**2.3 Biomaterial columns**

142 Biomaterials – Physics and Chemistry

It has been shown that carboxyl groups present in the cell walls of nonliving biomaterials contribute to metal-ion binding (Gardea-Torresdey, et al., 1999; 2001; Riddle, et al., 1997; Kelley, et al., 1999). Our group has used a variety of methods(Lin, et al., 2002; Drake, et al., 1996; Xia and Rayson, 1995; Drake, et al., 1996; 1997) to characterize the binding groups present in the cell walls of *Datura innoxia.* This plant is a member of the *Solanaceae* plant family and native to Mexico and the southwestern United States. To minimize variability of cell types investigated, the cell-wall fragments used are cultured anther cells of the plant. This plant was selected for study because it is a heavy metal resistant perennial that is both

Our group has concentrated primarily on nonviable biomaterials, specifically cell wall fragments from the cultured anther cells of *Datura innoxia.* The present study used frontal affinity chromatography with inductively coupled plasma optical emission spectroscopy (ICP-OES) detection for simultaneous monitoring both uptake and release of metal ions to both a chemically modified and native *D. innoxia* biomaterial (Williams and Rayson, 2003). The objective of the present study was to further investigate such sites through sequential exposure and subsequent stripping of three similar metal ions (Cd2+, Ni2+, and Zn2+) to both a modified and the native biosorbents, thus to study the role of carboxylate furface

It has been demonstrated (Drake, et al., 1996) that carboxylate-containing binding sites can be removed through the formation of the corresponding methyl esters by reaction with acidic methanol for 72 hours (Drake, et al., 1996). Undertaking a similar series of experiments with such a chemically modified sorbent enables the investigation of alternate

The cultured anther cells from *D. innoxia* were washed and prepared as described elsewhere (Drake, et al,, 1996; 1997). Only cell fragment aggregates with a mesh size greater than 200 (< 127 μm) were used for esterification. Following a method described elsewhere (Drake, et al., 1996), 10.0 grams of the biomaterial were suspended in 0.1 M HCl in methanol. The slurry was continuously heated at 60C and stirred for 72 hours. The biomaterial was then recovered through vacuum filtration, rinsed three times with 16.0-M water

In their native state, biomaterials have poor mechanical strength, low density, and a small particle size that can cause column clogging (Stark and Rayson, 2000). These characteristics can yield poor candidates for column-based water treatment applications. For this study native and modified *D. innoxia* biomaterials were each immobilized in a polysilicate matrix. The 40-60-mesh size fraction of the ground, and sieved immobilized biosorbents was then packed into columns. The process for immobilization has been described in detail elsewhere

Briefly, a suspension of 20 grams of the 100-200 mesh fraction of the washed biomaterial was generated with 300 mL of 5% v/v sulfuric acid adjusted to pH 2.0 by addition of a 6% (w/v) solution of Na2SiO35H2O. This suspension was stirred for 1 hour and the pH of the solution

(Barnstead,Millipore Ultrapure), freeze-dried, and set aside for later immobilization.

tolerant of arid climates and resistant to herbivory (Drake et al., 1996).

functionalities on passive metal ion binding of this material.

binding sites.

**2. Materials and methods 2.1 Esterification of biomaterial** 

**2.2 Immobilization of biomaterial** 

(Stark and Rayson, 2000).

The columns used have been described elsewhere (Williams and Rayson, 2003) and were constructed in-house from Plexiglas™ tubing (2.5 cm in length and 3 mm i.d.). Teflon™ tubing (0.8-mm i.d.) was used for all column connections. Interface of the column to the ICP-OES was accomplished by connecting the column outlet directly to the inlet of the crossflow type nebulizer using the minimum length of Teflon™ tubing (15 cm). Column effluent was monitored for each of 27 different metals simultaneously. Table 1 list the elements observed and their respective emission wavelengths.


Table 1. Elements and the corresponding emission wavelength used during monitoring of column effluents (elements of interests in this study indicated by boldface print).

Each column was packed with approximately 125 mg of the immobilized *D. innoxia* material and flow tested using distilled deionized water. Once packed and tested for leaks, each column was exposed to 20 mL of 1.0-M HCl using a peristaltic pump (Model Rabbit, Rainin) (1.0 mL/min for 20 min) and the effluent monitored for metals released from the biomaterial. Following the acid rinse, the columns were then exposed to 5 mL of distilled deionized water (1.0 mL/min for five minutes) to reestablish an ambient pH influent environment (~pH 6.2).

These studies involved, initially, the exposure of a small column (3.0 mm i.d., 10.0 mm in length) to an equimolar mixture of metal ions, specifically, Cd2+, Zn2+, and Ni2+, and exposure to solutions of each metal sequentially while continuously monitoring these (and other) metal species in the column effluent.

Comparative Metal Ion Binding to Native

0 10 20 30 40 50 Effluent Volume (mL)

**A**

0 10 20 30 40 50 Effluent Volume (mL)

**3.2 Modified material three-metal sequential study** 

of competitive metal ion binding.

material for these ions.

of the biomaterial.

0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06

0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06

Moles Metal in Effluent

Moles Metal in Effluent

material.

and Chemically Modified *Datura innoxia* Immobilized Biomaterials 145

the history of exposure appears to impact apparent steady state binding metal ion capacities

Moles Metal in Effluent

Moles Metal in Effluent

Fig. 1. Effluent profiles resulting from solutions of (A) 0.2mM Zn2+ (), (B) followed by 0.2

chemically modified *D. innoxia*.(D) Effluent profile of first 1.0M HCl wash of the metal-laden

Total amounts of metal bound to the native and modified materials at each stage of the sequence are listed in Table 2. Although the average amount of the fist metal ion bound was 24.5 µmol g-1, the average total metal ion bound after exposure to the second and third metals was 31.0 µmol g-1. This increase in the amount of metal captured by the biomass could be indicative of either the presence of metal ion-specific binding sites, or some degree

Simultaneous exposure of the biosorbent material to an equimolar solution of all three metal-ions (0.2 mM) was undertaken. Figure 2A shows the resulting effluent concentration profile. Even with a total metal ion concentration of 0.6 mM, the effluent concentration maximized at only 94% of the influent concentration (i.e., 0.19 mM). The modified biomaterial average capacity with simultaneous exposure was 41.72 mol g-1 biomaterial. Specifically, total amounts of metal ion bound were 13.24, 14.17, and 14.31 mol g-1 for Ni2+ Zn2+ Cd2+, respectively. This suggests no significant binding preference of the *D. innoxia* cell

Figure 2B shows the effluent concentration profile of the subsequent 1.0 M HCl metal-ion recovery/strip step. Total metal ion recovered from the two acid washes was 27.48 mol g-1.

mM Ni2+ (□), followed by 0.2mM Cd2+() pumped through a column packed with

**C**

0.00E+00 2.00E-07 4.00E-07 6.00E-07 8.00E-07 1.00E-06

0.00E+00

5.00E-07

1.00E-06

1.50E-06

0 10 20 30 40 50 Effluent Volume (mL)

**B** 

**D** 

0 0.5 1 1.5 2 2.5 Effluent Volume (mL)

#### **2.4 Frontal affinity chromatography with inductively coupled plasma atomic emission detection**

This technique has been described in detail elsewhere (Williams and Rayson, 2003). Briefly, the biomass packed column, having been exposed to 20 mL of 1.0M HCl to remove any metals remaining on the biomaterial (effluent monitored by ICPAES), was exposed to 5 mL of 16 M, distilled-deionized water. The influent was a metal-ion solution, 0.1mM-0.2mM, made from the nitrate salt of Cd2+, Ni2+, or Zn2+. Initially, the influent metal ion concentration increased as a step function.

Each influent was pumped through a column using a peristaltic pump (Rainin) at the rate of ~1.0 mL min-1 to a cross-flow type nebulizer and Scott-type double-pass spray chamber of the ICP-OES spectrometer (Jarrell-Ash, AtomComp700). The biomaterial in each column was exposed to each metal solution for 50 minutes. The effluent was monitored and resulting break-through curves were recorded for each metal ion (Figures 1A-C). Following exposure to the column, bound metal ions were stripped from the column using each of two exposures to a 1.0 M HCl solution. The first 150-second (~2.5 mL) exposure removed approximately 98% of the metal ions on the column (Figure 1D). The second 20 minute (~20 mL) exposure removed the remaining 2%. This was followed by a 5 minute (~5 mL) rinse with distilled deionized water to return the pH to f the biomaterial to that of the natural water (~6.2). Influent pH was not buffered to a predetermined pH to more accurately emulate conditions of a natural water supply within a remediation application.

With a three metal system there are six combinations that the metals can be sequentially exposed to the biomaterial (CdZnNi, CdNiZn, NiCdZn, NiZnCd, ZnCdNi, and ZnNiCd) and all six were performed on each column. Specifically, the ZnNiCd sequence involved exposure of a column packed with a biosorbents to a 0.20 mM Zn2+solution for 50 minutes (Figure 1A). The influent was changed to a 0.20 mM Ni2+ solution for another 50 minutes (Figure 1B). Similarly, a 0.20 mM Cd2+ solution was pumped through the same column for an additional 50 minutes (Figure 1C). The column was then exposed to 1.0 M HCl for 2.5 and 20 minutes to remove all bound metal ions (Figure 1D).

Simultaneous exposure of the three metals at the same molar concentration was also undertaken for each column with both the native (Figure 2) and modified (not shown) biomaterials. All determinations were performed in triplicate with three separate columns packed with each individual biosorbent.
