Zirconia Enrichment in Zircon Sand by Selective Fungus-MediatedBioleaching of Silica

Vipul Bansal,† Asad Syed,§ Suresh K. Bhargava,‡ Absar Ahmad,§ and Murali Sastry*,†,|

Nanoscience Group, Materials Chemistry and Biochemical Sciences DiVision, National ChemicalLaboratory, 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 zirconsand (ZrSixOy). We present here a completely green chemistry approach toward enrichment of zirconia in zircon sand;this is based on the reaction of the fungusFusarium oxysporumwith zircon sand by a process of selective extracellularbioleaching of silica nanoparticles. Since this reaction does not result in zirconia being simultaneously leached outfrom the sand, there is a consequent enrichment of the zirconia component in zircon sand. We believe that fungalenzymes specifically hydrolyze the silicates present in the sand to form silicic acid, which on condensation by certainother fungal enzymes results in room-temperature synthesis of silica nanoparticles. This fungus-mediated twofoldapproach might have vast commercial implications in low-cost, ecofriendly, room-temperature syntheses oftechnologically important oxide nanomaterials from potentially cheap naturally available raw materials like zirconsand.

IntroductionSilica is an important inorganic material1 extensively used in

a wide range of commercial applications such as resins, molecularsieves, catalyst supports, and fillers in polymeric items, as wellas in biomedical devices.2 Apart from demand for silica, thereis also a huge demand for the development of high dielectricmaterials for rapid scaling of silicon-based metal oxide semi-conductor field effect transistor (MOSFET) and advancedcomplementary metal oxide semiconductor (CMOS) devices inorder to achieve reduced effective oxide thickness (EOT) whilemaintaining the overall gate capacitance, so that the problem ofcurrent leakage in future devices can be avoided.3 Many highdielectric materials including Ta2O5 (ε ) 26), TiO2 (ε ) 80), andSrTiO3 (ε ) 175) have been developed; however, these materialsare not thermally stable in direct contact with silicon.4-7

Conversely, the metal oxide zirconia (ZrO2) as well as thecompound zirconium silicate ZrSiO4 (zircon) have been foundto be stable in direct contact with Si even at very hightemperatures.8,9 The pure oxide ZrO2 has high permittivity (ε )25) and is a promising candidate for such devices; however,

some potential concerns are that it tends to crystallize at lowtemperature and is an ionic conductor, and the heterointerfaceformed between the Si channel and ZrO2may degrade the electronchannel mobility in transistors.10Considering the thermal stabilityand the electrical results reported earlier,9bZrSixOy is a promisingcandidate to replace SiO2 as the gate dielectric material, sinceits permittivity value (ε ) 12.6) lies between those of its structuralcomponents SiO2 (ε ) 3.9) and ZrO2 (ε ) 25).11

The chemical syntheses of these high dielectric and silica-based materials are not only relatively expensive and ecohaz-ardous, but also often require extremes of temperature, pressure,and pH. For instance, silica and zirconia are produced com-mercially in the refractories at extremely high temperature usingsilica sand (white sand) and zircon sand that are rich in silicaand zirconia, respectively.12However, silicon impurity in zirconsand has always been a matter of concern in zirconia synthesisusing zircon sand.13 Similarly, zircon (zirconium silicate) isconventionally synthesized at extremely high temperatures(1600-2700 °C) in specially designed high-energy plasmareactors (20 kW), followed by various chemical treatments,wherein there is not much control over the zirconia content of* 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; AcademicPress: 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. ElectronDeVices 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. ElectronDeVices Meet.1998, 1038.

(6) McKee, R. A.; Walker, F. J.; Chisholm, M. F.Phys. ReV. Lett.1998, 81,3014.

(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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10.1021/la062535x CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 03/22/2007

zircon being synthesized due to thermokinetics limitations.12,14

Consequently, due to these problems in the development of highdielectric zirconia and zircon, there is a need to develop a protocolto selectively remove the low dielectric silica from zircon sandand hence enrich the high dielectric zirconia content in zirconsand. This, in turn, would help in the development of highdielectric zircon wherein dielectric values of the material can becontrollably shifted toward high dielectric zirconia.

In contrast to the extreme conditions employed in most of thesynthesis protocols for these materials, biosilicification by livingorganisms such as bacteria, diatoms, sponges, and plants proceedsunder mild physiological conditions, producing an amazingdiversity of complex and hierarchical biogenic silica nanostruc-tural frameworks.15,16Some polyamines, carbohydrates, proteins,and glycoproteins from diatoms, sponges, and plants have beenreported to be capable of polymerizing silicic acid at neutral toacidic pHs.17,18Fungal activity has also been reported to releasemetallic and silicate ions from minerals and rocks.19Bioleachinghas been explored previously as a significant tool for environ-mental friendly, low-cost commercial synthesis of various metalslike copper, iron, and gold from their precursors under ambientconditions.20

Despite the vast scientific literature on crystalline andamorphous silica synthesis by biological and biomimeticmethods,15-18 there have been no attempts at harnessing theenormous potential of these microorganisms to selectively leachout small amounts of silica present in cheap, naturally availableraw materials like zircon sand under ambient conditions. In thisarticle, we address this issue and describe our efforts to set upa biological model system for selective bioleaching of silicaimpurity present in naturally available zircon sand in the formof crystalline silica nanoparticles and simultaneous enrichmentof high dielectric zirconia in zircon sand, thereby convertinglow-cost raw materials to value-added raw materials for otherprocesses. More specifically, we show thatFusarium oxysporum,a plant pathogenic fungus, when exposed to zircon sand is capableof selectively leaching out silicon impurity of zircon sand in theform of silica nanoparticles of reasonable monodispersity underambient conditions. The silica bioleaching is fairly rapid andoccurs within 1 day of reaction of fungal biomass with zirconsand. It is interesting to note that, despite the in vitro studies of

various proteins or synthetic macromolecules in this context,21

only the F. oxysporumbased system was able to selectivelyleach out aforesaid silica structures for the conditions studied todate. In previous studies, we demonstrated thatF. oxysporumisan excellent microorganism for the biosynthesis of metal22 andmetal oxide23 nanoparticles from their precursor salts; this workalso develops significantly upon our earlier study on bioleachingof silica from sand24 and rice husk24 and shows the high levelof specificity of fungal enzymes toward the silica component inzircon sand.

Experimental Section

The plant pathogenic fungusFusarium oxysporumwas culturedas described elsewhere.22 After incubation, the fungal mycelia wereharvested and washed thoroughly under sterile conditions. For theextracellular bioleaching of silica from zircon sand (obtained fromEastern Ghats of Tamil Nadu, India) by the fungusF. oxysporum,the harvested fungal biomass (20 g wet weight) was resuspendedin 100 mL of sterile distilled water containing 10 g of zircon sandin 500 mL Erlenmeyer flasks and kept on a shaker (200 rpm) at 27°C. The reaction between the fungal biomass and sand was carriedout for a period of 24 h. The bioleached product was collected byseparating the fungal mycelia from the aqueous component byfiltration. The nanoparticle solution was evaporated under lowpressure to powder, which was then characterized before and aftercalcination at 400°C for 2 h.

Samples for transmission electron microscopy (TEM) wereprepared by drop-coating films of the bioleached nanoparticlepowders dispersed in water onto carbon-coated copper grids. Selectedarea electron diffraction (SAED) analysis was also carried out forthese samples. TEM and SAED patterns were obtained on a JEOL1200 EX instrument operated at an accelerating voltage of 120 kV.The extracellular products formed in the reaction were monitoredby Fourier transform infrared (FTIR) spectroscopy. The samples forFTIR analysis were taken in KBr pellets after thorough drying andanalyzed on a Perkin-Elmer Spectrum One instrument at a resolutionof 2 cm.-1 X-ray diffraction (XRD) measurements of drop-coatedfilms of the extracellularly synthesized biogenic silica before andafter calcination at 400°C for 2 h were carried out on a Phillips PW1830 instrument operated at a voltage of 40 kV and a current of 30mA with Cu KR radiation. XRD measurement of the zircon sandused as a precursor in this reaction was also performed.

X-ray photoemission spectroscopy (XPS) measurements of filmsof bioleached silica nanoparticles cast onto a Cu strip were carriedout on a VG MicroTech ESCA 3000 instrument at a pressure betterthan 1×10-9Torr. Moreover, in order to comprehend the enrichmentof the zirconium component in zircon sand as compared to its siliconcounterpart, XPS analysis of zircon sand before and after its exposureto the fungus was also performed. Samples for XPS measurementsfrom zircon sand were prepared by sticking the finely ground zirconsand onto Cu strips. The general scan and Si 2p, Zr 3d, and O 1score-level spectra for all the samples were recorded with un-monochromatized Mg KR radiation (photon energy) 1253.6 eV)at a pass energy of 50 eV and electron takeoff angle (angle betweenelectron emission direction and surface plane) of 60°. The overallresolution was∼1 eV for the XPS measurements. The core-level

(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 SilicateHigh-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 Perspec-tiVes; VCH: Weinheim, 1998. (e) Lowenstam, H.Science1981,211, 1126.

(16) (a) Simpson, T. L., Volcani, B. E., Eds.;Silicon and Siliceous Structuresin 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.Palaeontology1995, 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.Phytochemistry1996, 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.

(21) (a) Kroger, N.; Deutzmann, R.; Sumper, M.Science1999, 286, 1129. (b)Kroger, N.; Lorenz, S.; Brunner, E.; Sumper, M.Science2002, 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.Small2006, 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.

4994 Langmuir, Vol. 23, No. 9, 2007 Bansal et al.

spectra were background-corrected using the Shirley algorithm,25

and the chemically distinct species were resolved using a nonlinearleast-squares fitting procedure. The core-level binding energies (BEs)were aligned with the adventitious carbon binding energy of 285eV. A control experiment was performed wherein the zircon sandwas exposed to distilled water maintained at pH 3.5 for 24 h withoutadding the fungusF. oxysporum. The solution was further analyzedby FTIR and TEM.

In order to understand the bioleaching process, 10 g of zirconsand was exposed to 20 g of wet fungal biomass for 24 h. After 24h, the zircon sand was separated from biomass, and 9 g of pre-exposed zircon sand was re-exposed to 18 g of fresh fungal biomassfor the next 24 h. After this reaction, the zircon sand was againseparated from the biomass, and 8 g ofthis pre-exposed zircon sandwas further exposed to 16 g of fungal biomass for next 24 h. Thezircon sand obtained after exposure to fresh fungal biomass after 24,48, and 72 h of reaction was analyzed using an energy-dispersiveX-ray (EDX) instrument fitted to a Leica Stereoscan-440 scanningelectron microscope (SEM). The changes in the surface morphologyof zircon sand before and after the reaction were monitored usingSEM.

Results and Discussion

The bioleached product obtained from the fungus-zircon sandreaction mix was analyzed by TEM. Figure 1A,B shows therepresentative TEM images recorded from the film of theextracellular product obtained by the reaction ofFusariumoxysporumwith the zircon sand for 24 h (pH of the reactionmedium≈ 3.5). The particles embedded in the biomolecularmatrix are fairly regular in shape and depict an overall quasi-spherical morphology. A statistical analysis of 200 randomparticles indicated that the particle size range from 2 to 10 nm,with an average particle size of 5.5( 2 nm. SAED analysis ofthe particle assemblies (inset, Figure 1A) clearly indicates thatthey are crystalline in nature. The diffraction spots in the SAEDpattern could be indexed on the basis of the cristobalite polymorphof silica structure.26FTIR analysis of particles from the fungus-zircon sand reaction medium taken in KBr pellets showed thepresence of bands at ca. 1100 and 611 cm-1 (Figure 2A, curve1). The prominent 1100 cm-1 band can be assigned to the Si-O-Si27 antisymmetric stretching mode present in the leached-

out product. Another distinct vibrational mode detected around600 cm-1 is generally observed in sol-gel silica materials andcan be assigned to some cyclic structures present in the silicanetwork. Yoshino et al.28have assigned this IR vibration to cyclictetrameric siloxane species by referring to different types of cyclicsiloxanes and silicate minerals, and this attribution has also beensupported by molecular orbital calculations.29 Two absorptionbands at ca. 1650 and 1540 cm-1 (amide I and II bands,respectively; Figure 2A, curve 1) attest to the presence of proteinsin the quasi-spherical silica particles that have been released bythe fungus during reaction with zircon sand.

Additional evidence for the crystalline nature of the bioleachedsilica nanoparticles is provided by XRD analysis of the bioleachedproduct formed by the fungus-zircon sand reaction medium(Figure 2B, curve 1). The XRD spectrum of as-formed silicananoparticles shows well-defined Bragg reflection characteristicsof cristobalite polymorph of crystalline silica.26 The presence ofFTIR signatures corresponding to silica-entrapped proteins(Figure 2A, curve 1) and well-defined Bragg reflections in thesame sample (Figure 2B, curve 2) indicate that entrapped proteinsin the silica particles do not significantly interfere with theircrystallinity. Moreover, XRD spectra for zircon sand used as aprecursor in this study were also recorded as a control thatexactly matches with zirconium silicate (ZrSiO4) (SupportingInformation S1).

In order to preclude the possibility of silica leaching out dueto the acidic nature of the reaction medium, a control experimentwas performed wherein the zircon sand was kept in distilledwater maintained at an acidic pH of 3.5 for 24 h, and then thefiltrate was characterized by FTIR spectroscopy and TEM. Weobserved that characteristic Si-O-Si vibrational modes27 ofsilica as well as signatures from silicic acid (Si-OH vibrationalmodes)30 were clearly missing in the control zircon sand samplenot being exposed to the fungus (Figure 2A, curve 2). However,weak FTIR signatures were observed in the control sample at

(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 B2003, 107, 4711.

(28) Yoshino, H.; Kamiya, K.; Nasu, H.J. Non-Cryst. Solids1990, 126, 68.(29) Hayakawa, S.; Hench, L. L.J. Non-Cryst. Solids2000, 262, 264.(30) Silverstein, R. M.Spectrometric identification of organic compounds,

2nd ed.; John Wiley & Sons: New York, 1967; p 102.

Figure 1. TEM micrographs at different magnifications of silicananoparticles synthesized by the exposure of zircon sand to thefungusFusarium oxysporumbefore (A,B) and after (C,D) calcinationat 400°C for 2 h. The insets in A and D are the SAED patternsrecorded from representative silica nanoparticles.

Figure 2. (A) FTIR spectra recorded from the filtrate containingsilica particles synthesized by exposing zircon sand to the fungusFusarium oxysporumfor 24 h (curve 1) and from the filtrate obtainedby exposing zircon sand to water of pH 3.5 for 24 h (curve 2). (B)XRD patterns recorded from silica particles synthesized by theexposure of zircon sand to the fungusF. oxysporumbefore (curve1) and after (curve 2) calcination of particles at 400°C for 2 h.

Zirconia Enrichment in Zircon Sand Langmuir, Vol. 23, No. 9, 20074995

580 and 1050 cm-1 that might be due to the presence of a smallamount of organic impurities on the surface of zircon (Figure2A, curve 2). The fact that these signatures reside in the FTIRfingerprint region makes it extremely difficult to assign thesepeaks unequivocally. We believe that these peaks are not due toshifted Si-O-Si peaks, since zircon is considered to be anextremely resistant material and only plasma treatment of around2000°C or more is able to dissociate zircon into its components.12b

Moreover, the absence of any particles in the TEM micrographof drop-cast films from the control experiment further supportsour belief. In addition, the amide I and II signatures arising fromthe extracellular fungal proteins in the zircon sand exposed tothe fungus (Figure 2A, curve 1) were also missing from thefungus-deficient control sample (Figure 2A, curve 2). The controlexperiment and the TEM, SAED, FTIR, and XRD results of thefungus-zircon sand reaction medium clearly suggest thatF.oxysporumselectively leaches out the silicon component of zirconsand in the form of extracellular crystalline silica nanoparticlesand does not cause leaching of the zirconium counterpart ofzircon sand.

The FTIR results show the presence of proteins in the silicananoparticle powders (Figure 2A, curve 1). In order to removethe proteins that are intercalated/incarcerated in the silicastructures, calcination of the silica powder was performed at 400°C for 2 h. The calcined silica nanoparticle powder afterredispersion in water was analyzed by TEM (Figure 1C,D). Itis observed that the removal of incarcerated biomolecules bycalcination leads to sintering of silica nanoparticles andconsequently results in the formation of larger silica nanoparticlesranging in size 50-100 nm, with an average particle size of 80( 12 nm (Figure 1C,D). The SAED pattern recorded from thesesilica nanoparticles (inset, Figure 1D) clearly shows the crystallinenature of silica particles formed and could be indexed on thebasis of cristobalite polymorph of silica. In addition, thecrystallinity of these silica nanostructures was further confirmedby XRD (Figure 2B, curve 2). The XRD analysis of the calcinedpowder further shows well-defined Bragg reflections charac-teristic of a cristobalite polymorph of silica nanoparticles.26 Itappears that calcination, leading to removal of proteins from thesilica matrix, results in increased crystallinity of silica particles(Figure 2B, curve 2) as compared to as-synthesized silicananoparticles (Figure 2B, curve 1). However, the effect ofcalcination temperature on improved crystallinity of silica crystalscannot be neglected. In order to establish the effect of embeddedproteins on the crystallinity of silica particles, proteins frombioleached silica particles were removed using a phenol/chloroform mixture24a instead of calcination. An increase incrystallinity of silica particles after removal of proteins bychemical means was also observed (data not shown for brevity),which correlates well with our previous studies on bioleachingof silica from white sand, wherein removal of proteins by chemicalmeans was observed to increase the crystallinity of particles.24a

A chemical analysis of the nanoparticles bioleached from zirconsand was performed by XPS, which is known to be a highlysurface-sensitive technique (Figure 3). The Si 2p, Zr 3d, and O1s 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 fittedinto 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 excellentagreement with values reported for SiO2.31 In addition to the Si2p spectrum, the sample was also scanned for a Zr 3d signal;however, we could not detect any Zr 3d signal arising from the

sample (Figure 3B). Besides, an O 1s signal was also recordedin the sample (Figure 3C) that shows a single component withBE of 532.2 eV. Oxygen in the Si-O-Si environment is knownto show an O 1s BE component at 532.5 eV.3aSimilarly, oxygenin Si(OH)4 shows an O 1s BE component at 531.9 eV.15b Webelieve that both these components contribute to the XPS spectrashown in Figure 3C and hence illustrate an O 1s BE componentof ca. 532.2 eV. Notably, we do not observe any lower BEcomponent of ca. 530.1 eV arising from ZrO2.3aThe absence ofa Zr 3d signal and an O 1s signal corresponding to Zr-O furthersupports the selective bioleaching of silica nanoparticles fromzircon sand.

A chemical analysis of zircon sand before and after its exposureto the fungusF. oxysporumwas also performed by XPS. TheSi 2p and Zr 3d core-level spectra from finely ground zirconsand before (Figure 4A,C) and after (Figure 4B,D) its reactionwith 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 itsreaction with the fungus that could be fitted into a single spin-orbit pair (spin-orbit splitting∼0.6 eV)3awith 2p3/2BE of 102.2eV (Figure 4A), which is an excellent match to Si 2p3/2 BE inthe Zr-O-Si phase as reported previously.3aSi 2p XPS analysisof zircon sand after its reaction with the fungus (Figure 4B) leadsto the attribution of two distinct chemical species of Si atoms(31) Wagner, C. D.J. Vac. Sci. Technol.1978, 15, 518.

Figure 3. XPS data showing the Si 2p (A), Zr 3d (B), and O 1s(C) core-level spectra recorded from biologically synthesized silicananoparticle film cast onto a Cu substrate. The chemically resolvedcomponents are shown as solid lines in the figure and are discussedin 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 afterits exposure to the fungusFusarium oxysporum(B,D). The chemicallyresolved components are shown as solid lines in the figure and arediscussed in the text.

4996 Langmuir, Vol. 23, No. 9, 2007 Bansal et al.

by resolving the Si 2p spectra into two spin-orbit pairs with2p3/2 BEs of 98.4 and 102.1 eV, respectively (Figure 4B, curves1 and 2, respectively). Among these two Si 2p3/2BE components,the 102.1 eV3ais 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 98.4eV32(Figure 4B, curve 2) can be assigned to non-network-bondedSi atoms that apparently precipitate in the zirconium silicatenetwork.

Zircon sand was also analyzed for Zr 3d BEs before and afterits reaction with the fungus. Figure 4C shows the Zr 3d spectrumfrom zircon sand before its reaction with the fungus, which couldbe fitted into a single spin-orbit pair (spin-orbit splitting∼2.4eV)3awith 3d5/2 BE of 183.7 eV (Figure 4C). This 183.7 eV BEcomponent can be assigned to Zr 3d5/2 BE of Zr present in thesilica-zirconia network.15bThe Zr 3d spectrum from zircon sandafter its reaction with the fungus was also analyzed and couldbe 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 stateBE of Zr in zircon sand after silica leaching from sand as aconsequence of its reaction with fungal biomass. Our resultsmatch well with the previous reports where Zr 3d state BEs inZrSiO4 have been shown to be more than 0.5 eV larger than inZrO2.33 In addition, it has also been shown previously thatsuccessive reduction in silica content in SiO2-ZrO2alloys resultedin 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 thuscan be explained on the basis of the reduction in silica contentin zircon sand. The difference in the drop of Si 2p and Zr 3d BEsafter reaction (0.1 and 0.6 eV, respectively) is consistent withthe principle of electronegativity equalization, i.e., the chargetransfer out of Zr is larger in ZrSiO4 than in ZrO2, becauseelectronegativites of Si and O are each larger than that of Zr.34

The XPS analysis of zircon sand before and after its reactionwith fungal biomass clearly suggests enrichment of the zirconiacomponent in zircon sand, due to leaching out of silica fromzircon sand. In order to quantitatively comprehend enrichmentof the zirconium component in zircon sand, the Si/Zr ratios inzircon sand before (Figure 4A,C) and after (Figure 4B,D) itsreaction with fungal biomass were calculated, taking the integratedvalues of the respective fitted curves into account. The Si/Zrratios in zircon sand before and after its exposure to the fungusfor 24 h were found to be ca. 0.327 and 0.153, respectively. XPSresults therefore suggest that selective bioleaching of silica fromzircon sand results in ca. 53% reduction in the silica content ofzircon sand within 24 h of reaction. It is noteworthy that exposingthe zircon sand to the fungus for longer than 24 h does not resultin any further leaching of silica.

In order to understand the effect of fungal biomass on thesilica bioleaching process, zircon sand, initially exposed to fungalbiomass for 24 h, was re-exposed to a new batch of fungal biomassuntil 48 h, and then further exposed to fresh fungal biomass until72 h. The zircon sand obtained at the end of each reaction wasanalyzed using EDX, which is a semiquantitative technique. TheSi/Zr atomic percentage ratios in zircon sand after 0, 24, 48, and72 h of reaction were found to be ca. 0.353, 0.181, 0.100, and0.059, respectively. EDX results indicate that the exposure ofzircon sand to the fungus results in ca. 49%, 45%, and 42%

reduction in the silica content of zircon sand during the respectivefirst, second, and third cycles of exposure, which in turn leadsto 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 measurementsafter 24 h of reaction (53% silica leaching). We observe that,although the continuous exposure of zircon sand to the samebatch of fungus for more than 24 h does not lead to any increasein silica leaching, the exposure of already reacted zircon sandto a new batch of fungal biomass further leads to an almostsimilar amount of silica leaching during every new exposure (ca.40-50%). This suggests that the enzyme kinetics and the reactionequilibrium might be playing some role in limiting the silicaleaching to close to 50% during every exposure cycle.

Important information about the bioleaching process can beobtained by imaging the texture of the zircon sand particles beforeand after their reaction with the fungus. A few sand grains werefixed on a double-sided conducting tape and were imaged bySEM. Figure 5A,B shows the SEM images of zircon sand grainsbefore exposure to the fungusF. oxysporum, while Figure 5C,Dshows the SEM images of zircon sand after their exposure. It isevident from SEM images that the sand grain surface is relativelysmooth before exposure (Figure 5A,B), and becomes very roughand granular after exposure to the fungus for 24 h (Figure 5C,D).The roughening of the surface of the sand grain after reactionwith the fungus can be attributed to the leaching out of silicafrom zircon sand in the form of nanoparticles by the fungus.

In conclusion, we have demonstrated that the fungusFusariumoxysporummay be used for selective bioleaching of silica presentin zircon sand. The silica synthesized is in the form of crystallinenanoparticles capped by stabilizing proteins in the size range2-10 nm and is released into solution by the fungus. It appearsthat the fungal enzymes involved in the silica bioleaching actspecifically on Si precursors present in zircon sand and do notact on Zr precursors. We have previously observed that theextracellular cationic proteins released by the fungusFusariumoxysporumare involved in the hydrolysis of silica and zirconiaprecursors to form SiO223band ZrO2

23ananoparticles, respectively.However, in the case of zircon sand, where a mixed SiO2-ZiO2

system coexists, we believe that, in the vicinity of structurallysimilar substrates (silica/zirconia), the electrostatic interactionsbetween the cationic proteins and the anionic substrates mightplay 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 towardsilica, which might lead to selective leaching of silica particles.It would be interesting to perform detailed enzyme kinetics and

(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.

Figure 5. SEM micrograph of zircon sand grains before (A,B) andafter (C,D) their exposure to the fungusFusarium oxysporumfor24 h.

Zirconia Enrichment in Zircon Sand Langmuir, Vol. 23, No. 9, 20074997

modeling studies for a better understanding of this matter in thefuture studies. In addition, we have also shown previously thatthe proteins secreted by the fungusFusarium oxysporumact onsilicates to convert them into silicic acid, which on condensationby fungal proteins gets converted into silica nanoparticles.24a

We believe that silica nanoparticles from the zirconium silicatepresent in zircon sand are being leached out by a similarmechanism, which provides selectivity and specificity to thisreaction. Moreover, in this article, we have also shown that theselective bioleaching of silica from zircon sand also results insignificant enhancement of the zirconium component in zirconsand within 24 h of reaction. The room-temperature synthesisof oxide nanomaterials using microorganisms, starting frompotential cheap, naturally available materials is an exciting

possibility and could lead to an energy-conserving and economi-cally viable green approach to the large-scale synthesis ofnanomaterials.

Acknowledgment. V.B. thanks the Council of Scientific andIndustrial Research (CSIR), Government of India, for a researchfellowship.

Supporting Information Available: XRD pattern recordedfrom zircon sand (ZrSiO4) used as a raw material in the synthesis ofsilica nanoparticles by its reaction with the fungus,Fusarium oxysporum.Thismaterial isavailablefreeofchargeviatheInternetathttp://pubs.acs.org.

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4998 Langmuir, Vol. 23, No. 9, 2007 Bansal et al.