Zirconia Enrichment in Zircon Sand by Selective Fungus-Mediated

Langmuir 2007, 23, 4993-4998

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Zirconia Enrichment in Zircon Sand by Selective Fungus-Mediated Bioleaching of Silica Vipul Bansal,† Asad Syed,§ Suresh K. Bhargava,‡ Absar Ahmad,§ and Murali Sastry*,†,| Nanoscience Group, Materials Chemistry and Biochemical Sciences DiVision, National Chemical Laboratory, Pune - 411 008, India and School of Applied Sciences, Royal Melbourne Institute of Technology UniVersity, GPO BOX 2476V, Melbourne - 3001, Australia ReceiVed August 29, 2006. In Final Form: February 3, 2007 One of the important routes for the production of zirconia is by chemical treatment and removal of silica from zircon sand (ZrSixOy). We present here a completely green chemistry approach toward enrichment of zirconia in zircon sand; this is based on the reaction of the fungus Fusarium oxysporum with zircon sand by a process of selective extracellular bioleaching of silica nanoparticles. Since this reaction does not result in zirconia being simultaneously leached out from the sand, there is a consequent enrichment of the zirconia component in zircon sand. We believe that fungal enzymes specifically hydrolyze the silicates present in the sand to form silicic acid, which on condensation by certain other fungal enzymes results in room-temperature synthesis of silica nanoparticles. This fungus-mediated twofold approach might have vast commercial implications in low-cost, ecofriendly, room-temperature syntheses of technologically important oxide nanomaterials from potentially cheap naturally available raw materials like zircon sand.

Introduction Silica is an important inorganic material1 extensively used in a wide range of commercial applications such as resins, molecular sieves, catalyst supports, and fillers in polymeric items, as well as in biomedical devices.2 Apart from demand for silica, there is also a huge demand for the development of high dielectric materials for rapid scaling of silicon-based metal oxide semiconductor field effect transistor (MOSFET) and advanced complementary metal oxide semiconductor (CMOS) devices in order to achieve reduced effective oxide thickness (EOT) while maintaining the overall gate capacitance, so that the problem of current leakage in future devices can be avoided.3 Many high dielectric materials including Ta2O5 ( ) 26), TiO2 ( ) 80), and SrTiO3 ( ) 175) have been developed; however, these materials are not thermally stable in direct contact with silicon.4-7 Conversely, the metal oxide zirconia (ZrO2) as well as the compound zirconium silicate ZrSiO4 (zircon) have been found to be stable in direct contact with Si even at very high temperatures.8,9 The pure oxide ZrO2 has high permittivity ( ) 25) and is a promising candidate for such devices; however, * To whom correspondence is to be addressed. E-mail: msastry@ tatachemicals.com. † Nanoscience Group, Materials Chemistry Division, National Chemical Laboratory. ‡ Royal Melbourne Institute of Technology University. § Biochemical Sciences Division, National Chemical Laboratory. | Current Address: Tata Chemicals Innovation Centre, Anmol Pride, Baner Road, Pune - 411045, India. (1) Hubert, D. H. W.; Jung, M.; German, A. L. AdV. Mater. 2000, 12, 1291. (2) (a) Iler, R. K. The Chemistry of Silica; John Wiley & Sons: New York, 1979. (b) Kendall, T. Ind. Miner. 2000, 49. (c) Brinker, C. J.; Scherer, G. W. Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing; Academic Press: Boston, 1990. (d) Hench, L. L. J. Am. Ceram. Soc. 1991, 74, 1487. (3) (a) Seo, K. I.; McIntyre, P. C.; Kim, H.; Saraswat, K. C. Appl. Phys. Lett. 2005, 86, 082904. (b) Stathis, J. H.; DiMaria, D. J. Tech. Dig. Int. Electron DeVices Meet. 1998, 167. (c) Muller, D. A.; Sorsch, T.; Moccio, S.; Baumann, F. H.; Evans-Lutterodt, K.; Timp, G. Nature (London) 1999, 399, 758. (4) (a) Kizilyalli, I. C.; Huang, R. Y. S.; Roy, P. K. IEEE Electron DeVice Lett. 1998, 19, 423. (b) Park, D.; King, Y. C.; Lu, Q.; King, T. J.; Hu, C.; Kalnitsky, A.; Tay, S. P.; Cheng, C. C. IEEE Electron DeVice Lett. 1998, 19, 441. (5) He, B.; Ma, T.; Campbell, S. A.; Gladfelter, W. L. Tech. Dig. Int. Electron DeVices Meet. 1998, 1038. (6) McKee, R. A.; Walker, F. J.; Chisholm, M. F. Phys. ReV. Lett. 1998, 81, 3014.

some potential concerns are that it tends to crystallize at low temperature and is an ionic conductor, and the heterointerface formed between the Si channel and ZrO2 may degrade the electron channel mobility in transistors.10 Considering the thermal stability and the electrical results reported earlier,9b ZrSixOy is a promising candidate to replace SiO2 as the gate dielectric material, since its permittivity value ( ) 12.6) lies between those of its structural components SiO2 ( ) 3.9) and ZrO2 ( ) 25).11 The chemical syntheses of these high dielectric and silicabased materials are not only relatively expensive and ecohazardous, but also often require extremes of temperature, pressure, and pH. For instance, silica and zirconia are produced commercially in the refractories at extremely high temperature using silica sand (white sand) and zircon sand that are rich in silica and zirconia, respectively.12 However, silicon impurity in zircon sand has always been a matter of concern in zirconia synthesis using zircon sand.13 Similarly, zircon (zirconium silicate) is conventionally synthesized at extremely high temperatures (1600-2700 °C) in specially designed high-energy plasma reactors (20 kW), followed by various chemical treatments, wherein there is not much control over the zirconia content of (7) (a) Alers, G. B.; Werder, D. J.; Chabal, Y.; Lu, H. C.; Gusev, E. P.; Garfunkel, E.; Gustafsson, T.; Urdahl, R. S. Appl. Phys. Lett. 1998, 73, 1517. (b) Taylor, C. J.; Gilmer, D. C.; Colombo, D. G.; Wilk, G. D.; Campbell, S. A.; Roberts, J.; Gladfelter, W. L. J. Am. Chem. Soc. 1999, 121, 5220. (c) Wilk, G. D.; Wallace, R. M.; Anthony, J. M. Appl. Phys. Lett. 2001, 89, 5243. (d) Eisenbeiser, K.; Finder, J. M.; Yu, Z.; Ramdani, J.; Curless, J. A.; Hallmark, J. A.; Droopad, R.; Ooms, W. J.; Salem, L.; Bradshaw, S.; Overgaard, C. D. Appl. Phys. Lett. 2000, 76, 1324. (8) Wang, S. Q.; Mayer, J. W. J. Appl. Phys. 1998, 64, 4711. (9) (a) Wilk, G. D.; Wallace, R. M.; Anthony, J. M. J. Appl. Phys. 2000, 87, 484. (b) Wilk, G. D.; Wallace, R. M. Appl. Phys. Lett. 2000, 76, 112. (c) Qi, W. J.; Nieh, R.; Dhamarajan, E.; Lee, B. H.; Jeon, Y.; Kang, L.; Onishi, K.; Lee, J. Appl. Phys. Lett. 2000, 77, 1704. (d) Hubbard, K. J.; Schlom, D. G. J. Mater. Res. 1996, 11, 2757. (10) Kumar, A.; Rajdev, D.; Douglass, D. L. J. Am. Chem. Soc. 1972, 55, 439. (11) Blumenthal, W. B. The Chemical BehaVior of Zirconium; Van Nostrand: Princeton, NJ, 1958; p 201. (12) (a) Singh, B. P.; Bhattacharjee, S.; Besra, L. Ceram. Int. 2002, 28, 413. (b) Syamaprasad, U.; Bhattacharjee, J.; Galgali, R. K.; Mohapatra, B. K.; Mohanty, B. C. J. Mater. Sci. 1992, 27, 1762. (13) Arendt, R. H. (General Electrics Company, Schenectedy) U.S. Patent No. 4,361,542, 1982.

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zircon being synthesized due to thermokinetics limitations.12,14 Consequently, due to these problems in the development of high dielectric zirconia and zircon, there is a need to develop a protocol to selectively remove the low dielectric silica from zircon sand and hence enrich the high dielectric zirconia content in zircon sand. This, in turn, would help in the development of high dielectric zircon wherein dielectric values of the material can be controllably shifted toward high dielectric zirconia. In contrast to the extreme conditions employed in most of the synthesis protocols for these materials, biosilicification by living organisms such as bacteria, diatoms, sponges, and plants proceeds under mild physiological conditions, producing an amazing diversity of complex and hierarchical biogenic silica nanostructural frameworks.15,16 Some polyamines, carbohydrates, proteins, and glycoproteins from diatoms, sponges, and plants have been reported to be capable of polymerizing silicic acid at neutral to acidic pHs.17,18 Fungal activity has also been reported to release metallic and silicate ions from minerals and rocks.19 Bioleaching has been explored previously as a significant tool for environmental friendly, low-cost commercial synthesis of various metals like copper, iron, and gold from their precursors under ambient conditions.20 Despite the vast scientific literature on crystalline and amorphous silica synthesis by biological and biomimetic methods,15-18 there have been no attempts at harnessing the enormous potential of these microorganisms to selectively leach out small amounts of silica present in cheap, naturally available raw materials like zircon sand under ambient conditions. In this article, we address this issue and describe our efforts to set up a biological model system for selective bioleaching of silica impurity present in naturally available zircon sand in the form of crystalline silica nanoparticles and simultaneous enrichment of high dielectric zirconia in zircon sand, thereby converting low-cost raw materials to value-added raw materials for other processes. More specifically, we show that Fusarium oxysporum, a plant pathogenic fungus, when exposed to zircon sand is capable of selectively leaching out silicon impurity of zircon sand in the form of silica nanoparticles of reasonable monodispersity under ambient conditions. The silica bioleaching is fairly rapid and occurs within 1 day of reaction of fungal biomass with zircon sand. It is interesting to note that, despite the in vitro studies of (14) (a) Chang, J. P.; Lin, Y. S. Appl. Phys. Lett. 2001, 79, 3666. (b) Bruce, R. G., Jr. Spectroscopic Investigation of Local Bonding in Zirconium Silicate High-k Dielectric Alloys for Advanced Microelectronic Applications. Ph.D. Thesis, North Carolina State University, 2002. (15) (a) Mann, S. Nature (London) 1993, 365, 499. (b) Oliver, S.; Kupermann, A.; Coombs, N.; Lough, A.; Ozin, G. A. Nature (London) 1995, 378, 47. (c) Mann, S.; Ozin, G. A. Nature (London) 1996, 382, 313. (d) Mann, S., Webb, J., Williams, R. J. P., Eds. Biomineralization: Chemical and Biochemical PerspectiVes; VCH: Weinheim, 1998. (e) Lowenstam, H. Science 1981, 211, 1126. (16) (a) Simpson, T. L., Volcani, B. E., Eds.; Silicon and Siliceous Structures in Biological Systems; Springer-Verlag: New York, 1981. (b) Levi, C.; Barton, J. L.; Guillemet, C.; Le Bras, E.; Lehuede, P. J. Mater. Sci. Lett. 1989, 8, 337. (c) Westall, F.; Boni, L.; Guerzoni, E. Palaeontology 1995, 38, 495. (17) (a) Mitzutani, A. J.; Nagase, H.; Fujiwara, N.; Ogoshi, H. Chem. Soc. Jpn. 1998, 71, 2017. (b) Kroger, N.; Deutzmann, R.; Bergsdort, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14133. (c) Pohnert, G. Angew. Chem., Int. Ed. 2002, 41, 3167. (d) Hecky, R. E.; Mopper, K.; Kilham, P.; Degens, E. T. Mar. Biol. 1973, 19, 323. (e) Swift, D. M.; Wheeler, A. P. J. Phycol. 1992, 28, 202. (f) Shimizu, K.; Cha, J.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 6234. (g) Sumper, M.; Kroger, N. J. Mater. Chem. 2004, 14, 2059. (h) Cha, J. N.; Shimizu, K.; Zhou, Y.; Christiansen, S. C.; Chmelka, B. F.; Stucky, G. D.; Morse, D. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 361. (18) (a) Perry, C. C.; Keeling-Tucker, T. Colloid Polym. Sci. 2003, 281, 652. (b) Perry, C. C.; Keeling-Tucker, T. J. Biol. Inorg. Chem. 2000, 5, 537. (c) Perry, C. C.; Keeling-Tucker, T. Chem. Commun. 1998, 2587. (d) Harrison, C. C. Phytochemistry 1996, 41, 3642. (19) Moira, E.; Henderson, K.; Duff, R. B. J. Soil Sci. 1963, 14, 237. (20) Rawlings, D. E. J. Ind. Microbiol. Biotechnol. 1998, 20, 268.

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various proteins or synthetic macromolecules in this context,21 only the F. oxysporum based system was able to selectively leach out aforesaid silica structures for the conditions studied to date. In previous studies, we demonstrated that F. oxysporum is an excellent microorganism for the biosynthesis of metal22 and metal oxide23 nanoparticles from their precursor salts; this work also develops significantly upon our earlier study on bioleaching of silica from sand24 and rice husk24 and shows the high level of specificity of fungal enzymes toward the silica component in zircon sand. Experimental Section The plant pathogenic fungus Fusarium oxysporum was cultured as described elsewhere.22 After incubation, the fungal mycelia were harvested and washed thoroughly under sterile conditions. For the extracellular bioleaching of silica from zircon sand (obtained from Eastern Ghats of Tamil Nadu, India) by the fungus F. oxysporum, the harvested fungal biomass (20 g wet weight) was resuspended in 100 mL of sterile distilled water containing 10 g of zircon sand in 500 mL Erlenmeyer flasks and kept on a shaker (200 rpm) at 27 °C. The reaction between the fungal biomass and sand was carried out for a period of 24 h. The bioleached product was collected by separating the fungal mycelia from the aqueous component by filtration. The nanoparticle solution was evaporated under low pressure to powder, which was then characterized before and after calcination at 400 °C for 2 h. Samples for transmission electron microscopy (TEM) were prepared by drop-coating films of the bioleached nanoparticle powders dispersed in water onto carbon-coated copper grids. Selected area electron diffraction (SAED) analysis was also carried out for these samples. TEM and SAED patterns were obtained on a JEOL 1200 EX instrument operated at an accelerating voltage of 120 kV. The extracellular products formed in the reaction were monitored by Fourier transform infrared (FTIR) spectroscopy. The samples for FTIR analysis were taken in KBr pellets after thorough drying and analyzed on a Perkin-Elmer Spectrum One instrument at a resolution of 2 cm.-1 X-ray diffraction (XRD) measurements of drop-coated films of the extracellularly synthesized biogenic silica before and after calcination at 400 °C for 2 h were carried out on a Phillips PW 1830 instrument operated at a voltage of 40 kV and a current of 30 mA with Cu KR radiation. XRD measurement of the zircon sand used as a precursor in this reaction was also performed. X-ray photoemission spectroscopy (XPS) measurements of films of bioleached silica nanoparticles cast onto a Cu strip were carried out on a VG MicroTech ESCA 3000 instrument at a pressure better than 1 × 10-9 Torr. Moreover, in order to comprehend the enrichment of the zirconium component in zircon sand as compared to its silicon counterpart, XPS analysis of zircon sand before and after its exposure to the fungus was also performed. Samples for XPS measurements from zircon sand were prepared by sticking the finely ground zircon sand onto Cu strips. The general scan and Si 2p, Zr 3d, and O 1s core-level spectra for all the samples were recorded with unmonochromatized Mg KR radiation (photon energy ) 1253.6 eV) at a pass energy of 50 eV and electron takeoff angle (angle between electron emission direction and surface plane) of 60°. The overall resolution was ∼1 eV for the XPS measurements. The core-level (21) (a) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129. (b) Kroger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Science 2002, 298, 584. (c) Cha, J. N.; Stucky, G. D.; Morse, D. E.; Deming, T. J. Nature (London) 2002, 403, 289. (d) Patwardhan, S. V.; Clarson, S. J. Silicon Chem. 2002, 413, 291. (22) Mukherjee, P.; Senapati, S.; Mandal, D.; Ahmad, A.; Khan, M. I.; Kumar, R.; Sastry, M. ChemBioChem. 2002, 3, 461. (23) (a) Bansal, V.; Rautaray, D.; Ahmad, A.; Sastry, M. J. Mater. Chem. 2004, 14, 3303. (b) Bansal, V.; Rautaray, D.; Bharde, A.; Ahire, K.; Sanyal, A.; Ahmad, A.; Sastry, M. J. Mater. Chem. 2005, 15, 2583. (c) Bharde, A.; Rautaray, D.; Bansal, V.; Ahmad, A.; Sarkar, I.; Yusuf, S. M.; Sanyal, M.; Sastry, M. Small 2006, 2, 135. (d) Bansal, V.; Poddar, P.; Ahmad, A.; Sastry, M. J. Am. Chem. Soc. 2006, 128, 11958. (24) (a) Bansal, V.; Sanyal, A.; Rautaray, D.; Ahmad, A.; Sastry, M. AdV. Mater. 2005, 17, 889. (b) Bansal V.; Ahmad, A.; Sastry, M. J. Am. Chem. Soc. 2006, 128, 14059.

Zirconia Enrichment in Zircon Sand

Figure 1. TEM micrographs at different magnifications of silica nanoparticles synthesized by the exposure of zircon sand to the fungus Fusarium oxysporum before (A,B) and after (C,D) calcination at 400 °C for 2 h. The insets in A and D are the SAED patterns recorded from representative silica nanoparticles. spectra were background-corrected using the Shirley algorithm,25 and the chemically distinct species were resolved using a nonlinear least-squares fitting procedure. The core-level binding energies (BEs) were aligned with the adventitious carbon binding energy of 285 eV. A control experiment was performed wherein the zircon sand was exposed to distilled water maintained at pH 3.5 for 24 h without adding the fungus F. oxysporum. The solution was further analyzed by FTIR and TEM. In order to understand the bioleaching process, 10 g of zircon sand was exposed to 20 g of wet fungal biomass for 24 h. After 24 h, the zircon sand was separated from biomass, and 9 g of preexposed zircon sand was re-exposed to 18 g of fresh fungal biomass for the next 24 h. After this reaction, the zircon sand was again separated from the biomass, and 8 g of this pre-exposed zircon sand was further exposed to 16 g of fungal biomass for next 24 h. The zircon sand obtained after exposure to fresh fungal biomass after 24, 48, and 72 h of reaction was analyzed using an energy-dispersive X-ray (EDX) instrument fitted to a Leica Stereoscan-440 scanning electron microscope (SEM). The changes in the surface morphology of zircon sand before and after the reaction were monitored using SEM.

Results and Discussion The bioleached product obtained from the fungus-zircon sand reaction mix was analyzed by TEM. Figure 1A,B shows the representative TEM images recorded from the film of the extracellular product obtained by the reaction of Fusarium oxysporum with the zircon sand for 24 h (pH of the reaction medium ≈ 3.5). The particles embedded in the biomolecular matrix are fairly regular in shape and depict an overall quasispherical morphology. A statistical analysis of 200 random particles indicated that the particle size range from 2 to 10 nm, with an average particle size of 5.5 ( 2 nm. SAED analysis of the particle assemblies (inset, Figure 1A) clearly indicates that they are crystalline in nature. The diffraction spots in the SAED pattern could be indexed on the basis of the cristobalite polymorph of silica structure.26 FTIR analysis of particles from the funguszircon sand reaction medium taken in KBr pellets showed the presence of bands at ca. 1100 and 611 cm-1 (Figure 2A, curve 1). The prominent 1100 cm-1 band can be assigned to the SiO-Si27 antisymmetric stretching mode present in the leached(25) Shirley, D. A. Phys. ReV. B. 1972, 5, 4709. (26) The XRD and SAED patterns were indexed with reference to the crystal structures from the PCPDF charts: silica (PCPDF card nos. 03-0272, 32-0993, 45-0112, and 45-0131). (27) Innocenzi, P.; Falcaro, P.; Grosso, D.; Babonneau, F. J. Phys. Chem B 2003, 107, 4711.

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Figure 2. (A) FTIR spectra recorded from the filtrate containing silica particles synthesized by exposing zircon sand to the fungus Fusarium oxysporum for 24 h (curve 1) and from the filtrate obtained by exposing zircon sand to water of pH 3.5 for 24 h (curve 2). (B) XRD patterns recorded from silica particles synthesized by the exposure of zircon sand to the fungus F. oxysporum before (curve 1) and after (curve 2) calcination of particles at 400 °C for 2 h.

out product. Another distinct vibrational mode detected around 600 cm-1 is generally observed in sol-gel silica materials and can be assigned to some cyclic structures present in the silica network. Yoshino et al.28 have assigned this IR vibration to cyclic tetrameric siloxane species by referring to different types of cyclic siloxanes and silicate minerals, and this attribution has also been supported by molecular orbital calculations.29 Two absorption bands at ca. 1650 and 1540 cm-1 (amide I and II bands, respectively; Figure 2A, curve 1) attest to the presence of proteins in the quasi-spherical silica particles that have been released by the fungus during reaction with zircon sand. Additional evidence for the crystalline nature of the bioleached silica nanoparticles is provided by XRD analysis of the bioleached product formed by the fungus-zircon sand reaction medium (Figure 2B, curve 1). The XRD spectrum of as-formed silica nanoparticles shows well-defined Bragg reflection characteristics of cristobalite polymorph of crystalline silica.26 The presence of FTIR signatures corresponding to silica-entrapped proteins (Figure 2A, curve 1) and well-defined Bragg reflections in the same sample (Figure 2B, curve 2) indicate that entrapped proteins in the silica particles do not significantly interfere with their crystallinity. Moreover, XRD spectra for zircon sand used as a precursor in this study were also recorded as a control that exactly matches with zirconium silicate (ZrSiO4) (Supporting Information S1). In order to preclude the possibility of silica leaching out due to the acidic nature of the reaction medium, a control experiment was performed wherein the zircon sand was kept in distilled water maintained at an acidic pH of 3.5 for 24 h, and then the filtrate was characterized by FTIR spectroscopy and TEM. We observed that characteristic Si-O-Si vibrational modes27 of silica as well as signatures from silicic acid (Si-OH vibrational modes)30 were clearly missing in the control zircon sand sample not being exposed to the fungus (Figure 2A, curve 2). However, weak FTIR signatures were observed in the control sample at (28) Yoshino, H.; Kamiya, K.; Nasu, H. J. Non-Cryst. Solids 1990, 126, 68. (29) Hayakawa, S.; Hench, L. L. J. Non-Cryst. Solids 2000, 262, 264. (30) Silverstein, R. M. Spectrometric identification of organic compounds, 2nd ed.; John Wiley & Sons: New York, 1967; p 102.

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580 and 1050 cm-1 that might be due to the presence of a small amount of organic impurities on the surface of zircon (Figure 2A, curve 2). The fact that these signatures reside in the FTIR fingerprint region makes it extremely difficult to assign these peaks unequivocally. We believe that these peaks are not due to shifted Si-O-Si peaks, since zircon is considered to be an extremely resistant material and only plasma treatment of around 2000 °C or more is able to dissociate zircon into its components.12b Moreover, the absence of any particles in the TEM micrograph of drop-cast films from the control experiment further supports our belief. In addition, the amide I and II signatures arising from the extracellular fungal proteins in the zircon sand exposed to the fungus (Figure 2A, curve 1) were also missing from the fungus-deficient control sample (Figure 2A, curve 2). The control experiment and the TEM, SAED, FTIR, and XRD results of the fungus-zircon sand reaction medium clearly suggest that F. oxysporum selectively leaches out the silicon component of zircon sand in the form of extracellular crystalline silica nanoparticles and does not cause leaching of the zirconium counterpart of zircon sand. The FTIR results show the presence of proteins in the silica nanoparticle powders (Figure 2A, curve 1). In order to remove the proteins that are intercalated/incarcerated in the silica structures, calcination of the silica powder was performed at 400 °C for 2 h. The calcined silica nanoparticle powder after redispersion in water was analyzed by TEM (Figure 1C,D). It is observed that the removal of incarcerated biomolecules by calcination leads to sintering of silica nanoparticles and consequently results in the formation of larger silica nanoparticles ranging in size 50-100 nm, with an average particle size of 80 ( 12 nm (Figure 1C,D). The SAED pattern recorded from these silica nanoparticles (inset, Figure 1D) clearly shows the crystalline nature of silica particles formed and could be indexed on the basis of cristobalite polymorph of silica. In addition, the crystallinity of these silica nanostructures was further confirmed by XRD (Figure 2B, curve 2). The XRD analysis of the calcined powder further shows well-defined Bragg reflections characteristic of a cristobalite polymorph of silica nanoparticles.26 It appears that calcination, leading to removal of proteins from the silica matrix, results in increased crystallinity of silica particles (Figure 2B, curve 2) as compared to as-synthesized silica nanoparticles (Figure 2B, curve 1). However, the effect of calcination temperature on improved crystallinity of silica crystals cannot be neglected. In order to establish the effect of embedded proteins on the crystallinity of silica particles, proteins from bioleached silica particles were removed using a phenol/ chloroform mixture24a instead of calcination. An increase in crystallinity of silica particles after removal of proteins by chemical means was also observed (data not shown for brevity), which correlates well with our previous studies on bioleaching of silica from white sand, wherein removal of proteins by chemical means was observed to increase the crystallinity of particles.24a A chemical analysis of the nanoparticles bioleached from zircon sand was performed by XPS, which is known to be a highly surface-sensitive technique (Figure 3). The Si 2p, Zr 3d, and O 1s core-level spectra were collected and their binding energies (BEs) were aligned with the adventitious C 1s BE of 285 eV. Figure 3A shows the Si 2p XPS spectrum that could be fitted into a single spin-orbit pair (spin-orbit splitting ∼0.6 eV)3a with a 2p3/2 BE of 103.5 eV (Figure 3A), which is in excellent agreement with values reported for SiO2.31 In addition to the Si 2p spectrum, the sample was also scanned for a Zr 3d signal; however, we could not detect any Zr 3d signal arising from the (31) Wagner, C. D. J. Vac. Sci. Technol. 1978, 15, 518.

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Figure 3. XPS data showing the Si 2p (A), Zr 3d (B), and O 1s (C) core-level spectra recorded from biologically synthesized silica nanoparticle film cast onto a Cu substrate. The chemically resolved components are shown as solid lines in the figure and are discussed in the text.

Figure 4. XPS data showing the Si 2p (A,B) and Zr 3d (C,D) core-level spectra recorded from zircon sand before (A,C) and after its exposure to the fungus Fusarium oxysporum (B,D). The chemically resolved components are shown as solid lines in the figure and are discussed in the text.

sample (Figure 3B). Besides, an O 1s signal was also recorded in the sample (Figure 3C) that shows a single component with BE of 532.2 eV. Oxygen in the Si-O-Si environment is known to show an O 1s BE component at 532.5 eV.3a Similarly, oxygen in Si(OH)4 shows an O 1s BE component at 531.9 eV.15b We believe that both these components contribute to the XPS spectra shown in Figure 3C and hence illustrate an O 1s BE component of ca. 532.2 eV. Notably, we do not observe any lower BE component of ca. 530.1 eV arising from ZrO2.3a The absence of a Zr 3d signal and an O 1s signal corresponding to Zr-O further supports the selective bioleaching of silica nanoparticles from zircon sand. A chemical analysis of zircon sand before and after its exposure to the fungus F. oxysporum was also performed by XPS. The Si 2p and Zr 3d core-level spectra from finely ground zircon sand before (Figure 4A,C) and after (Figure 4B,D) its reaction with the fungus were recorded, and their binding energies (BEs) were aligned with respect to the adventitious C 1s BE of 285 eV. Figure 4A shows the Si 2p spectrum from zircon sand before its reaction with the fungus that could be fitted into a single spinorbit pair (spin-orbit splitting ∼0.6 eV)3a with 2p3/2 BE of 102.2 eV (Figure 4A), which is an excellent match to Si 2p3/2 BE in the Zr-O-Si phase as reported previously.3a Si 2p XPS analysis of zircon sand after its reaction with the fungus (Figure 4B) leads to the attribution of two distinct chemical species of Si atoms

Zirconia Enrichment in Zircon Sand

by resolving the Si 2p spectra into two spin-orbit pairs with 2p3/2 BEs of 98.4 and 102.1 eV, respectively (Figure 4B, curves 1 and 2, respectively). Among these two Si 2p3/2 BE components, the 102.1 eV3a is predominant and can be assigned to the Si 2p3/2 BE of Si present in the silica-zirconia network (Figure 4B, curve 1), whereas the extremely feeble BE component at 98.4 eV32 (Figure 4B, curve 2) can be assigned to non-network-bonded Si atoms that apparently precipitate in the zirconium silicate network. Zircon sand was also analyzed for Zr 3d BEs before and after its reaction with the fungus. Figure 4C shows the Zr 3d spectrum from zircon sand before its reaction with the fungus, which could be fitted into a single spin-orbit pair (spin-orbit splitting ∼2.4 eV)3a with 3d5/2 BE of 183.7 eV (Figure 4C). This 183.7 eV BE component can be assigned to Zr 3d5/2 BE of Zr present in the silica-zirconia network.15b The Zr 3d spectrum from zircon sand after its reaction with the fungus was also analyzed and could be fitted into a single spin-orbit pair with 3d5/2 BE of 183.1 eV (Figure 4D). We observed a 0.6 eV reduction in the Zr 3d state BE of Zr in zircon sand after silica leaching from sand as a consequence of its reaction with fungal biomass. Our results match well with the previous reports where Zr 3d state BEs in ZrSiO4 have been shown to be more than 0.5 eV larger than in ZrO2.33 In addition, it has also been shown previously that successive reduction in silica content in SiO2-ZrO2 alloys resulted in consecutive reduction of Si 2p and Zr 3d binding energies.15b The reduction of Si 2p and Zr 3d BEs by 0.1 and 0.6 eV, respectively, after reaction of zircon sand with the fungus thus can be explained on the basis of the reduction in silica content in zircon sand. The difference in the drop of Si 2p and Zr 3d BEs after reaction (0.1 and 0.6 eV, respectively) is consistent with the principle of electronegativity equalization, i.e., the charge transfer out of Zr is larger in ZrSiO4 than in ZrO2, because electronegativites of Si and O are each larger than that of Zr.34 The XPS analysis of zircon sand before and after its reaction with fungal biomass clearly suggests enrichment of the zirconia component in zircon sand, due to leaching out of silica from zircon sand. In order to quantitatively comprehend enrichment of the zirconium component in zircon sand, the Si/Zr ratios in zircon sand before (Figure 4A,C) and after (Figure 4B,D) its reaction with fungal biomass were calculated, taking the integrated values of the respective fitted curves into account. The Si/Zr ratios in zircon sand before and after its exposure to the fungus for 24 h were found to be ca. 0.327 and 0.153, respectively. XPS results therefore suggest that selective bioleaching of silica from zircon sand results in ca. 53% reduction in the silica content of zircon sand within 24 h of reaction. It is noteworthy that exposing the zircon sand to the fungus for longer than 24 h does not result in any further leaching of silica. In order to understand the effect of fungal biomass on the silica bioleaching process, zircon sand, initially exposed to fungal biomass for 24 h, was re-exposed to a new batch of fungal biomass until 48 h, and then further exposed to fresh fungal biomass until 72 h. The zircon sand obtained at the end of each reaction was analyzed using EDX, which is a semiquantitative technique. The Si/Zr atomic percentage ratios in zircon sand after 0, 24, 48, and 72 h of reaction were found to be ca. 0.353, 0.181, 0.100, and 0.059, respectively. EDX results indicate that the exposure of zircon sand to the fungus results in ca. 49%, 45%, and 42% (32) Carriere, B.; Brion, D.; Escard, J.; Deville, J. P. J. Electron. Spectrosc. Relat. Phenom. 1977, 10, 85. (33) Guittet, M. J.; Crocombette, J. P.; Gautier-Soyer, M. Phys. ReV. B 2001, 63, 125117. (34) Sanderson, R. T. Chemical Bonds and Bond Energy; Academic Press: New York, 1971; Chapter 2.

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Figure 5. SEM micrograph of zircon sand grains before (A,B) and after (C,D) their exposure to the fungus Fusarium oxysporum for 24 h.

reduction in the silica content of zircon sand during the respective first, second, and third cycles of exposure, which in turn leads to a total of 83% reduction in silica content within 72 h of reaction. EDX results obtained after 24 h of reaction (49% silica leaching) matches closely with those obtained from XPS measurements after 24 h of reaction (53% silica leaching). We observe that, although the continuous exposure of zircon sand to the same batch of fungus for more than 24 h does not lead to any increase in silica leaching, the exposure of already reacted zircon sand to a new batch of fungal biomass further leads to an almost similar amount of silica leaching during every new exposure (ca. 40-50%). This suggests that the enzyme kinetics and the reaction equilibrium might be playing some role in limiting the silica leaching to close to 50% during every exposure cycle. Important information about the bioleaching process can be obtained by imaging the texture of the zircon sand particles before and after their reaction with the fungus. A few sand grains were fixed on a double-sided conducting tape and were imaged by SEM. Figure 5A,B shows the SEM images of zircon sand grains before exposure to the fungus F. oxysporum, while Figure 5C,D shows the SEM images of zircon sand after their exposure. It is evident from SEM images that the sand grain surface is relatively smooth before exposure (Figure 5A,B), and becomes very rough and granular after exposure to the fungus for 24 h (Figure 5C,D). The roughening of the surface of the sand grain after reaction with the fungus can be attributed to the leaching out of silica from zircon sand in the form of nanoparticles by the fungus. In conclusion, we have demonstrated that the fungus Fusarium oxysporum may be used for selective bioleaching of silica present in zircon sand. The silica synthesized is in the form of crystalline nanoparticles capped by stabilizing proteins in the size range 2-10 nm and is released into solution by the fungus. It appears that the fungal enzymes involved in the silica bioleaching act specifically on Si precursors present in zircon sand and do not act on Zr precursors. We have previously observed that the extracellular cationic proteins released by the fungus Fusarium oxysporum are involved in the hydrolysis of silica and zirconia precursors to form SiO223b and ZrO223a nanoparticles, respectively. However, in the case of zircon sand, where a mixed SiO2-ZiO2 system coexists, we believe that, in the vicinity of structurally similar substrates (silica/zirconia), the electrostatic interactions between the cationic proteins and the anionic substrates might play a greater role. Since the isoelectric point of silica (pI ≈ 2) is much lower in comparison with that of zirconia (pI ≈ 4), cationic proteins would expectedly show a higher affinity toward silica, which might lead to selective leaching of silica particles. It would be interesting to perform detailed enzyme kinetics and

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modeling studies for a better understanding of this matter in the future studies. In addition, we have also shown previously that the proteins secreted by the fungus Fusarium oxysporum act on silicates to convert them into silicic acid, which on condensation by fungal proteins gets converted into silica nanoparticles.24a We believe that silica nanoparticles from the zirconium silicate present in zircon sand are being leached out by a similar mechanism, which provides selectivity and specificity to this reaction. Moreover, in this article, we have also shown that the selective bioleaching of silica from zircon sand also results in significant enhancement of the zirconium component in zircon sand within 24 h of reaction. The room-temperature synthesis of oxide nanomaterials using microorganisms, starting from potential cheap, naturally available materials is an exciting

Bansal et al.

possibility and could lead to an energy-conserving and economically viable green approach to the large-scale synthesis of nanomaterials. Acknowledgment. V.B. thanks the Council of Scientific and Industrial Research (CSIR), Government of India, for a research fellowship. Supporting Information Available: XRD pattern recorded from zircon sand (ZrSiO4) used as a raw material in the synthesis of silica nanoparticles by its reaction with the fungus, Fusarium oxysporum. This material is available free of charge via the Internet at http://pubs.acs.org. LA062535X