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Heavy-Metal Remediation by a Fungus as a Means of Production of Lead and Cadmium Carbonate Crystals Ambarish Sanyal,† Debabrata Rautaray,† Vipul Bansal,† Absar Ahmad,‡ and Murali Sastry*,† Nanoscience Group, Materials Chemistry and Biochemical Sciences Division, National Chemical Laboratory, Pune - 411 008, India Received November 22, 2004. In Final Form: May 18, 2005 We show here that reaction of the fungus, Fusarium oxysporum, with the aqueous heavy-metal ions Pb2+ and Cd2+ results in the one-step formation of the corresponding metal carbonates. The metal carbonates are formed by reaction of the heavy-metal ions with CO2 produced by the fungus during metabolism and thus provide a completely biological method for production of crystals of metal carbonates. The PbCO3 and CdCO3 crystals thus produced have interesting morphologies that are shown to arise because of interaction of the growing crystals with specific proteins secreted by the fungus during reaction. An additional advantage of this approach is that the reaction leads to detoxification of the aqueous solution and could have immense potential for bioremediation of heavy metals. Under conditions of this study, the metal ions are not toxic to the fungus, which readily grows after exposure to the metal ions.

Introduction Heavy-metal pollution represents an important environmental problem because of the toxic effects of metals, and their accumulation throughout the food chain leads to serious ecological and health problems.1 It is well-known that heavy metals can be extremely toxic as they damage nerves, liver, and bones and also block functional groups of vital enzymes. The toxicity of cadmium and lead present in drinking water has been well-known for a long time. Cadmium and lead can affect all types of body cells; they are absorbed in the body slowly but since their rate of excretion is extremely slow, the effects caused by these two well-known heavy-metal ions are extremely severe. Many technologies such as precipitation, coprecipitation, electrodeposition and electrocoagulation, membrane separation, solvent exchange, adsorption, and biosorption are in practice for the removal of these toxic metal ions from industrial effluents.2,3 Of these techniques, bioremediation of heavy metals is of much topical interest. There have been reports of biosorption of heavy metals such as Pb and Cd using marine organisms (Sargassam sp. is one example).4 Biosorption has also been accomplished by biosorbents such as bacteria and brown algal biomass5 which have been very effective in the removal of Pb2+, Cu2+, Cd2+, and Zn2+ ions from an aqueous solution. It has been recently demonstrated in this laboratory that metal carbonate crystals of interesting morphology may be obtained by the reaction of metal ions with fungi.6-8 * Author to whom correspondence should be addressed. E-mail: [email protected]. † Nanoscience Group, Materials Chemistry. ‡ Biochemical Sciences Division. (1) Malik, A. Environ. Int. 2004, 30, 261. (2) Meunier, N.; Laroulandie, J.; Blais, J. F.; Tyagi, R. D. Bioresour. Technol. 2003, 90, 255-263. (3) Klimmek, S.; Stan, H. J. Environ. Sci. Technol. 2001, 35, 4283. (4) Sheng, X. P.; Ting, P.-Y.; Chen, P. J.; Hong, L. J. Colloid Interface Sci. 2004, 275, 131. (5) Davis, T. A.; Volesky, B.; Mucci, A. Water Res. 2003, 37, 4311. (6) Rautaray, D.; Ahmad, A.; Sastry, M. J. Am. Chem. Soc. 2003, 125, 14656. (7) Rautaray, D.; Ahmad, A.; Sastry, M. J. Mater. Chem. 2004, 14, 2333.

We have shown that CO2 released by fungi and actinomycetes during their metabolism may be used to form crystals of CaCO3,6 BaCO3,7 and SrCO38 thereby leading to a completely biological method for the synthesis of these important biominerals. The formation of metal carbonates is of considerable interest in biological and materials sciences. In particular, the chemical and biological precipitation of CaCO3 polymorphs have been studied in great detail.9 The popular chemical methods for the synthesis of CaCO3 include crystallization at Langmuir monolayers,10 on self-assembled monolayers (SAMs),11 within lipid bilayer stacks,12 on functionalized polymer surfaces,13 in vesicles,14 in block copolymers,15 and in biomacromolecules extracted from organisms.16 However, fewer investigations have dealt with other alkaline earth carbonates.17-19 Very recently Yu et al. have shown that complex higher order superstructures of various metal carbonate minerals such as CaCO3, BaCO3, CdCO3, MnCO3, and PbCO3 can be obtained by the influence of (8) Rautaray, D.; Sanyal, A.; Adyanthaya, S. D.; Ahmad, A.; Sastry, M. Langmuir 2004, 20, 6827. (9) (a) Kuther, J.; Nelles, G.; Seshadri, R.; Schaub, M.; Butt, H.-J.; Tremel, W. Chem. Eur. J. 1998, 4, 1834. CaCO3: (b) Aizenberg, J.; Black, A. J.; Whitesides, G. M. J. Am. Chem. Soc. 1999, 121, 4500. (c) Travaille, A. M.; Donners, J. J. J. M.; Gerritsen, J. W.; Sommerdijk, N. A. J. M.; Nolte, R. J. M.; Kempen, H. V. Adv. Mater. 2002, 14, 492-495. (10) (a) Damle, C.; Kumar, A.; Bhagwat, M.; Sainkar, S. R.; Sastry, M. Langmuir 2002, 18, 6075. (b) Rautaray, D.; Sainkar, S. R.; Sastry, M. Chem. Mater. 2003, 15, 2809. (11) (a) Falini, G.; Gazzano, M.; Ripamonti, A. Adv. Mater. 1994, 6, 46. (b) Donners, J. J. J. M.; Nolte, R. J. M.; Sommerdijk, N. A. J. J. Am. Chem. Soc. 2002, 124, 9700-9701. (12) Walsh, D.; Mann, S. Nature 1995, 377, 320. (13) Qi, L.; Li, J.; Ma, J. Adv. Mater. 2002, 14, 300. (14) (a) Sondi, I.; Matijevic, E. Chem. Mater. 2003, 15, 1322. (b) Grajeda, J. P. R.; Zuniga, D. J.; Batina, N.; Salazar, M. S.; Moreno, A. J. Cryst. Growth 2002, 234, 227. (c) Qi, L.; Ma, J.; Cheng, H.; Zhao, Z. J. Phys. Chem. B 1997, 101, 3460. (15) Yu, S.-H.; Colfen, H.; Antonietti, M. J. Phys. Chem. B 2003, 107, 7396. (16) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67. (17) Yu, S.-H.; Colfen, H.; Xu, A. W.; Dong, W. Cryst. Growth Des. 2004, 4, 33. (18) Refat, M. S.; Teleb, S. M.; Sadeek, S. A. Spectrochim. Acta, Part A 2004, 60, 2803. (19) Couriol, C.; Amrane, A.; Prigent, Y. J. Biosci. Bioeng. 2001, 91, 570.

10.1021/la047132g CCC: $30.25 © 2005 American Chemical Society Published on Web 06/30/2005

Heavy-Metal Remediation by a Fungus

double hydrophilic block copolymer (DHBCs) with different functionalities such as carboxyl and partially phosphonated and phosphorylated groups.17 Metal carbonates such as BaCO3, SrCO3, CoCO3, and PbCO3 have been synthesized in the presence of polyelectrolytes,17 urea,14a and silica matrix14b and in reverse micelles.14c As our fungus-based biological method for the synthesis of biominerals evolved,6-8 it became clear to us that metal carbonate formation by fungi could be a potentially exciting mechanism for the removal of toxic heavy-metal ions from aqueous solutions. In this paper, we present details of our investigation into the reaction of aqueous Pb2+ and Cd2+ ions with the fungus Fusarium oxysporum and into the formation of the corresponding metal carbonates. The utility of this approach is underlined by the fact that these metal ions are not toxic to the fungus and it continues to regenerate after Pb2+ and Cd2+ ion exposure. This study illustrates beautifully an underlying unifying feature of our studies into the reaction of metal ions with eukaryotic organisms such as fungi:20 the byproducts from the process of metal ion detoxification are often interesting nanomaterials/biominerals and show enormous potential for development. Presented below are details of the investigation. Experimental Details A plant pathogenic fungus, Fusarium oxysporum, was isolated from a plant, Rosa sp., that was infected by a natural biological attack of this fungus. Thereafter, the fungus isolated was maintained on potato-dextrose-agar (PDA) slants. Stock cultures were 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 preparation of the PbCO3 and CdCO3 crystals, the fungus was grown in 500-mL Erlenmeyer flasks containing 100 mL MGYP medium, which is composed of malt extract (0.3%), glucose (1%), yeast extract (0.3%), and peptone (0.5%). After adjusting the pH of the medium 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 each of 10-3 M Pb(NO3)2 and CdCl2 solutions in 500-mL Erlenmeyer flasks. The flasks were then plugged with cotton and put into a shaker at 27 °C (200 rpm), and the reaction was carried out for a period of 24 h. Thereafter, the biomass was separated from the filtrate and both the fungal biomass and the filtrate were subjected to different chemical analyses as mentioned below to investigate the biotransformation of the electrolyte salts into the respective carbonates. The residue after 1 day of reaction was washed thoroughly several times using copious amounts of double-distilled water, and the washed biomass was cast in the form of films onto Si(111) substrate for further analysis. The filtrate was subjected to centrifugation at 10000 rpm, and the pellets obtained were washed repeatedly (20) (a) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Ramani, R.; Pasricha, R.; Ajaykumar, P. V.; Alam, M.; Sastry, M.; Kumar, R. Angew. Chem., Int. Ed. 2001, 40, 3585. (b) Ahmad, A.; Mukherjee, P.; Mandal, D.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. J. Am. Chem. Soc. 2002, 124, 12108. (c) Mukherjee, P.; Senapati, S.; Mandal, D.; Ahmad, A.; Khan, M. I.; Kumar, R.; Sastry, M. ChemBioChem 2002, 3, 461. (d) Ahmad, A.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. Langmuir 2003, 19, 3550. (e) Ahmad, A.; Senapati, S.; Khan, M. I.; Kumar, R.; Ramani, R.; Srinivas, V.; Sastry, M. Nanotechnology 2003, 14, 824. (f) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parischa, R.; Ajaykumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Nano. Lett. 2001, 1, 515. (g) Sastry, M.; Ahmad, A.; Khan, M. I.; Kumar, R. In Nanobiotechnology; Niemeyer, C. M., Mirkin, C. A., Eds.; Wiley-VCH: Weinheim, Germany, 2004; p 126.

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Figure 1. Pictures showing growth of fungus Fusarium oxysporum after exposure to cadmium (plate to the left) and lead ions for a period of 10 days (plate to the right). with copious amounts of double-distilled water. The purified pellets were solution-cast in the form of films onto different solid supports for further analysis. The biological PbCO3 and CdCO3 crystals thus obtained were analyzed by scanning electron microscopy (SEM), energy-dispersive analysis of X-rays (EDAX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). For SEM analysis, drop-coated films of the biological metal carbonate crystals were made on Si(111) substrates. SEM and EDAX measurements were performed on a Leica Stereoscan-440 instrument equipped with a Phoenix EDAX attachment. XRD measurements of drop-coated films of the biological CdCO3 and PbCO3 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 biological crystal powders taken in KBr pellets were carried out on a Perkin-Elmer Spectrum One instrument at a resolution of 4 cm-1. To determine the nature of proteins responsible for the morphology variation of PbCO3 and CdCO3 crystals, the extracellular proteins secreted by the fungus in the filtrate obtained in the aforesaid experiment in the absence of lead and cadmium ions were salted out overnight at 4 °C by ammonium sulfate precipitation followed by centrifugation. The proteins thus obtained were dissolved in a minimal volume of deionized water and were dialyzed (using 12 kDa cutoff dialysis membrane). The dialyzed protein fraction containing a mixture of proteins was further purified by ion-exchange columns. The various fractions obtained from CM-Sephadex column were checked for their activity in crystal morphology control by reacting them separately with CdCl2 and Pb(NO3)2 aqueous solutions during bubbling of CO2 to yield the respective metal carbonates. The protein fraction responsible for the control of CdCO3 crystal morphology was analyzed by 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) carried out at pH 8.2 according to the procedure published by Laemmli.21 In a separate experiment, a higher amount of the fungal biomass (40 g weight of the mycelia) was added to the same amount of CdCl2, and following the procedures outlined above, samples of CdCO3 crystals were drop-cast onto Si(111) wafers for further microscopic characterization. To test whether the lead and cadmium ions are toxic to the fungus or not, the fungus was extracted from the metal ion-fungus reaction medium after 1 day of reaction and was grown further in culture media. The fungal growth was monitored for a period of 10 days.

Results and Discussion As mentioned above, heavy-metal ions such as lead and cadmium are considered to be toxic to all types of cells, and hence it is important to establish whether the fungus Fusarium oxysporum survives the exposure to the Pb2+ and Cd2+ ions. To answer this crucial question, the fungus from the reaction medium containing lead and cadmium ions after 1 day of reaction was separately washed and plated in a Petri plate containing MGYP medium. Pictures of the fungus after exposure to cadmium (Petri plate to (21) Laemmli, U. K. Nature 1970, 227, 680.

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Figure 4. XRD patterns recorded from PbCO3 (curve 1) and CdCO3 (curve 2) crystals obtained by the reaction of aqueous Pb2+ ions and Cd2+ ions with the fungus Fusarium oxysporum, respectively. Figure 2. (A-D) SEM micrographs at different magnifications of PbCO3 crystals formed after 1 day of reaction of aqueous Pb2+ ions with the fungus Fusarium oxysporum.

Figure 5. (A and B) SEM micrograph at different magnifications of CdCO3 crystals obtained after reaction of CdCl2 with the fungus Fusarium oxysporum.

Figure 3. (A) Spot-profile EDAX spectra recorded from films of PbCO3 (curve 1) and CdCO3 (curve 2) crystals synthesized using the fungus Fusarium oxysporum. (B) FTIR spectra showing the amide I and II bands recorded from PbCO3 (curve 1) and CdCO3 (curve 2) crystals obtained by the reaction of aqueous Pb2+ ions and Cd2+ ions with the fungus Fusarium oxysporum, respectively.

the left) and lead ions (Petri plate to the right) and growth in the MGYP medium for a period of 10 days are shown in Figure 1. It can be clearly seen that the fungus survives the exposure to the Pb2+ and Cd2+ ions and grows quite readily in the nutrient medium. Figure 2 shows representative SEM images recorded from solution-cast films of the aqueous Pb(NO3)2 solution after exposure to Fusarium oxysporum for 1 day. It is observed from the SEM image that the sample mainly consists of large, highly irregular structures of size greater than 10 µm (Figure 2A). At progressively higher magnifications (Figure 2B-D), these structures are seen to be aggregates of smaller particles that are uniform in size and highly spherical in morphology. The size of the individual spherical crystallites in the large assemblies ranges between 120 and 200 nm. It would be instructive to understand the chemical composition of these spherical crystallites synthesized using Fusarium oxysporum. This is conveniently done by spot-profile EDAX measurement of one of the spherical crystallites grown using the fungus (curve 1, Figure 3A). In addition to the expected Pb, C, and O signals from PbCO3, we observe the presence of N and S signals in the PbCO3 crystals. (The Si signal is from the substrate.) The N and S signals in the EDAX spectrum indicate the presence of proteins within and on the

spherical PbCO3 crystallites. That these signals are likely to be due to proteins secreted by the fungus is supported by the FTIR measurement of the PbCO3 crystals (Figure 3B, curve 1) which clearly shows the presence of amide I and II bands at 1542 and 1633 cm-1, respectively.22 This observation indicates that the PbCO3 crystals in the spherical morphology are present with proteins that are possibly occluded into the crystals or are bound to the surface of the crystals thereby acting as a sort of glue holding the spherical crystallites together in the superstructure. That the proteins do play an important role in directing the morphology of the PbCO3 crystals can also be attributed to the fact that the spherical morphology obtained here is very different from that generally observed for cerrusite (PbCO3) grown in solution by Antonietti et al. in the presence of DHBCs. They have observed the formation of disklike plates (in the presence of PEG-b-PMAA) and flat hexagonal plates of PbCO3 (in the presence of PEG-b-[(2-[4-dihydroxyphosphoryl]-2oxabutyl) acrylate ethyl ester]), whereas poorly defined starlike PbCO3 platelets were obtained in the absence of block copolymers.15 XRD analysis of the biological PbCO3 crystals formed by the reaction of aqueous Pb2+ ions with Fusarium oxysporum for 1 day was performed and the diffraction pattern obtained is shown as curve 1 in Figure 4. Several Bragg reflections are identified and have been indexed with reference to the unit cell of the PbCO3 structure (a ) 5.178 Å, b ) 8.515 Å, c ) 6.146 Å; space group Pmcn).23 The broad Bragg reflections in the XRD spectrum indicate (22) (a) Dong, A.; Huang, P.; Caughey, W. S. Biochemistry 1992, 31, 182-189. (b) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66-76. (c) Kumar, C. V.; McLendon, G. L. Chem. Mater. 1997, 9, 863-870. (23) The XRD patterns were indexed with reference to the unit cell of the PbCO3 structure (a ) 5.178 Å, b ) 8.515 Å, c ) 6.146 Å; space group Pmcn, ASTM chart card no. 47-1734) and CdCO3 structure (a ) 4.929 Å, c ) 16.30 Å; space group R3c, ASTM chart card no. 42-1342) from ASTM chart.

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Figure 6. SEM micrographs of PbCO3 (A) and CdCO3 (B) crystals grown using protein fractions 1 and 2, respectively, extracted from the fungus Fusarium oxysporum. (C) SEM micrograph of PbCO3 crystals obtained by the reaction of Pb(NO3)2 with the protein fraction 2. Inset in C shows a higher magnification SEM image of one of the crystallites shown in the main figure. The scale bar in the inset of C corresponds to 2 µm. (D) SDS-PAGE analysis of the protein fraction responsible for the needlelike morphology of CdCO3 crystals (lane 2). Lane 1 shows standard protein molecular weight markers with the corresponding molecular weights in kDa. The arrow highlighting the band in lane 2 indicates the protein responsible for the morphology control of CdCO3 crystals.

that the PbCO3 crystals are rather small and are consistent with the SEM analysis that revealed the crystallites to be of 120-200 nm dimensions (Figure 2). Figure 5A and B shows representative SEM images recorded from extracellularly grown CdCO3 crystals after reaction of aqueous Cd2+ ions with the fungus Fusarium oxysporum for 1 day. The CdCO3 crystals exhibit a uniform morphology that is completely different from that reported earlier for crystals grown in solution and in the presence of DHBCs.15 The solution growth of CdCO3 crystals by simple CO2 infusion exhibits rhombohedral shapes which are structurally homologous to calcite whereas the presence of DHBCs as a crystal modifier resulted in the formation of dumbbell-sphere-shaped CdCO3 crystals.15 A large number of very uniform needle-shaped crystals of CdCO3 are observed in the SEM micrograph (Figure 5A). Viewed at higher magnification (Figure 5B), the CdCO3 needles can be seen in greater detail. The length of the needles varies from 2 to 6 µm while the width ranges from 100 to 300 nm. The EDAX spectrum recorded from the biological CdCO3 crystals is shown in Figure 3A, curve 2. In addition to Cd, C, and O signals, strong N and S signals are observed in this case as well indicating the presence of proteins within and on the needlelike crystals. The presence of proteins in the biological CdCO3 crystals is also indicated in the FTIR spectrum from this sample (curve 2, Figure 3B) where prominent amide I and II bands from the proteins are seen. The XRD pattern recorded from the CdCO3 needlelike crystals is shown as curve 2 in Figure 4. Several Bragg reflections are identified and have been indexed with reference to the unit cell of the CdCO3 structure (a ) 4.929 Å, c ) 16.30 Å; space group R3c).23 In the above experiments, the important facts stand out. First, the metal ions are not toxic to the fungus, at least up to 10-2 M concentration of the metal ions. Second, the lead and cadmium carbonate crystals are formed by reaction of the corresponding metal ions with CO2 produced by the fungus during metabolism; this is an important point of deviation from earlier biomimetic approaches to metal carbonate growth where an external

source of CO2 was used.6-8 The fact that the fungus also acts as a source of CO2 makes this a truly biological method for the synthesis of minerals and is thus not merely biomimetic. That a noncalcareous microorganism such as a fungus should be capable of crystal growth and engineering at a high level of sophistication opens up the exciting possibility that other microorganisms when challenged with metal ions may lead to similar exciting results. Third, the morphology of the PbCO3 and CdCO3 crystals is very different from that reported in other studies15 suggesting that the proteins secreted by the fungus during mineralization play a crucial role in directing the morphology. To determine the nature of proteins responsible for the crystal morphology control, the extracellular proteins secreted by the fungus in the filtrate obtained in the aforesaid experiment in the absence of lead and cadmium ions were salted out overnight at 4 °C using ammonium sulfate precipitation followed by centrifugation. The proteins obtained thereafter were dissolved in minimal volume of deionized water and were dialyzed (using 12 kDa cutoff dialysis membrane). The dialyzed protein fraction containing a mixture of proteins was further purified by ion-exchange columns. The various fractions obtained from CM-Sephadex column were checked for their activity. Out of various fractions obtained, only two fractions showed positive results in modulating the morphology of the lead and cadmium carbonate crystals. Out of these two protein fractions, one was responsible for the shape control of PbCO3 (protein fraction 1) while the second protein fraction (protein fraction 2) was responsible for shape control of CdCO3 crystals. These two protein fractions are anionic in nature suggesting that the interaction of the proteins with the lead and cadmium carbonate crystals could occur electrostatically with the metal ions exposed at specific crystallographic faces. SEM images of PbCO3 and CdCO3 crystals grown in the presence of protein fractions 1 and 2, respectively, after bubbling CO2 are shown in Figure 6A and B, respectively. The PbCO3 and CdCO3 crystals obtained in

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these control experiments are very similar to those obtained when the reaction of the lead and cadmium metal ions was carried out directly in the presence of the fungus Fusarium oxysporum indicating that these specific proteins secreted by the fungus are responsible for the shape control of the respective metal carbonate crystals. Figure 6C shows the PbCO3 crystals grown in the presence of protein fraction 2; clearly the prismatic structure of the large PbCO3 crystals grown in this control experiment is completely different from the spherical morphology observed in the PbCO3 crystals grown by reaction of lead ions directly with the fungus (Figure 2). To identify better the individual proteins in each of the fractions (protein fractions 1 and 2) leading to morphology control, gel electrophoresis analysis of the two protein fractions was carried out. While the proteins in protein fraction 1 could not be easily separated by SDS-PAGE, meaningful results were obtained for protein fraction 2 that is responsible for the morphology control of CdCO3 crystals (lane 2, SDSPAGE data shown in Figure 6D). A comparison of the protein bands in this fraction with the markers (Figure 6D, lane 1) indicates that the CdCO3 shape-modulating protein has a molecular weight of ∼68 kDa. We caution that though this protein contributes to a major extent in controlling the CdCO3 crystal morphology, contributions of other minor proteins in the crude extract cannot be ruled out. Future investigations are in progress to understand the sequence of this protein and the nature of interaction of this protein (and the protein in fraction 1) with different crystallographic faces of lead and cadmium carbonate crystals. It would be quite informative to understand the nature of the morphology change of the metal carbonates with change in the amount of the biomass taken. A higher amount of biomass taken would result in a larger amount of protein secretion as well as CO2 production which might lead to a different morphology of the crystals altogether. Figure S1A and B in the Supporting Information shows representative SEM images at different magnifications of CdCO3 grown using 40 g of fungal biomass while retaining the concentration of the Cd2+ salt used in the earlier experiment. There appears to be no change in the overall spindle-shaped morphology of the CdCO3 crystals (Figure S1B). Although the crystal density is reasonably high (Figure S1A), a large amount of protein is observed in the sample that obscures the crystals unless imaged at much higher magnification (Figure S1B). That a large excess of protein is present in the sample prepared with a higher amount of fungal biomass is confirmed from the spot profile EDAX spectrum (Figure S1C) taken from the

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edge of one of the spindle-shaped crystals (marked with an arrow in Figure S1B) which shows an intense signal of S along with that of Cd, C, N, and O. Comparison of this EDAX spectrum with that recorded from CdCO3 crystals grown with a smaller amount of fungal biomass (Figure 3A, curve 2) clearly shows that the S signal is significantly higher in the former case; the strong S signal can only come from a large excess of protein. We term this mineral formation process “total biosynthesis” since the source of carbonate ions is the microorganism itself and is thus at variance with other biomimetic methods wherein an external source of CO2 was used to grow the minerals. In the reaction of these metal ions with the microorganisms, we have shown that the morphology of the minerals grown in solution is directed by specific proteins secreted by the microorganism and is independent to a large extent of the concentration of the proteins secreted. Our finding that minerals of such interesting morphology can be grown by a completely biological process using microorganisms that are not normally (if ever) exposed to such metal ions is exciting with important implications in crystal engineering and associated applications. From the point of view of scale-up of production of minerals, the use of a renewable source of CO2 and crystal-modifying proteins from microorganisms is obvious. In conclusion, we have shown that heavy-metal ions such as Pb2+ and Cd2+ can be removed from aqueous solutions by exposure to the fungus Fusarium oxysporum. The detoxification occurs by a process of metal carbonate formation. The metal carbonates are useful byproducts and possess morphologies that are directed by proteins secreted by the fungus. The metal ions are not toxic to the fungus under the experimental conditions of this study and thus highlight the potential of this process in bioremediation and large-scale mineral growth. Acknowledgment. A.S., V.B., and D.R. thank the Council of Scientific and Industrial Research (CSIR) and Department of Science and Technology (DST), Government of India, for research fellowships. Supporting Information Available: SEM micrographs of CdCO3 crystals at different magnifications (Figure S1A and B) grown using 40 g wet weight of the harvested mycelial mass of the fungus Fusarium oxysporum after reaction with 10-3 M aqueous CdCl2. Spot-profile EDAX spectrum (Figure S1C) recorded from one of the edges of biologically grown CdCO3 crystals synthesized using the fungus Fusarium oxysporum. This information is available free of charge via the Internet at http://pubs.acs.org. LA047132G