Biological Synthesis of Strontium Carbonate Crystals Using the Fungus

fungus Fusarium oxysporum is responsible for modulating the morphology of strontianite crystals and directing their hierarchical assembly into higher ...
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Langmuir 2004, 20, 6827-6833

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Biological Synthesis of Strontium Carbonate Crystals Using the Fungus Fusarium oxysporum Debabrata Rautaray,† Ambarish Sanyal,† Suguna D. Adyanthaya,† Absar Ahmad,*,‡ and Murali Sastry*,† Materials Chemistry and Biochemical Sciences Division, National Chemical Laboratory, Pune - 411 008, India Received March 23, 2004. In Final Form: May 21, 2004 The total biological synthesis of SrCO3 crystals of needlelike morphology arranged into higher order quasi-linear superstructures by challenging microorganisms such as fungi with aqueous Sr2+ ions is described. We term this procedure “total biological synthesis” since the source of carbonate ions that react with aqueous Sr2+ ions is the fungus itself. We believe that secretion of proteins during growth of the fungus Fusarium oxysporum is responsible for modulating the morphology of strontianite crystals and directing their hierarchical assembly into higher order superstructures.

Introduction Living systems exercise considerable control over the crystallization of certain inorganic materials. The presence of inorganic materials in biological organisms has broad implications in guiding specific functions in their bodies. The structures of biocomposites are highly controlled from the nanometer to the macroscopic levels, resulting in complex architectures that provide multifunctional properties.1 Development of strategies to grow crystals of controllable structure, size, and morphology and superstructures of predefined organizational order is an important goal in crystal engineering with tremendous implications in the ceramic industry.2,3 In this context, biominerals have served as an inspiration to crystal engineers. Biominerals are essentially inorganic salts, which serve a variety of biological purposes. Control over their formation is often manipulated by the organism involved to achieve specific purposes. In biological systems, almost all mineralized tissues contain a distinctive assemblage of acidic proteins, glycoproteins, or both which are capable of influencing crystal growth. One of the mechanisms used for achieving this is the intercalation of some of the acidic macromolecules into the crystal lattice.4 Elucidation of general biomineralization principles has followed from studies into the growth of minerals in biological organisms1,5 and on biomacromolecules extracted from organisms.4 Much attention has been focused on growth of CaCO3 crystals by biomimetic methods such as those presented by Langmuir monolayers,6 self-assembled monolayers * To whom correspondence should be addressed. Phone: + 91 20 25893044. Fax: +91 20 25893952/25893044. E-mail: sastry@ ems.ncl.res.in; [email protected]. † Materials Chemistry. ‡ Biochemical Sciences Division. (1) Sarikaya, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14183. (2) Heuer, A. H.; Fink, D. J.; Laraia, V. J.; Arias, J. L.; Calvert, P. D.; Kendall, K.; Messing, G. L.; Blackwell, J.; Rieke, P. C.; Thompson, D. H.; Wheeler, A. P.; Veis, A.; Caplan, A. I. Science 1992, 255, 1098. (3) Bunker, B. C.; Rieke, P. C.; Tarasevich, B. J.; Campbell, A. A.; Fryxell, G. E.; Graff, G. L.; Song, L.; Liu, J.; Wirdem, J. W.; McVay, G. L. Science 1994, 264, 48. (4) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67. (5) Berman, A.; Hanson, J.; Leiserowitz, L.; Koetzle, T. F.; Weiner, S.; Addadi, L. Science 1993, 259, 776.

(SAMs),7 lipid bilayer stacks,8 functionalized polymer surfaces,9 bicontinuous emulsions,10a and block copolymers.10b Insofar as growth of SrCO3 is concerned, templated crystallization of SrCO3 has been achieved on centered rectangular SAMs where the terminal functionality of the self-assembling molecule (anthracene-terminated thiol, ANTH) self-assembled on Au(111) was tailored to force assembly into a rectangular lattice.7a Ordered SAMs prepared from p-mercaptophenol induced needlelike strontianite growth around a pseudohexagonal axis,11 while gold nanoparticles coated with a p-sulfanylphenol SAM resulted in strontianite spherules.12 Crystallization of SrCO3 has also been achieved in thermally evaporated lipid bilayer stacks by suitable ion entrapment methods.13 Very recently, Matijevic et al. have investigated the influence of urease on the precipitation of BaCO3 and SrCO3 crystals.14 They have shown the effects of the enzyme on the morphological and structural characteristics of these minerals by reacting Sr and Ba ions with carbonate ions obtained by the decomposition of urea at 90 °C and by enzyme-catalyzed decomposition of urea by urease at lower temperatures.14 Urease played a major role in the formation of initially solid-phase spheroidal particles of SrCO3 which, on prolonged aging, transformed to the more commonly observed needlelike morphology.14 Laboratory processes for the synthesis of SrCO3 crystals have hitherto relied on an external source of CO2 for reaction with Sr2+ ions and synthetic molecules or organized surfaces to achieve morphology control. Many (6) (a) Litvin, A. L.; Valiyaveettil, S.; Kaplan, D. L.; Mann, S. Adv. Mater. 1997, 9, 124. (b) Heywood, B. R.; Mann, S. Adv. Mater. 1994, 6, 9. (7) (a) Kuther, J.; Nelles, G.; Seshadri, R.; Schaub, M.; Butt, H.-J.; Tremel, W. Chem.-Eur. J. 1998, 4, 1834. (b) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500. (8) Damle, C.; Kumar, A.; Bhagwat, M.; Sainkar, S. R.; Sastry, M. Langmuir 2002, 18, 6075. (9) (a) Feng, S.; Bein, T. Science 1994, 265, 1839. (b) Falini, G.; Gazzano, M.; Ripamonti, A. Adv. Mater. 1994, 6, 46. (10) (a) Walsh, D.; Mann, S. Adv. Mater. 1997, 9, 658. (b) Qi, L.; Li, J.; Ma, J. Adv. Mater. 2002, 14, 300. (11) Kuther, J.; Bartz, M.; Seshadri, R.; Vaughan, G. B. M.; Tremel, W. J. Mater. Chem. 2001, 11, 503. (12) Kuther, J.; Seshadri, R.; Tremel, W. Angew. Chem., Int. Ed. 1998, 37, 3044. (13) (a) Sastry, M.; Kumar, A.; Damle, C.; Sainkar, S. R.; Bhagwat, M.; Ramaswami, V. CrystEngComm 2001, 21. (b) Rautaray, D.; Sainkar, S. R.; Sastry, M. Langmuir 2003, 19, 888. (14) Sondi, I.; Matijevic, E. Chem. Mater. 2003, 15, 1322.

10.1021/la049244d CCC: $27.50 © 2004 American Chemical Society Published on Web 07/02/2004

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fungi are known to produce reasonable amounts of CO2 during growth.15 We take advantage of this feature and show in this study that CO2 and characteristic proteins released from the fungus Fusarium oxysporum16 may be reacted with aqueous Sr2+ ions to produce truly biogenic SrCO3 crystals. Very recently, we have shown that biogenic CaCO3 crystals of complex morphology may be grown by simple exposure of aqueous Ca2+ ions to a fungus, Fusarium sp., and an actinomycete, Rhodococcus sp.,17 and this report represents an important advance in developing this strategy to encompass other mineral compositions. Strontianite crystals of highly branched needlelike structures that are in turn arranged in quasilinear superstructures are observed, indicating that the proteins secreted by the fungus play a crucial role in directing the morphology of the SrCO3 crystals. To our knowledge, the use of microorganisms such as fungi in the complete biological synthesis of SrCO3 crystals has not been demonstrated thus far. The interesting morphology variation in the strontianite crystals produced as a result of proteins secreted by organisms such as fungi which are not normally (if ever) exposed to such metal ions is an exciting outcome and underlines the untapped potential of biological methods in expanding the scope of crystal engineering. Experimental Details A plant pathogenic fungus, Fusarium oxysporum, was maintained on potato-dextrose-agar (PDA) slants. Stock culture was maintained by subculturing at monthly intervals. After growing at pH 7 and 27 °C for 4 days, the slants were preserved at 15 °C. From an actively growing stock culture, subcultures of the fungus were made on fresh slants and after 4 days of incubation at pH 7 and 27 °C were used as the starting materials for fermentation experiments. For the isolation of SrCO3 crystals, the fungus was grown in 500 mL Erlenmeyer flasks containing 100 mL of MGYP medium which is composed of malt extract (0.3%), glucose (1%), yeast extract (0.3%), and peptone (0.5%). After the pH of the medium was adjusted to 7, the culture was grown under continuous shaking on a rotary shaker (200 rpm) at 27 °C for 96 h. After 96 h of fermentation, mycelium of the fungus was separated from the culture broth by centrifugation (5000 rpm) at 20 °C for 20 min, and then the mycelia were washed thrice with sterile distilled water under sterile conditions. The harvested mycelia mass (20 g wet wt of mycelia) of Fusarium oxysporum was then resuspended in 100 mL of 10-3 M SrCl2 solution in a 500 mL Erlenmeyer flask. The flask was then plugged with cotton and put into a shaker at 27 °C (200 rpm), and the reaction was carried out for a period of 72 h. The biotransformation of Sr2+ ions into SrCO3 was monitored by separating the fungal mycelia from the reaction medium by filtration and subjecting both the filtrate and mycelia to analysis as described below. That formation of SrCO3 had occurred could be inferred from the fact that the filtrate became turbid and milky white after roughly 3 days of reaction. The residue after 3 days of reaction was washed thoroughly several times using copious amounts of doubledistilled water, and the washed biomass was cast in the form of film onto a Si(111) substrate for further analysis. The filtrate was subjected to centrifugation at 10 000 rpm, and the pellets obtained were washed repeatedly with copious amounts of doubledistilled water. The purified pellets were solution-cast in the form of films onto different solid supports for further analysis. Considerable care was taken to wash the SrCO3 crystals synthesized using the fungus Fusarium oxysporum prior to analysis. This precaution was taken to remove uncoordinated proteins in the reaction medium from the biogenic SrCO3 crystals. The biogenic SrCO3 crystals obtained were analyzed by X-ray (15) Couriol, C.; Amrane, A.; Progent, Y. J. Biosci. Bioeng. 2001, 91, 570. (16) Ahmad, A.; Mukherjee, P.; Mandal, D.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. J. Am. Chem. Soc. 2002, 124, 12108. (17) Rautaray, D.; Ahmad, A.; Sastry, M. J. Am. Chem. Soc. 2003, 125, 14656.

Rautaray et al. diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive analysis of X-rays (EDAX), and thermogravimetric analysis (TGA). XRD measurements of drop-coated films of the biogenic SrCO3 crystals on glass substrates 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. FTIR spectroscopy measurements of the purified and dried biogenic SrCO3 crystal powders taken in KBr pellets were carried out on a Perkin-Elmer Spectrum One instrument at a resolution of 4 cm-1. TGA analysis of the purified and dried biogenic SrCO3 powder was done on a TGA-7 Perkin-Elmer instrument in the temperature range of 0-800 °C at a scan rate of 10 °C/min. For SEM analysis, drop-coated films of biogenic SrCO3 crystals were made on Si(111) substrates. SEM and EDAX measurements were performed on a Leica Stereoscan440 instrument equipped with a Phoenix EDAX attachment. A control experiment was performed wherein the fungus Fusarium oxysporum was soaked in water for 3 days. The aqueous component with secreted proteins was separated by filtration, and SrCl2 was added to this solution to attain a concentration of 10-3 M. CO2 was bubbled very slowly through this solution, and the SrCO3 crystals formed were analyzed by SEM and EDAX. To realize the location of proteins in the biogenic SrCO3 crystals synthesized using Fusarium oxysporum, the aqueous solution containing biogenic SrCO3 crystals was treated with 4% sodium hypochlorite (NaOCl) solution. Thereafter, the solution containing NaOCl-treated biogenic SrCO3 crystals was subjected to centrifugation and the pellets obtained were washed repeatedly with copious amounts of double-distilled water. The purified pellet was solution-cast in the form of films onto different solid supports and was analyzed by FTIR, UV-vis spectroscopy, XRD, TGA, SEM, and EDAX. UV-vis spectroscopy measurements were carried out on a JASCO dual-beam spectrophotometer (model V-570) operated at a resolution of 2 nm. In another control experiment, the crystallization of SrCO3 was accomplished directly in solution by bubbling CO2 in an aqueous solution of SrCl2 under electrolyte concentrations identical to that mentioned above. The SrCO3 crystals formed in the control experiment were collected and subjected to SEM analysis. To identify the proteins bound to the SrCO3 crystals that are possibly responsible for the strontianite shape control, the biogenic SrCO3 crystals were washed thoroughly and were treated with dilute acetic acid solution. This mild acid treatment resulted in the dissolution of SrCO3 crystals, and the free proteins detached from the SrCO3 crystals in solution were separated by centrifugation and analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) carried out at pH 8.2 according to standard procedures.18

Results and Discussion Figure 1 shows SEM images recorded from solutioncast films of the aqueous SrCl2 solution after exposure to Fusarium oxysporum for 3 days. Highly dense branching of SrCO3 needles populating the substrate surface is observed in Figure 1A. The SrCO3 needles appear to grow outward and radially from central primary crystals and are often greater than 12 µm in length. Figure 1B shows a higher magnification SEM image of SrCO3 needles branching out from a central point. Figure 1C,D clearly shows the structure of the individual SrCO3 needles in greater detail. It can clearly be seen that the individual SrCO3 needles are porous with the pores dotting the whole crystal surface quite uniformly. The pores were estimated to range in size from 10 to 50 nm. The morphology of SrCO3 needlelike crystals and their assembly into higher order superstructures observed in this study are very much different from the strontianite growth observed in previous studies.7a,11-14 SAMs prepared from p-mercaptophenol induced needlelike strontianite growth,11 whereas psulfanylphenol SAMs assembled on the surface of gold (18) Laemmli, U. K. Nature 1970, 227, 680.

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Figure 1. (A-D) SEM micrographs at different magnifications showing SrCO3 crystals formed after 3 days of reaction of aqueous Sr2+ ions with Fusarium oxysporum.

nanoparticles resulted in the growth of strontianite spherules.12 SrCO3 crystal growth carried out in thermally evaporated lipid bilayer stacks such as stearic acid thin films by a method of ion exchange resulted in a flowerlike assembly of strontianite needles,13a while aerosol OT thin films by a similar ion-exchange mechanism yielded an unusual thin-ribbon-like morphology of the crystals.13b The effects of the enzyme-catalyzed decomposition of urea by urease led to the formation of spheroidal particles of SrCO3 which, on prolonged aging, transformed into the more commonly observed needlelike morphology.14 As will be demonstrated below, proteins are occluded into the SrCO3 crystalline needles and it is quite likely that these specific proteins secreted by Fusarium oxysporum play a crucial role in modulating the formation of SrCO3 crystals and also in their interesting assembly. It would be instructive to understand the chemical composition of the different features observed in the needlelike SrCO3 assemblies obtained from Fusarium oxysporum. This is conveniently done by spot-profile EDAX of the SrCO3 needles (Figure 2A). Curve 1 in this figure corresponds to the EDAX spectrum recorded from one of the SrCO3 needles shown in Figure 1D. In addition to the expected Sr, C, and O signals, strong N and S signals are also observed. The presence of N and S signals is clearly indicative of the presence of proteins in the SrCO3 needles. These signals are believed to arise from proteins either on the surface of the SrCO3 crystals or occluded into the crystals. That these signals are likely to be due to proteins secreted by the fungus is supported by the FTIR results (Figure 2B, curve 1) which clearly show the presence of amide I and II bands at 1640 and 1548 cm-1, respectively. To understand better the nature of protein occlusion into the SrCO3 crystals, TGA measurements of the purified

Figure 2. (A) Spot-profile EDAX spectra recorded from films of SrCO3 crystals synthesized using Fusarium oxysporum (curve 1), the biogenic SrCO3 crystals after calcination at 300 °C (curve 2), and the biogenic SrCO3 crystals after NaOCl treatment (curve 3). (B) FTIR spectra recorded from biogenic SrCO3 crystals obtained by the reaction of aqueous Sr2+ ions with the fungus Fusarium oxysporum before (curve 1) and after calcination at 300 °C (curve 2) and the biogenic SrCO3 crystals after NaOCl treatment (curve 3). The amide I and II bands are identified in the figure.

biogenic SrCO3 powder were done. Curve 1 in Figure 3A shows the TGA curve obtained from purified powder of biogenic SrCO3 crystals. It is observed that there are two prominent weight losses of ca. 40 and 30% at 100 and 230 °C, respectively. The first weight loss is clearly due to release of water entrapped in protein-SrCO3 biocomposite crystals, whereas the higher temperature weight loss is attributed to decomposition/desorption of proteins bound to the strontianite crystals.

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Figure 3. (A) TGA data obtained from purified biogenic SrCO3 crystal powder (curve 1) and the biogenic SrCO3 crystals after treatment with NaOCl. (B) XRD patterns recorded from solution-cast films on glass substrates of SrCO3 crystals synthesized using Fusarium oxysporum before (curve 1) and after calcination at 100 °C (curve 2) and 300 °C (curve 3) and the XRD pattern recorded from the biogenic SrCO3 crystals after NaOCl treatment (curve 4).

XRD analysis of the biogenic SrCO3 crystals formed by the reaction of aqueous Sr2+ ions with Fusarium oxysporum for 3 days was performed, and the diffraction pattern obtained is shown as curve 1 in Figure 3B. A number of Bragg reflections are identified and have been indexed with reference to the unit cell of the strontianite structure (a ) 5.107 Å, b ) 8.414 Å, c ) 6.029 Å; space group Pmcn).19 XRD analysis thus confirms the formation of the crystalline strontianite by the reaction of Sr2+ ions with Fusarium oxysporum. The formation of quasi-linear superstructures of strontianite needles by the reaction of aqueous Sr2+ ions with Fusarium oxysporum (Figure 1A-D) indicates that specific proteins secreted by these plant organisms are responsible for directing the assembly of SrCO3 crystals. While the exact nature of binding of the proteins with specific crystallographic faces of strontianite needs further study, the location of the proteins in the crystals (surface adsorption vs uniformly intercalated in the crystals) may be indirectly determined by removal of the proteins by calcination. Based on the TGA results, the biogenic SrCO3 crystals were calcined at temperatures of 100 and 300 °C for 3 h. Figure 4A,B shows representative SEM images of biogenic SrCO3 crystals grown using Fusarium oxysporum after calcination at 100 and 300 °C. Upon heating at 100 °C, it is observed that there is little (if any) disturbance to the overall structure of the originally porous biogenic SrCO3 crystals (Figure 4A). After calcination at 300 °C, the biogenic SrCO3 crystals show some degree of collapse in their internal structure but yet appear to be quite compact. The possibility of recrystallization of SrCO3 at some stage during heat treatment thus cannot be ruled out (Figure 4B). The spot-profile EDAX spectrum recorded from the biogenic strontianite crystals after calcination at 300 °C is shown as curve 2 in Figure 2A. As expected, strong Sr, C, and O signals are observed but this is accompanied by complete disappearance of N and S signals from the strontianite needles indicating removal of the occluded proteins. The complete disappearance of N and S signals after calcination is also observed in the FTIR spectrum (curve 2 in Figure 2B), which clearly shows the (19) The XRD patterns were indexed with reference to the unit cell of the strontianite structure from the ASTM chart (a ) 5.107 Å, b ) 8.414 Å, c ) 6.029 Å; space group Pmcn, ASTM chart card no. 5-0418).

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absence of the amide bands further to calcination. The XRD spectra recorded from the biogenic strontianite crystals after calcination at 100 °C (curve 2) and 300 °C (curve 3) are shown in Figure 3B. Loss of water (curve 2) and eventually occluded proteins (curve 3) from the biogenic crystals does not lead to loss in the strontianite structure of the crystals. The SEM image (Figure 4B) along with the XRD result (curve 3 in Figure 3B) of the SrCO3 crystals after calcination clearly indicates the reduction in crystalline properties of the strontianite needles upon removal of proteins. Even though there is a significant amount of trapped water in the strontianite needles (to the extent of 40 wt %), removal of water does not destabilize the linear superstructure (Figure 4A), thus underlining the very important role played by the proteins in stabilizing the superstructure. It is clear that removal of proteins from the strontianite crystals affects the crystallinity, morphology, and growth pattern of the crystals. A possible explanation for the formation of porous biogenic strontianite needles could be the following. Further to addition of Sr2+ ions to the fungal biomass, the reaction of the Sr2+ ions with carbonate ions in the presence of secreted proteins produced by Fusarium oxysporum leads to the growth of strontianite crystals yielding a porous, templated strontianite needle. It is also possible that the pores on the crystal surface are due to the loss of trapped water in the strontianite needles (as evidenced from TGA results) during drying/exposure to a vaccum during SEM analysis. The ca. 40% weight loss due to water entrapped in the strontianite framework would explain the large density of pores in the crystals. To understand whether the pores on the crystal surface are due to loss of water during drying or due to the presence of proteins which trap a large amount of water within the crystals, a control experiment was done where the crystallization of SrCO3 was accomplished directly in solution without mediation of any proteins by bubbling CO2 into the aqueous solutions of SrCl2. This control experiment resulted in the formation of compact SrCO3 crystals in a needlelike morphology with no visible signs of porosity on the crystal surface (Figure 5A). This suggests that the numerous pores observed in the biogenic SrCO3 needles are most likely due to the loss of trapped water associated with the presence of proteins. To understand the nature of proteins that were either deposited on the surface of the crystals or intercalated within the crystals, the solution containing biogenic SrCO3 crystals was treated with 4% sodium hypochlorite (NaOCl) solution to remove any surface-adsorbed proteins while preserving the proteins intercalated within the crystals. NaOCl treatment is known to be an efficient method to denature any surface-bound proteins due to its oxidizing capability. Figure 5B shows an SEM image recorded from solution-cast films of the biogenic SrCO3 crystals after NaOCl treatment. It is observed that the individual SrCO3 needles become highly porous (Figure 5B) upon NaOCl treatment in comparison with the original SrCO3 crystals (Figure 1A-C). The inset in the Figure 5B shows a higher magnification SEM image of the NaOCl-treated biogenic SrCO3 crystals. It is observed that there is an increase in the average pore size uniformly over the crystal surface after NaOCl treatment, indicating removal of proteins from the original strontianite crystals. The pores in this case were estimated to be in excess of 100 nm as compared with the 10-50 nm pore size observed in the case of untreated biogenic SrCO3 crystals (Figure 1D). This suggests that most of the proteins, which were present on the surface of the crystalline framework, have been removed upon addition of NaOCl and thus the porosity

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Figure 4. (A,B) SEM micrographs of biogenic SrCO3 crystals after calcination at 100 and 300 °C for 3 h, respectively. (C,D) SEM micrographs at different magnifications showing SrCO3 crystals grown using extracts from Fusarium oxysporum (see the text for details).

increases on the crystal surface. The EDAX spectrum recorded from the NaOCl-treated biogenic SrCO3 crystals is shown in Figure 2A, curve 3. In addition to the expected Sr, C, and O signals, the reduction of S and almost complete disappearance of N signals observed in the spectrum indicates removal of a large amount of proteins from the biogenic SrCO3 crystals. The removal of large amounts of proteins from the biogenic SrCO3 crystals upon NaOCl treatment as evidenced from the EDAX results was confirmed by FTIR and TGA measurements. The FTIR spectrum shows a broad band around 1620 cm-1 with no distinct amide signatures (curve 3 in Figure 2B). The TGA curve shows an overall loss in weight of 35% (curve 2 in Figure 3A) after NaOCl treatment of biogenic SrCO3 crystals, which is much smaller than the total weight loss (74%) recorded for the untreated biogenic SrCO3 crystals (curve 1 in Figure 3A). This confirms that after treatment with NaOCl, a large percentage of surface-bound proteins were removed from the biogenic SrCO3 crystals. The removal of surface-bound proteins was also confirmed by UV-vis spectroscopy (Figure 5D) where a comparative study of the as-prepared SrCO3 crystals and those after treatment with NaOCl at different time intervals was performed. Curve 1 in Figure 5D shows the UV-vis spectrum of the as-prepared SrCO3 crystals synthesized using Fusarium oxysporum. An absorption peak is observed at ca. 260 nm, indicating the presence of proteins bound to the SrCO3 crystals. After NaOCl treatment for 1 h, it is observed that the absorption peak shifted to 288 nm, indicating the detachment of surface-bound proteins from the SrCO3 crystals and their dissolution (curve 2). After 3 h of reaction, the intensity of the peak at 288 nm has reduced considerably (curve 3), and after 24 h, the protein absorption band has almost completely disap-

peared (curve 4). The UV-vis spectroscopy results thus indicate that the denaturation and oxidation of the strontianite-bound proteins occur over a period of 1 day during NaOCl treatment. The XRD pattern recorded from the NaOCl-treated biogenic SrCO3 crystals is shown as curve 4 in Figure 3. The XRD spectrum shows no discernible change (curve 4) in comparison with the XRD spectrum recorded from the biogenic strontianite crystals after calcination at 300 °C (curve 3). It is thus clear that consequent to protein removal, there is no loss in the strontianite structure (either by calcination or by NaOCl treatment) except for a collapse in the internal structure as observed in SEM. It is also observed that upon protein removal many of the intense peaks are reduced with some unaffected Bragg peaks. Since SrCO3 crystallizes in the strontianite crystallographic form, the possibility of a mixture of phases may be discounted. The presence of some unaffected Bragg peaks even after protein removal may be because of the growth of a few SrCO3 crystallites possibly in solution without mediation of any proteins, or it is also possible that preferred orientation is likely the most common cause of intensity variations in XRD powder experiments. A small amount of proteins intercalated within the SrCO3 crystalline framework remains after NaOCl treatment of the biogenic SrCO3 crystals as evidenced from the EDAX (curve 3 in Figure 2A) and FTIR (curve 3 in Figure 2B) measurements. The NaOCl-treated biogenic SrCO3 crystals were then calcined at 300 °C. Upon calcination, the originally porous strontianite crystals collapsed (Figure 5C), similar to results observed for the as-prepared strontianite crystals (without any NaOCl treatment) after calcination at 300 °C (Figure 4B). This indicates that the intercalated proteins stabilize the crystal structure to a

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Figure 5. (A) SEM micrograph of SrCO3 crystals obtained by reacting SrCl2 with CO2 in solution without mediation of any proteins. (B,C) SEM micrographs of biogenic SrCO3 crystals after treatment with NaOCl solution and the same sample after calcination at 300 °C, respectively. The inset in image B shows a magnified view of a particular region of SrCO3 needles shown in the main figure. The scale bar in the inset of image B corresponds to 300 nm. (D) Time-dependent UV-vis spectra of the SrCO3 solution after treatment with NaOCl. Curve 1: as-prepared SrCO3 synthesized using Fusarium oxysporum without NaOCl treatment. Curves 2, 3, and 4: NaOCl-treated SrCO3 crystals after 1, 3, and 24 h of reaction, respectively. (E) SDS-PAGE data showing the proteins that were originally bound to the surface of the SrCO3 crystals after dissolution of the crystals by mild acidic treatment. The arrows highlighting the bands in lane 1 indicate the proteins bound to SrCO3 crystals and their approximate molecular weights. Lane 2 shows standard protein molecular weight markers with the corresponding molecular weights in kiloDaltons indicated by arrows (see the text for details).

significant extent and only upon their removal is complete loss in the crystal morphology observed. The important role played by the proteins secreted by Fusarium oxysporum in directing the strontianite needle morphology may be highlighted by means of a simple control experiment. In this experiment, Fusarium oxysporum was soaked in water for 3 days and the aqueous component was separated by filtration. The aqueous fraction containing proteins released by the fungus was taken with 10-3 M SrCl2, and CO2 was bubbled very slowly through this solution. The SrCO3 crystals formed in this manner were analyzed by SEM (Figure 4C,D). The SEM images of these strontianite crystals indicate that they are remarkably similar in overall morphology (porous and quasi-linear) to those obtained by the direct reaction of Sr2+ ions with Fusarium oxysporum (Figure 1), indicating that the proteins secreted by Fusarium oxysporum into solution are responsible for the strontianite morphology control and, more importantly, that the growth of the crystals is not likely to occur within specific reaction sites in the fungal biomass. To test this hypothesis, the fungal biomass during various stages of reaction with Sr2+ ions was analyzed by SEM and XRD, and at no stage did we find evidence of the presence of strontianite within the fungal biomass (data not shown). Clearly, the growth of strontianite occurs extracellularly. Having determined that crystal growth occurs in solution, the peculiar, branched nature of the crystallites that indicate radial growth from central primary crystals requires elaboration (Figure 1A,B). As briefly mentioned earlier, reaction of Sr2+ ions with proteins secreted by the fungus resulted in the interesting morphology control and assembly. It is

possible that these proteins bind to specific crystallographic faces of the nucleating strontianite crystals, thereby inhibiting their growth along specific directions. Such branched structures may also occur due to carbonate ion diffusivity variation.20 These radial structures could also be due to isotropic growth in solution modulated by proteins secreted by the fungus; free-growing crystals have been observed to commonly form sheaves or radially assembled structures. A more or less uniform supply of ions to the growing surface leads to preferred growth of crystals. The exact mechanism leading to the shape control of the SrCO3 crystals observed in this study is yet to be fully elucidated for this microorganism. As a first step in this direction, we have analyzed the major proteins bound to the surface of the biogenic SrCO3 crystals by mild acetic acid treatment of the purified biogenic SrCO3 crystals. Mild acid treatment leads to the dissolution of SrCO3 crystals leaving behind the crystal-bound proteins in solution. Preliminary gel electrophoresis measurements of the proteins detached by acid treatment (Figure 5E) indicate two prominent protein bands having molecular weights of ∼33 and 50 kDa. We believe that either of these two proteins most likely contributed in modulating the crystal morphology of SrCO3, while contribution of other minor proteins in the crystal growth cannot be ruled out. Future investigations are in progress to understand the amino acid sequence of this protein, the nature of the interaction of this protein with the strontianite surface, the possibility of epitaxy, and so forth. (20) (a) McGrath, K. M. Adv. Mater. 2001, 13, 989. (b) Dickinson, S. R.; McGrath, K. M. J. Mater. Chem. 2003, 13, 928.

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To conclude, the total biological synthesis of SrCO3 crystals of needlelike morphology by challenging the fungus Fusarium oxysporum with aqueous Sr2+ ions has been described. This study is different from other biomineralization strategies in that the source of carbonate ions is the microorganisms themselves. Proteins secreted by the fungus play a very important role in directing the morphology and assembly of the strontianite crystals grown in solution. Our finding that minerals of such complex morphology can be grown by a completely biological process using microorganisms that are not normally (if ever) exposed to metal ions is exciting, with

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important implications in crystal engineering and associated applications. Acknowledgment. D.R. and A.S. thank the Department of Science and Technology (DST), Government of India, and the Council of Scientific and Industrial Research (CSIR), Government of India, for research fellowships. We acknowledge the SEM/EDAX and XRD facilities of the Center for Materials Characterization, NCL Pune. We thank Mr. Vipul Bansal and Mr. Atul Bharde for assistance with the gel electrophoresis measurements. LA049244D