Adsorption Behavior of Mercury on Functionalized Aspergillus

The adsorption characteristics of mercury on Aspergillus versicolor mycelia have been studied under varied environments. The mycelia are functionalize...
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Langmuir 2009, 25, 360-366

Adsorption Behavior of Mercury on Functionalized Aspergillus Wersicolor Mycelia: Atomic Force Microscopic Study Sujoy K. Das,† Akhil R. Das,‡ and Arun K. Guha*,† Department of Biological Chemistry, and Polymer Science Unit, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed August 22, 2008. ReVised Manuscript ReceiVed October 24, 2008 The adsorption characteristics of mercury on Aspergillus Versicolor mycelia have been studied under varied environments. The mycelia are functionalized by carbon disulfide (CS2) treatment under alkaline conditions to examine the enhance uptake capacity and explore its potentiality in pollution control management. The functionalized A. Versicolor mycelia have been characterized by scanning electron microscopy-energy dispersive X-ray analysis (SEMEDXA), attenuated total reflection infrared (ATR-IR), and atomic force microscopy (AFM) probing. SEM and AFM images exhibit the formation of nanoparticles on the mycelial surface. ATR-IR profile confirms the functionalization of the mycelia following chemical treatment. ATR-IR and EDXA results demonstrate the binding of the sulfur groups of the functionalized mycelia to the mercury and consequent formation metal sulfide. AFM study reveals that the mycelial surface is covered by a layer of densely packed domain like structures. Sectional analysis yields significant increase in average roughness (Rrms) value (20.5 ( 1.82 nm) compared to that of the pristine mycelia (4.56 ( 0.82 nm). Surface rigidity (0.88 ( 0.06 N/m) and elasticity (92.6 ( 10.2 MPa) obtained from a force distance curve using finite element modeling are found to increase significantly with respect to the corresponding values of (0.65 ( 0.05 N/m and 32.8 ( 4.5 MPa) of the nonfunctionalized mycelia. The maximum mercury adsorption capacity of the functionalized mycelia is observed to be 256.5 mg/g in comparison to 80.71 mg/g for the pristine mycelia.

Introduction In the world of environmental science and technology, the removal of toxic heavy metals from wastewater by adsorption1,2 is receiving increasing attention over the currently used methodology3,4 because of the eco-friendly characteristics of the phenomenon. However, the commonly used adsorbents5-8 usually suffer from lack of adequate affinity9 and adsorption capacity, requiring prolonged equilibration time. In view of the vital roles played by surface and interfaces in numerous areas ranging from material to biological sciences,10-12 the modification of substrate surfaces has emerged as a very important field toward enhanced specificity and desired recognition while retaining bulk phase characteristic properties. In recent years, although considerable efforts have been directed toward biological adsorbents,13-17 specificities of these materials for cationic species in most cases * Corresponding author. E-mail: [email protected]. Fax: +91 33 2473 2805. Phone: +91 33 2473 4971/5904 Ext. 502. † Department of Biological Chemistry. ‡ Polymer Science Unit. (1) Moreno-Castilla, C.; Alvarez-Merino, M. A.; Lo´pez-Ramo´n, M. V.; RiveraUtrilla, J. Langmuir 2004, 20, 8142–8148. (2) Qadeer, R.; Khalid, N. Sep. Sci. Technol. 2005, 40, 845–859. (3) Chiarle, S.; Ratto, M.; Rovatti, M. Water Res. 2000, 34, 2971–2978. (4) Patterson, J. W.; Passono, R. Metals Speciation-Separation and RecoVery; Lewis Publisher: New York, 1990. (5) Velev, O. D.; Jede, T. A.; Lobo, R. F.; Lenhoff, A. M. Nature 1997, 389, 447–448. (6) Kadirvelu, K.; Kavipriya, M.; Karthika, C.; Vennilamani, N.; Pattabhi, S. Carbon 2004, 42, 745–752. (7) Holland, B. T.; Blanford, C. F.; Stein, A. Science 1998, 281, 538. (8) He, J.; Kunitake, T.; Nakao, A. Chem. Mater. 2003, 15, 4401–4406. (9) Pe´rez-Quintanilla, D.; del Hierro, I.; Fajardo, M.; Sierra, I. J. Mater. Chem. 2006, 16, 1757–1764. (10) Berdunov, N.; Mariotto, G.; Balakrishnan, K.; Murphy, D.; Shvets, I. V. Surf. Sci. 2006, 600, L287-L290. (11) Kasemo, B. Surf. Sci. 2002, 500, 656–677. (12) Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; LappinScott, H. M. Annu. ReV. Microbiol. 1995, 49, 711–745. (13) Davis, T. A.; Mucci, A.; Volesky, B. Water Res. 2003, 37, 4311–4330. (14) Tsezos, M.; Volesky, B. Biotechnol. Bioeng. 1982, 24, 385–401. (15) Schneider, I. A. H.; Rubio, J. EnViron. Sci. Technol. 1999, 33, 2213– 2217.

are found to be rather poor.12,18 Chemical modification of adsorbent materials19-23 having desired functionality is considered as an effective and feasible alternative for enhancing the adsorption capacity. Consequently, exploring the characterization of such surfaces is considered a prerequisite for an in-depth understanding of the relevant phenomenon. However, lack of appropriate methodologies have arrested desired progress toward comprehending detailed physicochemical properties of the surface following the process of functionalization. Recently, atomic force microscopy (AFM) has been successfully used to probe nanomechanical properties inherent to bacterial cells, mammalian cells and biomolecules, including analysis of cellular mechanical strain and elasticity, due to appropriate application of low forces to cells with minimal disruption.24-29 Although AFM is used to probe cellular mechanics under native and ambient conditions,30-32 to date, no report is available on (16) Darnell, D. W.; Greene, B.; Henzl, M. T.; Hosea, J. M.; McPherson, R. A.; Sneddon, J.; Alexander, M. D. EnViron. Sci. Technol. 1986, 20, 206–208. (17) Maruyama, T.; Matsushita, H.; Shimada, Y.; Kamata, I.; Hanaki, M.; Sonokawa, S.; Kamiya, N.; Goto, M. EnViron. Sci. Technol. 2007, 4, 1359–1364. (18) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P. Nat. ReV. Microbiol. 2004, 2, 95–108. (19) Zu¨rcher, S.; Wa¨ckerlin, D.; Bethuel, Y.; Malisova, B.; Textor, M.; Tosatti, S.; Gademan, K. J. Am. Chem. Soc. 2006, 128, 1064. (20) Sanghvi, A. B.; Miller, K, P-H.; Belcher, A. M.; Schmidt, C. E. Nat. Mater. 2005, 4, 496–502. (21) Reddy, A. R.; Reddy, K. H. J. Appl. Polym. Sci. 2004, 91, 1932–1936. (22) Xio, B.; Thomas, K. M. Langmuir 2005, 21, 3892–3902. (23) Lu, Y.-K.; Yan, X.-P. Anal. Chem. 2004, 76, 453–457. (24) Dufrene, Y. F. Nat. ReV. Microbiol. 2004, 2, 451–460. (25) Camesano, T. A.; Natan, M. J.; Logan, B. E. Langmuir 2000, 16, 4563– 4572. (26) Yao, X.; Walter, J.; Burke, S.; Stewart, S.; Jericho, M. H.; Pink, D.; Hunter, R.; Beveridge, T. J. Colloids Surf., B 2002, 23, 213–230. (27) Razatos, A.; Ong, Y. L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11059–11064. (28) Pelling, A. E.; Sehati, S.; Gralla, E. B.; Valentine, J. S.; Gimzewski, J. K. Science 2004, 305, 1147–1150. (29) Rotsch, C.; Jacobson, K.; Radmacher, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 921–926. (30) Zhao, L.; Schaefer, D.; Xu, H.; Modi, J. S.; LaCourse, W. R.; Marten, M. R. Biotechnol. Prog. 2005, 21, 292–299.

10.1021/la802749t CCC: $40.75  2009 American Chemical Society Published on Web 12/02/2008

Adsorption BehaVior of Hg on A. Versicolor Mycelia

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the alteration of the nanomechanical properties of fungal cell wall following the chemical functionalization process. This manuscript deals with an attempt to study the characterization of functionalized mycelia and their adsorption characteristics with respect to mercury following the introduction of xanthate group on the surface of Aspergillus Versicolor mycelia by covalent linkage of sulfur groups.

Scheme 1. (a) Functionalization of the Mycelia and (b) Adsorption of Mercuric Ions on the Functionalized Mycelia

Experimental Section

4000-350 cm-1 region. The IR spectra were recorded with 500 scans at a resolution of 2 cm-1. AFM Imaging. To monitor the morphological changes due to the xanthate functionalization as well as binding of heavy metal ions on the adsorbent surface, AFM images of fungal mycelia in both topographic and phase imaging mode were recorded using a multimode AFM (Veeco Metrology, Autoprobe CP-II, model no. AP0100). The details of the experimental procedure are described in the Supporting Information. Fluorescence Microscopic Images. The fluorescence microscopic images of mercury-adsorbed functionalized mycelia were recorded on a fluorescence microscope (Olympus BX-UCB). Regeneration of Adsorbent. The functionalized A. Versicolor mycelia obtained after adsorption with 50 mg/L metal ion solution were used for the desorption experiment. The metal-adsorbed mycelia (50 mg) were incubated with 25 mL of water having pH adjusted to 1.0-5.0 under shaking at 130 rpm for 60 min. On completion of the incubation period, the concentration of metal ions eluted from the loaded mycelia was measured by atomic absorption spectroscopy.

Chemical. HgCl2 was purchased from E-Merck, Germany. All the other reagents used were of analytical reagent grade and procured from Merck, India. Metal Ion Solution and Analysis. Aqueous solution (1000 mg/ L) of mercury was prepared by dissolving the required amount of HgCl2 in double distilled water and diluted to the desired concentration. The concentration of the metal ions was measured by cold vapor atomic absorption spectrometer (Varian Spectra AA 55) using appropriate standard solution for calibration. Preparation of the Adsorbent and Thiol Functionalization of the Adsorbent Template. A. Versicolor used in this study was maintained and cultivated in potato dextrose (20% potato extract and 2% dextrose) slant and broth, respectively. Preparation of A. Versicolor biomass is described in the Supporting Information. Xanthate functionalization of the adsorbent template was carried out according to the method of Wing et al.33 A. Versicolor mycelia (5 g) was treated with carbon disulfide (20 mL) and NaOH (25 mL of 14% aq. solution) and stirred (24 h at 10 °C). The resulting orange yellow product was filtered at different intervals and washed repeatedly with deionized water until neutral. The functionalized A. Versicolor mycelia was further washed with acetone and diethyl ether and dried at room temperature. Batch Experiment. Adsorption experiments were conducted in the batch process to study the effect of pH, adsorption kinetics, and the equilibrium adsorption isotherm of metal ions. To a 25 mL solution of mercuric ions (50 mg/L) 50 mg of pristine or functionalized adsorbent was added, except for in control flasks without any adsorbent. The flasks were incubated at 30 °C for 300 min under shaking (130 rpm) unless otherwise stated. The adsorbent was separated by centrifugation (10 000 rpm for 10 min) at the end of incubation, and the concentration of the metal ions in the supernatant was measured as described above, while the concentration of adsorbed metal ions was calculated using the mass balance equation. The details of the experimental procedures are given in the Supporting Information. Characterization of the Adsorbent. Zeta Potential Measurement. The surface charge characteristic of the functionalized A. Versicolor mycelia at different pH values was determined from zeta potential measurement by a Zetasizer (Malvern Zetasizer) following the protocol of Li and Bai.34 Electron Microscopic Study. Scanning electron micrographs of the adsorbents before and after adsorption were taken on a JEOL JSM 6700F field-emission scanning electron microscope operating at 8 kV equipped with an energy dispersive X-ray spectrometer (FESEM-EDXA). Samples were coated with platinum before FESEM-EDXA analysis. For transmission electron microscopy (TEM) mycelia were thin sectioned in ultramicrotome, and then micrographs were recorded on a high-resolution TEM (HRTEM; JEOL JEM 2010) instrument. Experimental details are described in the Supporting Information. Infrared Spectroscopy. Infrared spectra of the pristine, functionalized, and metal-adsorbed mycelia were recorded on a Bruker IFS 66v/S attenuated total reflection infrared (ATR-IR) spectrometer with a mercury-cadmium-telluride (MCT) detector, in the (31) Ma, H.; Snook, L. A.; Kaminskyj, S. G. M.; Dahms, T. E. S. Microbiology 2005, 151, 3679–3688. (32) Volle, C. B.; Ferguson, M. A.; Aidala, K. E.; Spain, E. M.; Nu´n˜ez, M. E. Langmuir 2008, 24, 8102–8110. (33) Wing, R. E.; Doane, W. M.; Russell, C. R. J. Appl. Polym. Sci. 1975, 19, 847–854. (34) Li, N.; Bai, R. Sep. Purif. Technol. 2005, 42, 237–247.

Results and Discussion Characterization of Xanthate Functionalization. In order to increase the mercury adsorption capacity, we functionalized the fungal mycelia by incorporating coordinative ligands containing xanthate groups (Scheme 1). The EDXA analysis of the pristine (Figure 1A) and functionalized (Figure 1B) mycelia were recorded to measure the chemical components present in the mycelia. The spectra show the peaks of C, N, and O corresponding to the precursor of carbohydrate and protein molecules of the mycelia. Additional peaks of S and Na (Figure 1B), recorded in the functionalized mycelia, indicate the incorporation of these groups following the functionalization process. Elemental analysis studies show that the functionalized mycelia contain 6.15% sulfur. The quantity of xanthate groups attached to the surface of the mycelia has been evaluated from the percentage of sulfur in the functionalized mycelia on the basis of the following expression (eq 1):9

L0 )

%S × 10 atomic weight of sulfur

(1)

and the value is found to be 1.92 mmol/g. ATR-IR spectra of the pristine and functionalized mycelia were recorded to obtain information on the chemical functionalization of the adsorbent surface. ATR-IR spectrum of the pristine mycelia (Supporting Information Figure S1A, curve 1) shows peaks at 3242.5, 2926.1, 2851.4, 1635.7, 1552.5, 1377.4, 1152.5, 1081.4, 1020.2, and 931.7 cm-1. The broad and strong band ranging from 3200 to 3700 cm-1 is assigned to the overlapping of the hydroxyl and amine groups of various polysaccharides present in the mycelia.35-37 This was consistent with peaks at 1000-1350 cm-1, due to the -OH bending and -C-O and C-N stretching vibrations, thus accounting for the presence of (35) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 4th ed.; Wiley: New York, 1981; p 166. (36) Jin, L.; Bai, R. Langmuir 2002, 18, 9765–9770. (37) Sankararamakrishnan, N.; Sanghi, R. Carbohydr. Polym. 2006, 66, 160– 167.

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Das et al.

Figure 1. EDXA of the pristine (A) and functionalized (B) mycelia recorded in spot profile mode.

Figure 2. AFM images of the pristine (A) and functionalized (B-D) mycelia taken under tapping mode in air. Topographic (top panel) and phase contrast (bottom panel) AFM images (scan area 1 µm × 1 µm). Mycelia was harvested from the reaction mixture after (B) 5 h, (C) 10 h, and (D) 20 h. AFM cross-sectional profile of the pristine (E) mycelia indicate relatively smooth surface compared to the functionalized (F) mycelia.

hydroxyl and amine groups on the mycelia. The bands at 2926.1 and 2851.4 cm-1 are attributed to the -CH stretching of the alkyl groups. The peak in the region of 1650 at 1550 cm-1 stands for of the acetyl carbonyl group of chitin and N-H bending vibration of the chitosan moiety, respectively.37 The weak peak at 931.7 cm-1 is attributed to the glycosidic bonds in the polysaccharide structure. Thus different functional groups such as hydroxyl and amine groups35-37 are present in the mycelia. The spectrum of the functionalized mycelia (Supporting Information Figure S1A, curve 2) exhibits remarkable spectral change compared to the pristine one in the region of 3200-3700 cm-1. The shifting of the broad peak at 3242.5 cm-1 of the pristine species toward higher frequency (3267.4 cm-1) in the functionalized mycelia suggests inter- and intrachain hydrogen bonding during grafting of xanthate functionality through the hydroxyl groups.35 The shifting of the stretching frequency from 1552.5 cm-1 along with the appearance of a new peak at 1115.7 cm-1 indicates the involvement of hydroxyl and amine groups in the thiofunctionalization process. Enhancement of intensity and bandwidth as well as peak shifting in the region of 800-1200 cm-1 are also found in the functionalized mycelia. It exhibits

xanthate characteristic peaks at 655.4, 1039.9, 1052.2, 1081.4, 1102.7 and 1225.2 cm-1 (Figure S1B, curve 2 in the Supporting Information), corresponding to γc-s, γcds, γcss(a), γc-o-c and γcss(s), respectively.38,39 The functionalization of the mycelia (Scheme 1) thus finds support form the shifting of the peak and the appearance of xanthate characteristic peaks. It has been reported that the adsorption capacity can be increased through chemical functionalization19-23 of the materials; however, no information regarding the change of surface properties is available. We investigated the morphological alterations of the mycelia on functionalization using AFM in both topographic as well as phase imaging mode. AFM micrographs of the mycelia after surface functionalization (Figure 2B-D) show a conspicuous change with respect to the pristine mycelia (Figure 2A), the mycelial diameter increasing from 2.8-3.2 µm to 3.6-3.8 µm (Supporting Information Figure S2). The topographic images exhibit the relatively smooth surfaces of the pristine A. Versicolor mycelia (Figure 2A, top panel) with (38) Sundholm, G.; Talonen, P. J. Electroanal. Chem. 1995, 380, 261–267. (39) Hellstro¨m, P.; Oberg, S.; Fredriksson, A.; Holmgren, A. Spectrochim. Acta, Part A. 2006, 65, 887–895.

Adsorption BehaVior of Hg on A. Versicolor Mycelia

rounded features distributed randomly throughout the surface. The phase image of the pristine mycelia (Figure 2A, bottom panel) depicts distinct characteristics in comparison with the corresponding topographic image, showing rounded surface features. The cell surface depicts a few undulated structures, which may develop as a result of depressions in the cell wall. Similar type of surface features were also observed by Ma et al.31 in A. nidulans. The phase contrast among the different cell surface components is found to be almost similar, indicating homogeneous distribution of the cell surface. Enhanced contrast in the phase images compared to that of the topographic ones is attributed to the viscoelastic differences of the various components of the cell. On functionalization, the mycelial surface is covered by a layer of densely packed material comprising segregated domains (Figure 2B). With increasing incubation period, the surface coverage increases (Figure 2C,D) along with the disappearance of rounded features noted in the pristine mycelia (Figure 2A). The domain like structures on the functionalized A. Versicolor mycelia are resolved more clearly to the corresponding phase image (Figure 2B-D, bottom panel) due to the increase in phase shifting of the different components. Sectional analysis demonstrates the relatively smooth surface of the pristine mycelia (Figure 2E) compared with the functionalized species (Figure 2F). On functionalization, the average roughness values (Rrms) increase significantly to 20.5 ( 1.82 from 4.56 ( 0.82 (pristine mycelia). The AFM images and the surface roughness profiles thus reveal structural alterations of the cell surface topography on functionalization. FESEM like the AFM images also delineate that the diameter of the mycelia increases on functionalization from 2.8-3.2 µm (pristine mycelia) to 3.6-3.8 µm (see Supporting Information, Figure S3). The alterations of the nanomechanical properties of the cell wall of A. Versicolor on chemical functionalization were obtained employing force spectroscopy (FS).28-31,40,41 Fungal mycelia were immobilized on the glass coverslip and used for the determination of local cell wall elasticity or stiffness (Young’s modulus, E) and relative rigidity by measuring force curves on the cell wall. Probe responses during the approach-retract cycle in the subsequent FS measurement summarized as force curves, can be used to measure the unit force required to indent a surface at a given distance. The extension half of the force cycle reflects the advance of the tip toward the cell, the initial cell contact, and the deflection of the tip as it is pressed into the surfaces. The shape and slope of the curve depends on the surface characteristics. The extension curve for the glass coverslip (Figure 3A) shows two regimes, a flat horizontal approach and a steep linear deflection, whereas for pristine A. Versicolor mycelia (Figure 3B) demonstrate three regimes. In between the horizontal approach and linear deflection, a nonlinear transition (marked by circle in the figure) is observed. The nonlinear region (sometimes referred to as the “repulsive” region)42 of the extension curve is caused by repulsive electrostatic and van der Waals forces between the cell surface and AFM tip. A similar phenomenon is also observed by Volle et al.32 in the case of E. coli. The extension curve for the functionalized mycelia (Figure 3C) also has three regimes, much like the pristine mycelia, but the nonlinear deflection is less than that of the pristine ones, indicating that the tip-cell surface interaction is different for the functionalized mycelia compared to the pristine mycelia. The ultimate linear portion of the approach curve for both pristine and functionalized (40) Ma, H.; Snook, L. A.; Tian, C.; Kaminskyj, S. G. M.; Dahms, T. E. S. Mycol. Res. 2006, 110, 879–886. (41) Hoh, J. H.; Schoenenberger, C. J. Cell. Sci. 1994, 107, 1105–1114. (42) Camesano, T. A.; Logan, B. E. EnViron. Sci. Technol. 2000, 34, 3374– 3362.

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Figure 3. Force distance curve of the glass coverslip (A), pristine (B), and functionalized (C) A. Versicolor mycelia. (D) Changes of yield threshold values and elasticity with functionalization process. Data represent an average of five independent experiments ( SD shown by an error bar.

mycelia exhibits a breakthrough feature (arrows) corresponding to the penetration of the cell wall by the tip apex. The breakthrough force (also called yield threshold)43 occurs at ∼2.1 N in the functionalized mycelia, whereas in pristine mycelia it appears at ∼0.7 N, which is about 3 times lower. Figure 3D shows that the yield threshold values change with functionalization process depending on the incubation period. The last section of the retraction curve demonstrates the adhesion events where the tip pulls away from the cell. The glass coverslip, pristine, and functionalized mycelia exhibit different adhesion patterns, and it is observed that the pristine mycelia develop larger pull off forces than that of the functionalized mycelia. Several factors could be responsible for the adhesion characteristics. The adhesive forces are the sum of the capillary forces, due to Laplace pressure from the water meniscus formed between the AFM tip and the sample, and the direct adhesion between the two surfaces in contact.31 It is considered that the capillary effects remain same in the pristine as well as in the functionalized mycelia, whereas the changes of the chemical composition on functionalization affect the relative adhesion properties between the cell wall and AFM tip. On the basis of adhesion events recorded for glass coverslip, pristine, and functionalized mycelia, it is suggested that pristine mycelia interacts with tip more strongly than that of the functionalized one. The slope of the linear portion of each extension was used to determine the cell wall rigidity and elasticity. The spring constant (KF) of the cell was obtained from the slope of the linear portion (terminal portion of the approach cycle or initial portion of the retraction cycle) of the force distance curve using the formula (eq 2)26,44,45

KF ) -

KC·S (KC + S)

(2)

where KC is the cantilever spring constant and S is the negative slope. The spring constant associated with the cell wall is reported to be relatively rigid. The cell wall elasticity (Young’s modulus, E) was measured using finite element modeling (eq 3):30 (43) Garcia-Manyes, S.; Oneins, G.; Sanz, F. Biophys. J. 2005, 89, 1812– 1826. (44) Suo, Z.; Yang, X.; Avci, R.; Kellerman, L.; Pascual, D. W.; Fries, M.; Steele, A. Langmuir 2007, 23, 1365–1374. (45) Velegol, S. B.; Logan, B. E. Langmuir 2002, 18, 5256–5262.

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E ) 0.80

KF R h h

Das et al. 1.5

()

(3)

where E is the elastic modulus of the cell wall, R is the radius of the mycelia, and h is the thickness of the cell wall. Cell wall thickness (h) and hyphal radius (R) were determined from transmission electron micrographs as described earlier.46 The cell wall rigidity and elasticity of the fungal hyphae following the finite element modeling are found to be 0.65 ( 0.05 N/m and 32.8 ( 4.5 MPa, respectively. The elasticity of the fungal cell wall was measured from different positions of 5-6 hyphae grown in five independent experiments. For each experiment, 50 force-distance curves were recorded. The Young’s modulus was calculated from the mean value derived from those 50 curves, and it slightly varied between the individual force-distance curves (standard deviation ∼3%). In view of the close agreement of the estimated elasticity values, the obtained elastic modulus of A. Versicolor is considered reasonable. We compared the elasticity of A. Versicolor mycelia with other biological materials adopting similar model. The cell wall of A. Versicolor is stiffer than the phospholipid vesicles (∼2 MPa)47 but lower than that of A. nidulans reported by Zhao et al.30 and Ma et al.31 The lower elasticity of A. Versicolor compared to that of A. nidulans may be due to the variation of cell wall constituents (β-1,3-D-glucans, mannans, chitin, and chitosan, as well as glycoproteins and lipids).48,49 It is suggested that the polysaccharide composition of A. Versicolor differs considerably from that of A. nidulans; consequently, considerable difference in the cell wall elasticity is observed. On xanthate functionalization, the cell wall rigidity and elasticity increased significantly (p < 0.1) to 0.88 ( 0.05 N/m and 92.6 ( 10.2 MPa, respectively, from 0.65 ( 0.05 N/m and 32.8 ( 4.5 MPa of the corresponding pristine mycelia. The cell wall elasticity changes as a function of time during the functionalization process (Figure 3D), as we have already demonstrated that the surface coverage increases (Figure 2) with increasing incubation time, indicating increasing hardness of the cell surface on functionalization. The differences in the obtained stiffness indicate changes of the mechanical properties of the material due to the chemical functionalization process. Adsorption Characteristics of Mercury on the Functionalized Mycelia. Kinetics of Adsorption. The most important characteristic of the adsorbent is the rate at which the solid phase adsorbs ions from the aqueous solution and attains equilibrium. The adsorption kinetics of mercury on functionalized mycelia was carried out at the optimum pH 6.0 (see Supporting Information for details). The results indicate that the process is very fast in comparison with the pristine mycelia (300 min)46 and reaches equilibrium within 15 min (Figure 4A). The rigidity of the functionalized mycelia increases with time during the adsorption process (Figure 4B). The mercury adsorbed functionalized mycelia have higher rigidity (185.6 ( 12.6 MPa) compared to that of the unadsorbed functionalized mycelia (92.6 ( 10.2 MPa), resulting from the increase of the surface hardness. Figure 4C shows the representative topographic images of the functionalized mycelia on mercury adsorption as a function of incubation period. The images depict that the surface morphology of the mercuryadsorbed functionalized mycelia is conspicuously different from that of the unadsorbed functionalized mycelia and exhibit the formation of metal nanostructures on the surface. On increasing (46) Das, S. K.; Das, A. R.; Guha, A. K. EnViron. Sci. Technol. 2007, 41, 8281–8287. (47) Liang, X. M.; Mao, G. Z.; Ng, K. Y. S. Colloids Surf., B 2004, 34, 41–51. (48) Pessoni, R. A. B.; Freshour, G.; Figueiredo-Ribeiro, R. d. C. L.; Hahn, M. G.; Braga, M. R. Mycologia 2005, 97, 304–311. (49) Tischer, C. A.; Gorin, P. A. J.; de Souza, M. B.; Barreto-Bergter, E. Carbohydr. Polym. 2002, 49, 225–230.

Figure 4. (A) Adsorption kinetics of mercuric ions on the functionalized A. Versicolor mycelia; (B) changes of elasticity of functionalized mycelia on binding of mercuric ions at different incubation periods; (C) AFM images (scan area 1 µm × 1 µm) of functionalized mycelia on the adsorption of mercuric ions at different incubation periods. Data represent an average of five independent experiments ( SD shown by an error bar.

the incubation period, the surface coverage with metal nanostructures increases and reaches a saturation level within 15 min. The reasonably fast kinetics reflects good congregation of the metal ions to the binding sites of the functionalized mycelia. The enhanced uptake rates have significant practical advantage in terms of time and space over the conventional techniques. Adsorption Isotherm. The adsorption of metal ions from aqueous solution is usually considered to be a function of the surface chemistry of the adsorbent. The functional groups such as amino, carboxyl, phosphate, and so forth present on the adsorbent play key roles in the adsorption process,22,50 thus it is expected that the functionalization of the adsorbent19,20,51,52 will enhance the adsorption capacity. Maximum adsorption capacity with respect to a particular ion or molecule is an important characteristic of an adsorbent. In order to characterize the metalbinding properties of the functionalized adsorbent, stoichiometry and affinity toward Hg2+(aq) was determined from equilibrium adsorption isotherm studies. The maximum adsorption capacity (Figure 5) of the xanthate functionalized mycelia for Hg2+(aq) is found to be 256.5 mg/g, whereas, for nonfunctionalized mycelia, the corresponding values is 80.71 mg/g. The adsorption capacity of the functionalized mycelia in the case of mercury is much higher in comparison with most other adsorbents (see the Supporting Information, Table S1). The isotherm profiles of Hg (aq) species (Figure 5) on the functionalized mycelia are much steeper than that of the pristine species. The sorption behavior analyzed by Langmuir,53 and (50) Merifield, J. D.; Davids, W. G.; MacRac, J. D. Water Res. 2004, 38, 3132–3138. (51) Lagadic, I. L.; Mitchell, M. K.; Payne, B. D. EnViron. Sci. Technol. 2001, 35, 984–990. (52) Celis, R.; Hermosın, M. C.; Cornejo, J. EnViron. Sci. Technol. 2000, 34, 4593–4599. (53) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221–2295.

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Figure 5. Adsorption isotherm of mercuric ions on the pristine and functionalized A. Versicolor mycelia. Data represent an average of five independent experiments ( SD shown by an error bar. Table 1. Adsorption Parameters of Mercury on A. Wersicolor Myceliaa type of adsorbent functionalized mycelia pristine mycelia a

Qmax (mmol/g)

Kd (L/g)

KL (L/mol)

1.27 ( 0.15

0.34 ( 0.09 (3.57 ( 0.85) × 104

0.4 ( 0.11

0.08 ( 0.05 (2.57 ( 0.35) × 103

Data represent an average of five independent experiments ( SD.

Freundlich54 isotherm models for optimizing the sorption system follows the type-I isotherm state according to IUPAC classification.54,55 The adsorption of mercury on the functionalized species fitted very well with the linearized form of Langmuir isotherm model with the correlation coefficient of 0.999 against the Freundlich (0.938) model (see Supporting Information Figure S5). On the other hand, the type-V appearance of the isotherm profiles with regression coefficients (r) 0.975 and 0.946, respectively, for Langmuir and Freundlich models on the pristine mycelia indicate lack of energetic uniformity9,54 of the binding sites for metal species compared with that of the xanthate functionalized species. The ability of the adsorbent to remove metal ions from solution can be expressed in terms of distribution coefficient (Kd):9

Kd )

Q Ceq

(4)

where Q is the amount of metal species adsorbed (mmol/g), and Ceq is the equilibrium metal ion concentration (mmol/L). Table 1 summarizes the uptake capacity (Qmax), distribution coefficient (Kd), and the Langmuir adsorption constant (KL),53 related to the adsorption energy for Hg (aq) species. The Qmax, Kd, and KL values for mercury on the functionalized mycelia are more than 3 times higher (p < 0.1) than those on the nonfunctionalized mycelia, indicting high affinity of the functionalized mycelia for mercury. The high affinity of the xanthate

groups for mercury is attributed to the metal binding ability of the sulfur groups,56-58 providing significant binding capacity enhancement of the xanthate functionalized A. Versicolor mycelia. In addition, mercury and sulfur groups are soft acid and soft base, respectively,59 hence high adsorption of mercury by xanthate-functionalized mycelia are due to soft-soft interaction according to the Pearson rule.60 Mechanism of Interaction. The adsorption of metal ions on the functionalized materials may involve electrostatic interaction and/or ion exchange processes. AFM, SEM, as well as EDXA analysis were carried out to obtain information on the adsorption process, including the mechanism involved. Functionalized mycelia exhibits the C, N, O, Na, and S peaks in the EDXA spectrum (Figure 1B). However, the peaks due to Na are replaced by Hg on adsorption (Figure 6A). The disappearance of alkaline earth metal peaks and the presence of Hg on the xanthate functionalized mycelia suggest that the adsorption process mainly follows ion exchange mechanism, as represented schematically (Scheme 1). SEM images (Figure 6B) like those of AFM ones also demonstrate formations of metal nanostructures on the xanthate functionalized mycelia after adsorption of mercury. The mercuryadsorbed functionalized mycelia generates bright colors under a fluorescence microscope (Figure 6C). The development of fluorescence properties thus clearly indicates the formation of mercuric sulfide on the functionalized mycelia.61 The ATR-IR spectra of the functionalized species show perceptible changes on the adsorption of the metal ions. The downfield shifts of the wavenumber as well as the intensity reduction in the region of 800-1200 cm-1, 655.4, 1039.9, 1052.18, 1081.4, 1106.74, and 1225.2 cm-1 occur as a result of metal ion adsorption. The appearance of new peak at 362 cm-1 (Figure S6, in Supporting Information) on the adsorbed mycelia demonstrates the formation of a Hg-S62 bond. The IR spectrum thus indicates that the S2- group of the functionalized mycelia is the main binding site of the metal ions yielding metal sulfide nanostructures. The formation of metal sulfide nanostructures may also be responsible for increased uptake capacity compared to that of the pristine mycelia. A detailed characterization of the metal sulfide nanostructures is currently underway. Regeneration and Reuse of the Mycelia. Desorption of metal ions from the loaded mycelia is essential for regeneration and reuse. Adsorbed mercury from the loaded biomass was desorbed only by changing the pH of the solution. About 92% of the absorbed mercury was eluated from the mycelia with a solution of low pH (