Langmuir 2008, 24, 8643-8650
8643
Interaction of Chromium with Resistant Strain Aspergillus Wersicolor: Investigation with Atomic Force Microscopy and Other Physical Studies Sujoy K. Das,† Manabendra Mukherjee,‡ and Arun K. Guha*,† Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India, and Surface Physics DiVision, Saha Institute of Nuclear Physics, Bidhannagar, Kolkata 700 064, India ReceiVed March 27, 2008. ReVised Manuscript ReceiVed May 21, 2008 The interaction of chromium and a chromate resistant Aspergillus Versicolor strain has been studied by atomic force (AFM) and transmission electron (TEM) microscopies. The nanomechanical properties such as cell wall rigidity and elasticity were measured by force spectroscopy and found to be 0.61 ( 0.08 N/m, and 20.5 ( 2.1 MPa, respectively. On chromium binding, ultrastuctural changes of the cell wall along with the formation of layered structures on the cell wall were observed. TEM and AFM micrographs demonstrate the accumulation of chromium on the cell wall, which were rough and irregular compared with the smooth pristine mycelia. The surface roughness, cell wall rigidity and elasticity increased to 35.5 ( 3.5 nm, 0.88 ( 0.05 N/m, and 62.5 ( 3.5 MPa, respectively, from the corresponding values of 5.2 ( 0.68 nm, 0.61 ( 0.02 N/m, and 20.5 ( 2.1 MPa for the pristine mycelia. X-ray photoelectron spectroscopy and Fourier transform infrared studies suggest that bound chromium was reduced to its trivalent state by the cell wall components. The reduced chromium species on the cell surface further electrostatically bind chromate ions forming layered structure on the cell wall.
Introduction Chromium, especially in the hexavalent form, is considered to be one of the most hazardous elements and included in the list of priority pollutants1,2 of the Environmental Protection Agency. However, trivalent chromium is an essential micronutrient for organisms. The tri- and hexavalent states of chromium are known to be stable species. The hexavalent chromium species exist in aqueous solution as oxyanionic entities such as chromate (CrO42-), bichromate (HCrO4-), and dichromate (Cr2O72-), the relative proportion of which depends on the solution pH.3,4 In comparison, Cr(III) forms stable hydroxo complexes [e.g., Cr(OH)n(3-n)+] having strong affinity for particle surfaces yielding insoluble Cr(OH)3 at neutral pH, and becomes almost immobile in the environment.5,6 In view of toxicity and related environmental hazards, the level of chromium in wastewater must be reduced to a permissible limit before discharging into the water bodies. The removal of chromium employing conventional methodologies suffers from certain limitations.7 Biosorption is emerging as an alternative technology and has received increasing attention for the removal and recovery of heavy metals from effluents in recent years. Gadd8 and Brierley9 described a number * Corresponding author. E-mail:
[email protected]. Fax: +91 33 2473 2805. Phone: +91 33 2473 4971/5904 Ext. 502. † Indian Association for the Cultivation of Science. ‡ Saha Institute of Nuclear Physics. (1) Hamilton, J. N.; Wetterhan, K. E. In Handbook on Toxicity of Inorganic Compounds; Seiller, H. G., Sigel H., Eds.; Marcel Dekker, Inc.: New York, 1988; p 239. (2) Nies, D. H. Appl. Microbiol. Biotechnol. 1999, 51, 730–750. (3) Cieslak-Golonka, M. Coord. Chem. ReV. 1991, 109, 223–249. (4) Tandon, R. K.; Crisp, P. C.; Ellis, J.; Baker, R. S. Talanta 1984, 31, 227– 228. (5) Baes, C. F.; Mesmer, R. E. The Hydrolysis of Cations; New York: John Wiley & Sons, 1976. (6) Rai, D.; Sass, B. M.; Moore, D. Inorg. Chem. 1987, 26, 345–349. (7) Zhou, X.; Korenaga, T.; Moriwake, T.; Shinoda, S. Water Res. 1993, 27, 1049–1054. (8) Gadd, G. M. Heavy metal and radionuclide by fungi and yeasts. In Biohydrometallurgy; Norris, P. R., Kelly, D. P., Eds.; A. Rowe: Chippenham, U.K., 1988.
of ways by which bacteria, fungi, and algae can take up toxic pollutants. Because of widespread industrial applications,10–12 microbes resistant to chromium are often encountered in the effluent. Therefore, the response of microorganisms to toxic metal ions is important because of their association in the reclamation of polluted sites.13,14 Since cell surface plays a vital role in the adsorption process, characterization of microbial surfaces is a prerequisite for an indepth understanding of the relevant phenomenon. 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, because of the precise application of low forces to cells with minimal disruption.15–20 Although AFM is used to probe cellular mechanics under native and ambient conditions, only limited attention has been paid to the nanomechanical properties of fungal cell walls.21,22 This manuscript deals with an attempt to describe the cell wall properties of Aspergillus Versicolor, a chromium (9) Brierley, C. L. Geomicrobiol. J. 1990, 8, 201–223. (10) McLean, J. S.; Beveridge, T. J.; Phipps, D. EnViron. Microbiol. 2000, 2, 611–619. (11) Romanenko, V. I.; Koren’kov, V. N. Mikrobiologiya 1977, 46, 414–417. (12) Verma, T.; Srinath, T.; Gadpayle, R. U.; Ramteke, P. W.; Hans, R. K.; Garg, S. K. Bioresour. Technol. 2001, 78, 31–35. (13) Ohtake, H.; Fujii, E.; Toda, K. EnViron. Sci. Technol. 1990, 11, 663–668. (14) Tsezos, M. Can. Metal. Quart. 1985, 24, 141–144. (15) Dufrene, Y. F. Nat. ReV. Microbiol. 2004, 2, 451–460. (16) Camesano, T. A.; Natan, M. J.; Logan, B. E. Langmuir 2000, 16, 4563– 4572. (17) 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. (18) Razatos, A.; Ong, Y. L.; Sharma, M. M.; Georgiou, G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11059–11064. (19) Pelling, A. E.; Sehati, S.; Gralla, E. B.; Valentine, J. S.; Gimzewski, J. K. Science 2004, 305, 1147–1150. (20) Rotsch, C.; Jacobson, K.; Radmacher, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 921–926. (21) Zhao, L.; Schaefer, D.; Xu, H.; Modi, J. S.; LaCourse, W. R.; Marten, M. R. Biotechnol. Prog. 2005, 21, 292–299. (22) Ma, H.; Snook, L. A.; Kaminskyj, S. G. M.; Dahms, T. E. S. Microbiology 2005, 151, 3679–3688.
10.1021/la800958u CCC: $40.75 2008 American Chemical Society Published on Web 07/04/2008
8644 Langmuir, Vol. 24, No. 16, 2008
resistant (1000 mg/L) fungal strain in relation to adsorption of chromate ions employing AFM along with transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS).
Experimental Section Chemicals. Dehydrated microbiological media and ingredients were procured from Himedia, India. All other reagents were of analytical reagent grade and procured either from Merck (Germany) or Sigma (U.S.A.). Metal Ions Solution. A stock solution (2000 mg/L) of chromium was prepared by dissolving the required amount of potassium dichromate (K2Cr2O7) in double-distilled water and diluted to get the desired concentration. The concentrations of total chromium and its hexavalent species were measured by a flame atomic absorption spectrometer (Varian Spectra AA 55) and diphenyl carbazide, respectively. Isolation of Chromium Resistant Strain. Chromium resistant A. Versicolor used in this study was isolated from tannery industry effluent. A small volume of the effluent (0.2 mL) was spread over different potato-dextrose agar (PDA) plates containing chromium as K2Cr2O7 in the range of 100-1000 µg/mL. The plates were incubated at 30 °C for 5 days. The colonies that developed on the medium containing 1000 µg/mL chromium were isolated and transferred to PDA slants. The fungal strains were maintained on PDA slants and used for metal adsorption studies. Mycelia of A. Versicolor were grown in potato dextrose broth (75 mL) taken in different Erlenmeyer flasks (250 mL). The flasks containing the medium were inoculated with 0.1 mL spore suspension (∼4 × 107 spores/mL) of the organism and incubated at 30 °C for 5 days with shaking (130 rpm). The suspension of spores in saline was prepared previously by growing the organism in PDA slants for 10 days or until sporulation. On completion of incubation, mycelia were harvested by filtration, washed with deionized water, and dried by lyophilization. Binding of Chromate Ions with Mycelia. Twenty-five milliliter K2Cr2O7 solutions containing 100 mg/L of chromium were incubated with 0.1 g of lyophilized A. Versicolor mycelia at 30 °C (ambient temperature) for 48 h under shaking (130 rpm) unless otherwise stated. The control flask received no adsorbent. At the end of incubation, mycelia were separated by centrifugation (10 000 rpm for 10 min), and the concentration of chromium in the supernatant was determined as described above. The uptake of chromium by the mycelia was calculated using the mass balance equation.23 The influence of hydrogen ion concentration on the binding of chromium was monitored by suspending A. Versicolor mycelia in 25 mL citratephosphate buffer (50 mM, pH 2.0-8.0) containing 50, 100, and 1000 mg/L of Cr(VI) taken in 100 mL Erlenmeyer flasks with other conditions remaining unchanged. The equilibrium adsorption isotherm experiment was conducted at pH 3.0 by varying the Cr(VI) concentration from 10 to 1000 mg/L with other conditions remaining the same. Characterization of A. Wersicolor Mycelia. The experimental details of zeta potential measurement, TEM, and energy dispersive X-ray analysis (EDXA) are described in the Supporting Information. Immobilization of Fungal Mycelia for AFM Imaging. Fungal mycelia were harvested from the growth medium, washed with deionized water, and then conditioned (protonated) by suspending in citrate-phosphate buffer (50 mM, pH 3.0). Samples were then prepared by incubating the mycelial suspension with an ultrasonically cleaned glass coverslip for 30 min, followed by repeated washing with deionized and double-distilled water and then airdried. The surface topography of the chromium loaded fungal mycelia was compared with those of the pristine (protonated) mycelia. Atomic Force Microscopy. AFM images of A. Versicolor mycelia were recorded in air at ambient conditions in tapping mode using a multimode AFM (Veeco Metrology, Autoprobe CP-II, Model No (23) Das, S. K.; Bhowal, J.; Das, A. R.; Guha, A. K. Langmuir 2006, 22, 7265–7272.
Das et al. AP0100) (for details, see Supporting Information). On completing image recording, an offline section analysis was performed for each image to obtain information on the sample height and surface roughness. The surface roughness of the sample was measured using the ProScan Image Processing Program provided by the manufacturer. The root-mean-square average of the surface roughness (Rrms) was measured with the following expression:24
Rrms )
∑ (Zi - Zavg)2 Np
(1)
where Zi is the current Z value, Zavg is the average of the Z values within the given area, and Np is the number of point within the given area. Force-distance curves were recorded in contact mode to measure the cell wall elasticity and rigidity. A phosphorus (n)doped Si cantilever with a spring constant of 0.9 N m-1 was used for force measurement. In each experiment, the force curve was recorded on bare coverslip glass to serve as a reference. In every experiment, approximately 50 force curves were recorded. A single cantilever and tip were used for all force spectroscopy measurements as we measured the relative changes in cell wall elasticity. Since the present study deals with the changes of the surface properties along with the morphology of the fungal mycelia compared to that of the chromium-loaded species, imaging and force spectroscopy were recorded without any treatment (chemical fixation and/or gold coating). Standard errors were calculated for elastic modulus of pristine and chromium-loaded mycelia and a Student’s t test was used to assess the significant difference (SigmaStat 3.0.1). Fourier Transform Infrared (FTIR) and XPS study. The infrared and X-ray photoelectron spectra of the pristine and chromium loaded A. Versicolor mycelia were recorded on a Shimadzu FTIR spectrophotometer and an Omicron Multiprobe (Omicron NanoTechnology GmbH, U.K.) spectrometer fitted with an EA125 hemispherical analyzer, respectively. Standard procedures and literatures were followed to assign the peaks.25,26 The experimental details are described in the Supporting Information.
Results and Discussion The effluent collected from the tannery waste contains several chromium resistant microorganisms. The fungal strains resistant to high amount of Cr(VI) (1000 µg/mL) were isolated. The strain which showed maximum chromium binding capacity was identified as A. Versicolor from the Department of Botany, University of Calcutta. Adsorption Isotherm. The most important characteristic of an adsorbent is maximum uptake capacity with respect to a particular ion or molecule. To characterize the metal binding properties of the adsorbent, stoichiometries and affinities were obtained from equilibrium adsorption isotherm. The adsorption isotherm of chromium on A. Versicolor mycelia was carried out at an optimum pH value of 3.0 (see Supporting Information for details). The maximum chromium binding capacity of A. Versicolor mycelia in the present system obtained from the adsorption isotherm (Figure 1) is found to be 225.2 mg/g, which is much higher than the other biosorbents (see Supporting Information, Table S1) under the same conditions as those reported.27–31 The result demonstrates the higher binding capacity (24) Zhang, X.-Z.; Yang, Y.-Y.; Chung, T.-S.; Ma, K-X. Langmuir 2001, 17, 6094–6099. (25) Seah, M. P.; Briggs, D. Practical Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, 2nd ed.; Wiley & Sons: Chichester, U.K., 1992. (26) Nakamoto, K. Infrared Spectra of Inorganic and Co-ordination Compounds, 2nd ed.; Wiley Interscience: New York, 1970. (27) Kiran, B.; Kaushik, A.; Kaushik, C. P. J. Hazard. Mater. 2007, 141, 662–667. (28) Kiran, B.; Kaushik, A. Biochem. Eng. J. 2008, 38, 47–54.
Interaction of Chromium with A. Versicolor
Figure 1. Adsorption isotherm of chromium on A. Versicolor mycelia. Data represent an average of four independent experiments ( SD shown by the error bar.
of the cell wall of A. Versicolor mycelia compared with that of other biosorbents. The sorption behavior analyzed by different isotherm models such as Langmuir,32 Freundlich,33 and Brunauer-Emmett-Teller (BET)34 for optimizing the sorption system follows the type II isotherm model according to IUPAC classification.33 The present sorption process fitted very well with the BET isotherm model (Figure 1, inset) with a correlation coefficient of 0.992 against those of Langmuir (0.942) and Freundlich (0.962) models, respectively (see Supporting Information, Figure S2), indicates chemisorption and multilayer coverage of adsorbate on the adsorbent. Binding Mechanism. Transmission electron micrographs of the pristine mycelia along with the chromium loaded mycelia were recorded to determine the association of chromium with the cell. The micrographs conspicuously reveal the electrondense region/layer throughout the cell wall, only without any intracellular accumulation (Figure 2, B-D). These layers are composed of chromium, as indicated by EDXA (Figure 2F); however, no such peak of chromium was detected when the spectra were recorded on the cytosolic region of the treated cell (Figure 2E). The signals of carbon, nitrogen, and oxygen are characteristics of the polysaccharides and proteins present on the cell wall of the mycelia. Figure 2A depicts the control cells (protonated) free of metal ion accumulation. EDXA spectrum also exhibits the absence of chromium in the control cell. Thus, it may be concluded that the cell wall components of the mycelia contain the major binding sites for chromium, and the process of diffusion and/or transportation into cytoplasm is insignificant. The cell wall of A. Versicolor prevents chromate transportation into the cytoplasm, thereby providing a survival rout for avoiding the contact of chromate with the intracellular organelles. The control (protonated) cell wall (Figure 2A) shows sharp and smooth surface edges; however, the cell wall becomes rough and irregular on the sorption of chromium. The thickness of the electrondense layer increases with increase in the chromium concentration leading to the formation of multilayer coverage of chromium species on the cell wall (Figure 2, B-D). (29) Dias, M. A.; Lacerda, I. C. A.; Pimentel, P. F.; de Castro, H. F.; Rosa, C. A. Lett. Appl. Microbiol. 2002, 34, 46–50. (30) Gupta, V. K.; Shrivastava, A. K.; Jain, N. Water Res. 2001, 35, 4079– 4085. (31) Bai, R. S.; Abraham, T. E. Bioresour. Technol. 2003, 87, 17–26. (32) Langmuir, I. J. Am. Chem. Soc. 1916, 38, 2221–2295. (33) Glastone, S. Textbook of Physical Chemistry, 2nd ed.; MacMillan Publishing Co: New York, 1962; p 1196. (34) Brunauer, S.; Emmett, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309–319.
Langmuir, Vol. 24, No. 16, 2008 8645
Detailed information on the interaction of chromate ions with the A. Versicolor cell is more appropriately obtained using AFM technique. The AFM images along with the surface height profiles reveal the structural alteration of the cell surface topography on chromate ion binding. On binding the chromate ions with A. Versicolor, the cell surface is covered by domain-like materials (Figure 3A) composed of chromium, as confirmed by EDXA. The surface morphology was transformed to a regular cage-like pattern (Figure 3B) with increasing chromate ion concentrations. Multilayer growth formation on the adsorbed species was observed with further increase of chromium concentrations (Figure 3C-F). The surface of the pristine mycelia (depicted in the Supporting Information, Figure S3) was relatively homogeneous with Rrms value 5.2 ( 0.68 nm (Figure S3E) but developed heterogeneity on sorption of chromate ions. Phase imaging provides an indispensable method for measuring the sample heterogeneity. Thus, it is very difficult to separate the effect of mechanical, chemical, and topographic variations in the sample leading to changes in the phase response. The phase image of the pristine mycelia (SI Figure S3D) exhibits more features than the corresponding topographic image (SI Figure S3C), showing rounded surface features. Some pore-like structures are also noted on the cell surface, likely due to the depressions in the cell wall. Similar types of surface features were also observed in A. nidulans ascribed by Ma et al.22 The phase contrast among different components of the cell surface is almost similar, indicating homogeneous distribution of the cell surface. Enhanced contrast in the phase images compared to that of the topographic images is attributed to the viscoelastic differences among the different components of the cell. The surface develops heterogeneity on adsorption of chromate ions. The cell surface depicts lighter color in the phases revealing the increase in the hardness of the cell surface. The phase images show more characteristics (Figure 4A-F) than the topographic images (Figure 3A-F) indicating an increase in the hardness of the surface components after the adsorption of chromate ions. The present image resolution of the chromium-loaded mycelia (Figures 3A-F and 4A-F), except for the pristine one (SI Figure S3), is lower in comparison to fixed cells and/or gold-coated cells since the latter increases surface resilience.22,35 The relatively poor image resolution of the chromium loaded mycelia (Figures 3A-F and 4A-F) in comparison with that of the pristine mycelia (SI Figure S3) may be due to an increase in surface roughness. This appears to cause tip-sample interaction, resulting in inconsistent feedback. Sectional analyses were conducted to measure the surface profile of the chromium-loaded mycelia. The surface plot of the chromium-loaded mycelia demonstrates uniform distribution (Figure 5A) of chromium species throughout the surface at a certain concentration, with a roughness of 10.5 ( 2.1 nm. However, layer-by-layer depositions (Figure 5B) were noted during increasing concentrations of chromium, resulting in increments of Rrms values (20.3-35.5 nm) (Figure 5C). Force Spectroscopy of A. Wersicolor Cell Wall. Although surface ultrastructure and mechanical properties have been successfully resolved22,35,36 using AFM, the application of AFM to fungal cell walls as a function of metal ion sorption remains unexplored. The nanomechanical properties of the cell wall of A. Versicolor were determined employing force spectroscopy.19–22 Fungal mycelia were immobilized on the glass coverslip and used for the determination of local cell wall elasticity or stiffness (35) Kaminskyj, S. G. M.; Dahms, T. E. S. Micron 2008, doi: 10.1016/ j.micron.2007.10.023. (36) Ma, H.; Snook, L. A.; Tian, C.; Kaminskyj, S. G. M.; Dahms, T. E. S. Mycol. Res. 2006, 110, 879–886.
8646 Langmuir, Vol. 24, No. 16, 2008
Das et al.
Figure 2. Transmission electron micrographs of pristine (protonated) (A) and chromium adsorbed mycelia (B-D). Mycelia were adsorbed with different concentrations of chromium: (B) 100; (C) 500; and (D) 1000 mg/L. Scale bar 1 µm. EDXA spectrum of the pristine (E) and chromium loaded (F) mycelia.
(Young’s modulus, E)37 and relative rigidity22 by measuring force curves on the cell wall. Subsequent force spectroscopy (FS) measurements on the cell wall (Figure 6A) demonstrated that the cells were significantly more viscoelastic38 than the glass coverslip (Figure 6A, inset). The force curve associated with the mycelia shows hystereses in between loading and unloading curves, suggesting a plastic deformation. In fact, under the loading conditions used in this study, no elastic recovery was observed, similar to that associated with the hard materials, i.e., coverslip glass used. The spring constant (KF) of the A. Versicolor cells was obtained from the slope of the linear portion of the force distance curve using the formula17,39,40
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 of fungal mycelia is reported to be relatively rigid. The cell wall elasticity (Young’s modulus, E) was measured using finite element modeling (eq. 2).21,22
E ) 0.80
KF R h h
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 (Figure 2). Alternatively, hyphal diameter (2R) can also be measured from AFM topographic images (see Supporting Information, Figure S3B). Zhao et al.21 developed a model for the measurement of cell wall elasticity and evaluated the Young’s module for azide treated, dried, and rehydrated fungal hyphae. The cell wall rigidity and elasticity of the fungal hyphae following the finite element modeling are found to be 0.61 ( 0.02 N/m and 20.5 ( 2.1 MPa, respectively. 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. The Young’s modulus varied slightly between individual force-distance curves with a standard deviation value of ∼3%. The close elasticity indicates that the estimated value for the elastic modulus of A. Versicolor is reasonable. A. Versicolor cell wall has higher rigidity and elasticity compared to the reported values of bacterial and mammalian cells.17,41 Fungal cells have a thick cell wall, which accounts for their high local stiffness;37 however, the elasticity of A. Versicolor mycelia differs significantly from the reported values of A. nidulans described by Zhao et al.21 and Ma et al.22 Polysaccharides (mainly β-1,3-D-glucans, mannans, chitin, and chitosan) are the main constituents of fungal cell walls; some glycoproteins and lipids are also present in the cell wall.42,43 Composition of polysaccharides and proteins depends not only
Interaction of Chromium with A. Versicolor
Langmuir, Vol. 24, No. 16, 2008 8647
Figure 3. Atomic force topographic images (1 µm × 1 µm) of chromium-loaded A. Versicolor mycelia. Mycelia were adsorbed with different concentrations of chromium: (A) 50; (B) 100; (C) 200; (D) 500; (E) 800; and (F) 1000 mg/L.
Figure 4. Phase images (1 µm × 1 µm) of chromium-loaded A. Versicolor mycelia. Mycelia were adsorbed with different concentrations of chromium: (A) 50; (B) 100; (C) 200; (D) 500; (E) 800; and (F) 1000 mg/L.
on the fungal species but also on the growth conditions.42,43 It is suggested that the polysaccharide composition of A. Versicolor differs considerably from that of A. nidulans; hence a considerable difference in the cell wall elasticity is observed. On binding with chromium, the cell wall rigidity and elasticity of the mycelia increased significantly (p < 0.1) to 0.88 ( 0.05 N/m and 61.2 ( 3.5 MPa, respectively, from 0.61 ( 0.02 N/m and 20.5 ( 2.1 MPa of the corresponding values of pristine mycelia, due to increasing hardness of the cell surface. With increasing chromium
concentration on the mycelia, hardness of the cell surface increases, resulting in the cell wall rigidity and elasticity increment (Figure 6B). AFM techniques thus demonstrate that the binding of chromium with A. Versicolor changes the ultrastructural surface morphology and nanomechanical properties of the cell wall. XPS and FTIR spectra of the A. Versicolor mycelia were studied to understand the surface binding mechanism. FTIR spectra of the pristine mycelia show strong bands at 3309.6, 1656.2, 1648.5, 1635.6, and 1552.1 cm-1 due to the presence
8648 Langmuir, Vol. 24, No. 16, 2008
Figure 5. AFM cross section profile of Figure 3 showing the (A) uniform distribution of chromium throughout the surface at a concentration of 100 mg/L, (B) multilayer growth formation on the adsorbed species (1000 mg/L), and (C) surface roughness values of the mycelia as a function of chromium concentrations.
of amine, carboxyl, and hydroxyl groups26,44–48 of different polysaccharides and protein molecules on the cell surface. Chromium-loaded mycelia demonstrate conspicuous changes in the spectral position, particularly in the ranges of 3300-3400 cm-1 and 1600-1100 cm-1. The peaks present at 3309.6, 1656.2, 1648.5, 1635.6, and 1552.1 cm-1 in the pristine mycelia (given in the Supporting Information Figure S4A), appeared at 3400.3, 1651.2, 1643 and 1548 cm-1, respectively in the chromiumloaded mycelia (Supporting Information Figure S4B). The amide III band located around 1330 cm-1 in the pristine mycelia disappeared on chromium sorption. The altered vibrational spectra thus indicate the interaction of the amine groups of the mycelia with chromate ions. The disappearance of the band (1520, 1508.5, and 1410 cm-1) and accompanying shifting from 1456.6 to 1450.1 cm-1 are also observed in the FTIR profile, indicating the involvement of carboxyl groups in the sorption process. In addition, a new peak also appeared at 528 cm-1, suggesting the (37) Hoh, J. H.; Schoenenberger, C. J. Cell. Sci. 1994, 107, 1105–1114. (38) Touhami, A.; Nysten, B.; Dufrene, Y. F. Langmuir 2003, 19, 4539–4543. (39) Suo, Z.; Yang, X.; Avci, R.; Kellerman, L.; Pascual, D. W.; Fries, M.; Steele, A. Langmuir 2007, 23, 1365–1374. (40) Velegol, S. B.; Logan, B. E. Langmuir 2002, 18, 5256–5262. (41) Gaboriaud, F.; Bailet, S.; Dague, E.; Jorand, F. J. Bacteriol. 2005, 187, 3864–3868. (42) Pessoni, R. A. B.; Freshour, G.; Figueiredo-Ribeiro, R. d. C. L.; Hahn, M. G.; Braga, M. R. Mycologia 2005, 97, 304–311. (43) Tischer, C. A.; Gorin, P. A. J.; de Souza, M. B.; Barreto-Bergter, E. Carbohydr. Polym. 2002, 49, 225–230. (44) Jin, L.; Bai, R Langmuir 2002, 18, 9765–9770. (45) Heber, J. R.; Stevenson, R.; Boldman, O. Science 1952, 116, 111–116. (46) Guibal, E.; Roulph, C.; Cloirec, P. EnViron. Sci. Technol. 1995, 29, 2496– 2503. (47) Schmitt, J.; Flemming, H. C. Int. Biodeterior. Biodegrad. Sci. 1998, 41, 1–11. (48) Naja, G.; Mustin, C.; Berthelin, J.; Volesky, B. J. Colloid Interface Sci. 2005, 292, 537–543. (49) Xia, L.; McCreery, R. L. J. Electrochem. Soc. 1998, 145, 3083–3089.
Das et al.
Figure 6. (A) Force distance curve of A. Versicolor pristine (protonated) mycelia and a glass coverslip (inset). (B) Cell wall rigidity and elasticity as a function of chromium concentrations on the mycelia. Data represent an average of four independent experiments ( SD shown by error bar.
formation of Cr(OH)3 on the mycelia.49 With increasing chromium concentration on the mycelia (mycelia adsorbed with 200, 600, and 1000 mg/L chromium solution), two new bands corresponding to CrO4-2 also appeared at 905-950 cm-1 and 775-790 cm-1 (Supporting Information Figure S4C-D).50 XPS was used to characterize the oxidation state of chromium on the adsorbed mycelia. The binding energy for the N1s level of the pristine mycelia exhibits two peaks at ∼399.0 and 400.7 eV (Figure 7A), characteristics of the N atoms of amine groups.25,44,51 On sorption of chromium, the shifting of core level binding energy for the N1s is observed (Figure 7B). A new peak appeared at 399.6 eV, indicating participation of N atom of the amine groups in the sorption process.44,52,53 We could not record changes corresponding to C1s and O1s in the spectral profile since the samples were mounted on double-sided UHVgraded carbon tapes containing C and O along with the presence of O atoms of the chromate ion. Two asymmetric peaks due to the Cr2p core level appeared in the chromium-loaded mycelia at 576.3 and 579.7 eV, corresponding to Cr(III) and Cr(VI) species, respectively (Figure 7C).44,52,54 The asymmetric peaks corresponding to the Cr2p core level at 576.3 eV indicate the formation of chromic hydroxide.55 Thus it appears from both IR (50) Ko, Y. G.; Choi, U. S.; Kim, T. K.; Ahn, D. J.; Chun, Y. J. Macromol. Rapid Commun. 2002, 23, 535–539. (51) Neal, A. L.; Lowe, K.; Daulton, T. L.; Jones-Meehan, J.; Little, B. J. Appl. Surf. Sci. 2002, 202, 150–159. (52) Dambies, L.; Guimon, C.; Yiacoumi, S.; Guibal, E. Colloid Surf. A 2001, 177, 203–214. (53) Zhou, D.; Zhang, L.; Guo, S. Water Res. 2005, 39, 3755–3762. (54) Park, D.; Yun, Y.-S.; Jo, J. H.; Park, J. M. Water Res. 2005, 39, 533–540. (55) James, M. G.; Beattie, J. K.; Kennedy, B. J. Waste Manage. Res. 2000, 18, 380–385.
Interaction of Chromium with A. Versicolor
Langmuir, Vol. 24, No. 16, 2008 8649
Figure 7. XPS spectra of (N1s) pristine mycelia (A), chromium-loaded mycelia (B). Core level high resolution Cr2p spectra of the chromium-loaded mycelia (C,D). Mycelia were adsorbed with different concentrations of chromium: (C) 200; (D) 1000 mg/L. All peak positions were normalized with respect to the major peak in the C 1s spectra at 285.0 eV.
and XPS studies that Cr(VI) initially binds to the amine groups and then reduces to Cr(III) compounds, and consequently binds to the available carboxyl groups of the mycelia. However, it is interesting to note that, with increasing thickness of the layer [high chromium loaded mycelia (1000 mg/L)], the intensity of the Cr2p peak at higher energy (579.7 eV) increases compared to that at lower energy (576.3 eV) (Figure 7D). It is also observed that the N1s peak resulting from the pristine as well as low chromium-loaded mycelia disappeared in the case of high chromium-adsorbed mycelia. This strongly indicates that the presence of Cr(OH)3 on the mycelia serves as a template for further binding of chromate ions, leading to the formation of a multilayer. Since the spectral position of Cr2p level at 576.3 eV did not show any shifting in high chromium-loaded mycelia, it may be concluded that the binding of chromate ions with chromic hydroxide mainly occurs through electrostatic attraction.44 The insoluble Cr(OH)3 contains a number of surface hydroxyl groups available for binding with the negatively charged chromate ions, resulting in the formation of Cr(III)-O-Cr(VI) as presented in eq 4.
Cr(III)-OH (matrix) + CrO4-2(aq) + H+(aq) T Cr(III)O-Cr(VI) (matrix) + H2O (4) Cr(III)-OH (matrix) in eq 4 represents insoluble Cr(III) hydroxide matrix produced on the mycelial surface, CrO42- (aq) is solution phase hexavalent chromium, and Cr(III)-O-Cr(VI) (matrix) is the product formed on the A. Versicolor surfaces during multilayer formation. To prove this hypothesis, Cr(III) hydroxide matrix [mycelia after sorption with 100 mg/L Cr(VI) solution were collected, washed with deionized water, and dried by lyophilization] was added to Cr(VI) solution with different pH values. It was noted that sorption of Cr(VI) on Cr(OH)3 matrix favored at
Figure 8. Mechanism of multilayer formation of chromium on A. Versicolor: (1) initial binding of chromate ions on the cell wall followed by reduction to chromic hydroxide, and (2) chromic hydroxide electrostatically binds chromate ions based on eq 4, resulting in multilayer formation. Cr(VI) shown as CrO42-, similar reactions are also possible by using HCrO4- or Cr2O72-; only one bond of chromic hydroxide is shown to improve the clarity.
low pH value (data not shown). Thus, the positively charged Cr(OH)3 matrix electrostatically attracts negatively charged chromate ions. A probable mechanism of the phenomenon is represented schematically (Figure 8) on the basis of both microscopic and spectroscopic investigation as well as the findings of a study52 on the interaction of Cr(VI) ions with chromate conversion coating (CCC) film.
Conclusions The present study reveals new insight into the interaction of chromium with the cell surface of resistant strain A. Versicolor. The adsorption process occurs through chemisorption, yielding multilayer coverage of chromium species on the adsorbent. The binding sites on the cell wall components are mainly responsible for the adsorption of chromium without significant diffusion and transportation into the cytoplasm. AFM study manifests the alterations in the cell wall ultrastructural morphology along with the mechanical properties such as cell wall rigidity and elasticity
8650 Langmuir, Vol. 24, No. 16, 2008
on chromium binding. XPS suggests that the cell wall components reduce the hexavalent chromium to its trivalent state, which further binds chromate ions electrostatically to form a multilayered structure on the cell wall. Acknowledgment. We thank Dr. K. Acharya of the Department of Botany, Calcutta University, Kolkata, for his valuable suggestion during identification of fungal strain. Thanks are also
Das et al.
due to Mr. R. N. Banik of our Institute and Mr. S. Dey of the Indian Institute of Chemical Biology, Kolkata, for their cooperation during AFM and TEM analysis. Supporting Information Available: Additional table, figures, and experimental details. This material is available free of charge via the Internet at http: //pubs.acs.org. LA800958U
