cell wall fragments were washed for 1 h in 5.0% H2SO4 that was adjusted to pH 2 with 1 M NaOH. This process removed endogenous metals and soluble biomolecules from the biomaterial. Cell wall fragments were then washed repeatedly with nanopure H2O (Barnstead Thermolyne D4751) to remove the acid. The cell fragments were then freeze-dried. Stock solutions (1000 ppm) of Cd2+ [8.90 mM Cd(NO3)2‚ 4H2O], Cu2+ [15.74 mM CuSO4‚5H2O], and Sr2+ [11.41 mM Sr(NO3)2] were prepared in 0.1 M NaCl. Both the metalcontaining contact solutions and the standards were prepared in 0.1 M NaCl. The 0.1 M NaCl was used to control the ionic strength and also to ensure that the matrices of the contact solutions and standards match. Cell fragments of D. innoxia were subjected to esterification of carboxyl groups in a manner similar to that described by Gardea-Torresdey and co-workers (20). Briefly, 10.0 g of D. innoxia was suspended in 650 mL of anhydrous methanol. A total of 5.4 mL of concentrated HCl was added to the suspension to make the mixture 0.1 M in HCl. Three 3.0-mL aliquots were removed periodically over a 2-day period. These samples and the final product were washed repeatedly with nanopure H2O to remove excess HCl and CH3OH. The samples and the final product were freezedried and then stored for subsequent experiments. To test the extent of esterification, 2 mL of 0.25 M NaOH was added to 30 mg of each of the freeze-dried samples. Each sample was agitated for 15 min and then stored overnight at 4 °C. The samples were centrifuged at 3000 rpm with a bench-top centrifuge (Sorvall GLC-2B), and 200 µL of the supernatant was diluted to 1 mL with nanopure water. Methanol released during the hydrolysis procedure was determined via gas chromatographic analysis. Each sample was spiked with 1 mM 1-butanol prior to injection. 1-Butanol served as an internal standard. Both the esterified material and the native cell fragments of D. innoxia were subjected to base hydrolysis to determine the effects on metal ion uptake. Samples for metal binding studies were prepared by adding 1.00 g of biomaterial to 100 mL of 0.1 M NaCl. The mixture was then adjusted to pH 11 with 1 M NaOH and continuously agitated over a 2-h period. The mixture was then adjusted to pH 5 and allowed to equilibrate for 30 min. All pH measurements were obtained with a pH meter (Orion 710A) equipped with a gel-filled electrode (Beckman 39844). The product was centrifuged and the supernatant removed. The product was freeze-dried and stored for later use. All metal binding experiments were carried out in a “batch” mode. For these studies, 1.00 g of the native biomaterial or modified material was suspended in 100 mL of 0.1 M NaCl. The mixture was stirred and adjusted to the pH of interest with 1 M HCl or 1 M NaOH. After the samples equilibrated at the desired pH, three 3.0-mL aliquots were removed from the suspension and placed in polypropylene test tubes. The samples were centrifuged and the super-
natant removed. Two of the samples were then contacted with 3 mL of a 1 mM solution containing the metal of interest at the desired pH. The third sample was contacted with 3 mL of 0.1 M NaCl at the same pH. Samples were agitated for 1 h to allow for equilibration (19) and centrifuged, and the pH of the supernatant was rechecked and recorded. Bound metal was determined by subtracting the metal ion content of the supernatant from the initial metal content of the contact solution. The sample contacted with 0.1 M NaCl served as a blank for metal analysis and as a reference for IR studies. Instrumentation. The quantity of methanol released upon base hydrolysis was determined with a gas chromatograph (GC) (Hewlett-Packard 5880A). The GC was equipped with a flame ionization detector and an RSL-500 polar column. The GC was operated with an injector temperature of 250 °C, a column temperature of 40 °C, and a detector temperature of 270 °C. Metal ion analysis was carried out on an atomic absorption spectrometer (Perkin-Elmer 3030B). Either airacetylene or nitrous oxide-acetylene flames were used as an atomization source. The average and standard deviation of three absorption measurements were recorded for each sample. All infrared (IR) spectra were obtained with a Fourier transform infrared spectrometer (Perkin Elmer FTIR 1720X). The averaged spectra were obtained at a resolution of 2 cm-1 and 5 scans. For IR studies, 5 mg of D. innoxia was encapsulated in 150 mg of KBr. This ratio resulted in a better resolved IR spectra than the 1:100 typically recommended. Theory of Method. The esterification of carboxylic acids native to D. innoxia was accomplished with methanol:
RCOOH + CH3OH
H+
RCOOCH3 + H2O
(1)
Although it is well known that metal ions can coordinate to esters, the binding ability should be greatly reduced and a marked decrease in metal uptake should occur. The hydrolysis of esters is catalyzed by base as shown in OH-
RCOOCH3 9 8 RCOO- + CH3OH HO
(2)
2
The methanol released in reaction 2 allows for the determination of the extent of esterification. Reaction 2 also suggests that the addition of base to the biomaterial could result in the hydrolysis of esters that are native to D. innoxia. This would result in an increase in the number of carboxylate functionalities and possibly an increase in metal binding.
Results and Discussion Modified and native D. innoxia cell fragments were contacted with 1 mM Sr2+, Cu2+, and Cd2+ at pH 5 and pH 2. Each of these metal ions has a pH binding profile that is similar to Eu3+. Comparison to literature pKa values suggests that carboxylate groups are the dominant functional groups involved in adsorption of these metal ions to D. innoxia (16). This conclusion was further supported by competetive binding experiments with Eu3+ (13). Each of these metals reduces the luminescence of Eu3+, indicating
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FIGURE 1. Quantity of methanol released from D. innoxia after having been subjected to 0, 6, 12, 24, and 48 h of the esterification process. FIGURE 3. Infrared spectra of (A) native, (B) esterified, and (C) saponified cell wall fragments of D. innoxia at pH 2.
FIGURE 2. Infrared spectra of (A) native, (B) esterified, and (C) saponified cell wall fragments of D. innoxia at pH 5.
that each has an affinity for the carboxyl groups responsible for binding of Eu3+. The quantity of methanol released after saponification of the samples is shown in Figure 1. The results shown are for native cell fragments not subjected to esterification and for cell fragments removed after being subjected to 6, 12, 24, and 48 h of the esterification process. Although a majority of the reaction occurs in the first 12 h, the amount of methanol released continues to increase up to 48 h. This indicates that the process is incomplete after 2 days and that it is possible to further esterify the cell fragments. Native cell fragments also release methanol upon hydrolysis. These results are due to the hydrolysis of esters native to D. innoxia and may increase the uptake of metal ions that bind to carboxylate groups. Infrared Spectra. The IR spectra of modified and native cell fragements contacted with metal ions are the same as the blank samples. The IR spectra obtained at pH 5 and pH 2 after contacting with the blank are shown in Figures 2 and 3, respectively. Of particular interest is the carbonyl region occurring in the IR spectra between 1700 and 1750 cm-1 (23). For the native material, this region is pH dependent, and the inflection observed at pH 5 appears as a more pronounced shoulder at the lower pH. These observations suggest that this shoulder is due to the carbonyl stretch of un-ionized carboxylates. The decrease in the shoulder indicates that a substantial portion of the carboxylate groups are unprotonated at pH 5 and are capable of electrostatic attractions to the positive metal ions. This correlates well with the findings that metal ions bind in a
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FIGURE 4. First derivative spectra of the carbonyl region of native and saponified D. innoxia at pH 5. The decrease in the peak intensity at 1745 cm-1 in the spectra of the modified samples indicates the hydolysis of esters native to the plant material.
greater abundance at higher pH values (11-18) and further confirms that carboxlyate functionalities are important in metal ion uptake. D. innoxia samples treated with acidic methanol exhibit a peak at 1735 cm-1 that is not pH dependent in the ranges studied. Futhermore, this peak is removed when esterified samples are subjected to base hydrolysis. From these observations, this peak is attributed to the carbonyl stretch of the ester that is formed in the modification procedure. A comparison of the un-ionized carboxyl/ester region before and after the base hydrolysis of the native material is shown as the first derivative in Figure 4. The decrease in the slope intensity at ≈1750 cm-1 in the first derivative plot of the hydrolyzed native cell fragments indicates that ester functionalities native to the material are hydrolyzed. This is further supported by a decrease in the band centered at 1237 cm-1 upon saponification. For D. innoxia, this region is suspected to be comprised of overlapping vibrations from un-ionized carboxylates and C-O-C stretching vibrations from native esters (23). As compared to the native material, this band area increases upon esterification and decreases after the base hydrolysis of D. innoxia. This indicates a substantial portion of this peak is due to the C-O-C stretch of esters. Other areas of the fingerprint region are also changed in the modification processes, which suggests that there are conformational changes in the macromolecules com-
FIGURE 5. pH 5 (solid) and pH 2 (hatched). Average quantity of metal ions bound by native and modified cell wall fragments of D. innoxia after contacting with 1 mM (A) Cu2+, (B) Cd2+, and (C) Sr2+ at pH 5 and pH 2. Results are normalized to 1.0 g of biomaterial. Individual measurements between the duplicate samples varied by less than 10% for a given material.
prising D. innoxia. Recently, there have been several reports on the use of IR in the regions between 1200 and 1350 cm-1 (amide III band) for monitoring changes in the secondary structure of proteins after denaturation or metal binding (24-26). Although the cell wall fragments of D. innoxia are believed to contain protein type structures, the interpretation of changes in the fingerprint region of these spectra is difficult due to the convoluted nature of the IR spectra and the unknown chemical composition of D. innoxia. Further studies via NMR spectroscopy are currently underway which may prove more beneficial in determining more subtle structural changes. Metal Binding Studies. The results of the metal binding studies for modified and native D. innoxia cell fragments are shown in Figure 5. The percentage of the initial Cu2+, Cd2+, and Sr2+ bound to the native biomass at pH 5 is 56%, 31%, and 16%, respectively. These results are consistent with the trend shown in previous studies (12, 13). The trend in metal uptake is also consistent with the decrease observed in the pH as Cu (pH 5.0->4.3) and Cd (pH 5.0->4.8) ions are bound to the cell fragments at pH
5. This suggests that an ion-exchange process, where positive metal ions are competing with H+ (or other metal ions) for sites in or on the cell walls, is at least partially responsible for the binding of these metal ions. Since this process is an equilibrium process, the amount of metal ions removed from the contact solution and bound to the cell wall fragments would increase in a titration-type batch experiment, where the pH is maintained at a relatively constant value, or in a column type experiment, where both metal ion concentration and pH are maintained at a constant value (26-28). The addition of Sr2+ at pH 5 results in no change in the pH. The affinity of Na and Sr ions for binding sites on D. innoxia may be similar, and because of the Na ions that have been previously bound to the cell fragments and the 100-fold excess of Na ions in the solution, only a small amount of Sr ions are bound to the cell fragments. This results in an undetectable pH change. There is also the possibility that Sr2+ is being adsorbed by the biomaterial by a different mechanism. The presence of multiple mechanisms for metal ion adsorption to D. innoxia has been demonstrated for Ag+ (13). Ag ions are adsorbed to D. innoxia by at least two different mechanisms that are dependent upon the concentration of the metal ion. At low Ag+ concentrations, the adsorption is pH independent, but as the concentration is increased, the adsorption becomes pH dependent. For the esterified cell material, there is a decrease in metal binding at pH 5 and, within the uncertainty of the experiment, no change in metal ion uptake at pH 2. Cd2+ binding is decreased by 40%, Cu2+ by 21%, and Sr2+ by 18%. These results indicate that carboxylate groups are important in the uptake of these metal ions at pH 5, but the lack of change in metal binding at pH 2 supports the supposition that groups other than carboxylate functionalities are involved in metal ion uptake at pH e 3 (e.g., sulfate groups) (16). The mole decrease in Cu and Cd binding is ≈11 and 12 µmol/g of biomaterial, respectively. From competetive binding experiments involving Eu3+ (13), Cu ions are known to have a much higher affinity for carboxylate functionalities in the cell wall fragments of D. innoxia than Cd ions; thus, the larger decrease in Cd2+ uptake appears contradictory. Interestingly, the decrease in pH when Cu (pH 5.0->4.3) is bound to the esterified cell fragments is comparable to the change monitored when Cu is bound to the native cell fragments. If only carboxylate functionalities and the ion exchange process were important in Cu uptake, the amount of Cu ions bound should be the same for the esterified and native cell wall fragments. In contrast to results obtained for the algae species (20), the results reported here suggest that the esterification process may not simply be a “capping” technique and that other functionalities and mechanisms are also responsible for metal ion sorption. For the native material subjected to base hydrolysis, there is a slight increase (
