Synthesis of Platinum Nanoparticles by Reaction of Filamentous

Interaction of cyanobacteria (Plectonema boryanum UTEX 485) with aqueous platinum(IV)−chloride (PtCl4°) has been investigated at 25−100 °C for u...
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Synthesis of Platinum Nanoparticles by Reaction of Filamentous Cyanobacteria with Platinum(IV)-Chloride Complex Maggy F. Lengke,* Michael E. Fleet, and Gordon Southam Department of Earth Sciences, UniVersity of Western Ontario, London, Ontario, N6A 5B7, Canada ReceiVed March 31, 2006. In Final Form: June 13, 2006 Interaction of cyanobacteria (Plectonema boryanum UTEX 485) with aqueous platinum(IV)-chloride (PtCl4°) has been investigated at 25-100 °C for up to 28 days, and 180 °C for 1 day. The addition of PtCl4° to the cyanobacteria culture initially promoted the precipitation of Pt(II)-organic material as amorphous spherical nanoparticles (e0.3 µm) in solutions and dispersed nanoparticles within bacterial cells. The spherical Pt(II)-organic nanoparticles were connected into long beadlike chains by a continuous coating of organic material derived from the cyanobacterial cells, and aged to nanoparticles of crystalline platinum metal with increase in temperature and reaction time. The stepwise reduction for the formation of platinum nanoparticles in the presence of cyanobacteria was deduced to be Pt(IV) [PtCl4°] f Pt(II) [Pt(II)-organics] f Pt(0). Spherical platinum-bearing nanoparticles were not present in abiotic PtCl4° experiments conducted under similar conditions and duration.

Introduction Nanoparticles of free metals have been extensively researched because of their unique physical properties, chemical reactivity, and potential applications in catalysis,1,2 biosensing,3,4 biological labeling,5 electronics,6 optical devices,6 and controlled drug delivery.7 In particular, nanoparticles of platinum-group metals are widely used in industrial and automotive catalysts because they have high corrosion resistance and are stable to oxidation at high temperatures.8 The automobile catalysts in North America employ a combination of platinum, palladium, and rhodium to oxidize carbon monoxide and hydrocarbons to carbon dioxide and water, and to reduce nitrogen oxides to nitrogen gas. Currently, there are no acceptable substitutes for the platinum-group metals for the automobile catalyst application. The synthesis of platinum nanoparticles of different sizes, ranging from one to tens of nanometers, and shapes, including cubic, tetrahedral, hexagonal, polyhedral, irregular prismatic, spherical, icosahedral, and cuboctahedral, has been conducted in aqueous solutions.9-11 The usual synthetic route to prepare platinum nanoparticles involves the reduction of a platinum salt (usually a halide) in solution by citrate, hydrogen, or sodium borohydride.12-14 To maintain the formation of stable platinum * Corresponding author. E-mail: [email protected]. (1) Lewis, L. N. Chem. ReV. 1993, 93, 2693-2730. (2) Moreno-Manas, M.; Pleixats, R. Acc. Chem. Res. 2003, 36, 638-643. (3) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (4) Han, M.; Gao, X.; Su, J. Z.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. (5) Nicewarner-Pena, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pena, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137-141. (6) Kamat, P. V. J. Phys. Chem. 2002, B106, 7729-7744. (7) Rao, C. N. R.; Cheetham, A. K. J. Mater. Chem. 2001, 11, 2887-2894. (8) Lloys, S. M.; Lave, L. B.; Matthews, H. S. EnViron. Sci. Technol. 2005, 39, 1384-1392. (9) Ahmadi, T. S.; Wang, Z. L.; Green, T. C.; Henglein, A.; El-Sayed, M. A. Science 1996, 272, 1924-1926. (10) Sarathy, K. V.; Raina, G.; Kulkarni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1997, 101, 9876-9880. (11) Chen, S.; Kimura, K. J. Phys. Chem. B 2001, 105, 5397-5403. (12) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55. (13) Furlong, D. N.; Launikons, A.; Sasse, W. H. F. J. Chem. Soc., Faraday Trans. 1984, 80, 571-588. (14) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. Chem. Commun. 1994, 801-802.

nanoparticles, stabilizers such as polymers, polyelectrolytes, and thiol groups have been used.9,15 Other methods, including photochemical, pulse-radiolytical, and thermal methods, have also been applied to produce platinum nanoparticles.13 Many studies have been conducted to synthesize platinum nanoparticles using chemical methods.9,13,16-18 However, the formation of platinum nanoparticles by biological methods including living and nonliving organisms has not been investigated to date. Biological processes that lead to the formation of nanoscale inorganic materials are appealing as possible environmentally friendly nanofactories. In this study, we investigate the synthesis of platinum nanoparticles by reacting platinum(IV)-chloride (PtCl4°) complex with a microorganism, the cyanobacterium Plectonema boryanum (strain UTEX 485). Cyanobacteria, known as blue-green algae, form one of the largest and most important groups of bacteria on earth and are commonly found in natural environments. Various species of cyanobacteria and algae have been known to adsorb and take up heavy metal ions.19-22 The carboxyl groups on dead algae (algae biomass) apparently bind to various metal ions,23 while intracellular polyphosphates as well as extracellular polysaccharide appear to participate in metal sequestration by live algae.24,25 Although previous studies on metal removal by cyanobacteria and algae have been conducted for bioremediation and biorecovery purposes, this approach could also be extended for the development and application of nanoparticles. Therefore, the goals of this study were to investigate the formation of platinum nanoparticles by the reaction of PtCl4° with cyanobacteria and (15) Duff, D. G.; Edward, P. P. J. Phys. Chem. 1995, 99, 15934-15944. (16) Miyazaki, A.; Nakano, Y. Langmuir 2000, 16, 7109-7111. (17) Petroski, J. M.; Wang, Z. L.; Green, T. C.; El-Sayed, M. A. J. Phys. Chem. B 1998, 102, 3316-3320. (18) Petroski, J. M.; Green, T. C.; El-Sayed, M. A. J. Phys. Chem. A 2001, 105, 5542-5547. (19) Gadd, G. M. Experientia 1990, 46, 834-840 (20) Kuyucak, N.; Volesky, B. Biosorption of HeaVy Metals; Volesky, B., Ed.; CRC Press: Boca Raton, FL, 1990; pp 173-198. (21) Bender, J. Water EnViron. Res. 1994, 66, 679-683. (22) Hameed, A.; Hasnain, S. Chin. J. Oceanol. Limnol. 2005, 23, 433-441. (23) Gardea-Torresdey, J. L.; Becker-Hapak, J. M.; Hosea, J. M.; Darnell, D. W. EnViron. Sci. Technol. 1990, 19, 1372-1379. (24) Kaplan, D. D.; Christiaen, D.; Arad, S. M. Appl. EnViron. Microbiol. 1987, 12, 2953-2956. (25) Zhang, W.; Majimi, V. EnViron. Sci. Technol. 1994, 28, 1577-1581.

10.1021/la060873s CCC: $33.50 © 2006 American Chemical Society Published on Web 07/13/2006

Pt Nanoparticles Synthesized by Cyanobacteria

to understand the effect of complex organic/inorganic reactions on the shape and size of platinum nanoparticles. Experimental Section Cyanobacteria Cultures. The filamentous cyanobacterium, Plectonema boryanum, strain UTEX 485 (obtained from the culture collection at the University of Texas at Austin, Austin, Texas), was grown in batch cultures in BG-11 medium26 and buffered with 10 mM HEPES at a control temperature of 29 °C under ambient CO2 conditions. The pH of the medium was adjusted to pH 8.0 using 1 M NaOH solution. Before experimentation, the culture was transferred (20% [v/v] of inoculum) and grown to a stationary “growth” phase for approximately 6-8 weeks. It was then centrifuged and washed three times with distilled, deionized water to remove salts and trace metals from the medium. Cyanobacterial and Abiotic Experiments. The cyanobacterial experiments were conducted to examine the role of cyanobacteria in the synthesis of platinum nanoparticles from aqueous solutions of PtCl4° [PtCl4; Alfa Aesar Company, Ward Hill, Massachusetts]. To initiate the experiments, 5 mL of platinum solution (∼500 mg/L) was added to 5 mL of cyanobacteria culture (∼8 mg dry weight). The experiments were conducted at 25, 60, 80, and 100 °C for up to 28 days, and 180 °C for 1 day after the incubation period with platinum solution. The range of temperatures from 25 to 180 °C was chosen to investigate the changes in the morphology of platinum nanoparticles with temperature. The experiments were maintained in the dark because of the instability of platinum solution exposed to light. For experiments at 25, 60, 80, and 100 °C, reactants were contained in sealed borosilicate vials in temperature-regulated laboratory ovens (Blue M Electric Company and Fisher Isotemp Oven, respectively). For the 180 °C experiments, the mixtures were contained in an acid digestion bomb (Parr 4749) and heated in a box furnace (Thermolyne 1500). pH, Eh, total platinum, and cyanobacteria population were measured with time. All experiments were conducted in duplicate. Abiotic control experiments consisting of ∼500 mg/L of platinum were conducted using PtCl4° solution without the presence of cyanobacteria. Bacterial Viability and Total Bacterial Counts. The effects of PtCl4° on the cyanobacteria were monitored during the course of the cyanobacterial experiments by determining bacterial viability using the LIVE/DEAD BacLightTM bacterial viability kit (L-7007, Molecular Probes, Inc., Eugene, Oregon). When viewed by a fluorescence microscope, viable bacteria stained with the LIVE/ DEAD BacLightTM reagents appear green, whereas dead bacteria appear red. The total number of bacteria in the cultures was determined by the direct counting method using a Petroff-Hauser counting chamber and a phase contrast light microscope (Nikon Labophot microscope). Chemical Analyses. The pH and Eh were monitored using a Denver Instrument Basic pH/oxygen reduction potential (ORP)/ temperature meter. The pH electrode was calibrated using buffer solutions 4, 7, and 10 with analytical uncertainties in measurements of pH of (0.05 pH units. The Eh was measured using an ORP electrode and calibrated using ZoBell’s solution.27 Total platinum concentrations were measured over the course of the experiments with a Perkin-Elmer 3300-DV inductively coupled plasma optical emission spectrometer (ICP-OES). The uncertainty in measured platinum is e5%, with detection limits of 0.1 mg/L for platinum. Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM). Unstained whole sample mounts of cyanobacteria and platinum nanoparticles from the experiments were examined with a Phillips CM-10 transmission electron microscope (TEM) operated at 80 kV and a Phillips EM400T TEM with an energy-dispersive X-ray spectrometer (EDS) operated at 100 kV. The whole mounts were prepared by floating Formvar carbon-coated 200 mesh copper grids on a drop of culture for several minutes to (26) Rippka, R.; Deruelles, J.; Waterbury, J.; Herdman, M.; Stanier, R. Y. J. Gen. Microbiol. 1979, 111, 1-61. (27) Nordstrom, D. K. Geochim. Cosmochim. Acta 1977, 41, 1835-1841.

Langmuir, Vol. 22, No. 17, 2006 7319 allow the bacteria and any fine-grained particles to attach to the grid. The mounts were then washed with distilled, deionized water and allowed to air-dry. Selected-area electron diffraction (SAED) patterns of the precipitated solids were obtained by TEM from unstained whole mounts to ascertain the crystallinity of solids. The precipitated platinum and cyanobacteria cells were also examined using a LEO 1540 XB focus ion beam scanning electron microscope equipped with an energy dispersive spectrometer (FIBSEM-EDS). All samples for FIB-SEM-EDS were carbon-coated before analysis. X-ray Photoelectron Spectroscopy (XPS). The reaction products obtained from the cyanobacterial experiments at 25, 80, and 100 °C and the abiotic experiments at 100 °C were analyzed by XPS to investigate the oxidation state of platinum and sulfur. All samples were dried in the XPS vacuum chamber. A modified Surface Science Laboratories SSX-100 spectrometer, with a monochromatized Al KR X-ray source and an analytical chamber with a base pressure of 10-9 Torr, was used to collect XPS spectra. The spectrometer work function was adjusted to 71.20 ( 0.05 eV [with peak full width at half-maximum (fwhm) of 2.50 ( 0.05 eV] for the Pt 4f7/2 peak. The energy dispersion was set to give an energy difference of 857.5 ( 0.1 eV between the Cu 2p3/2 and Cu 3p1/2 lines of metallic Cu. Internal referencing of spectra was made to the C 1s peak of graphite-like C at 285.00 eV to compensate for the charging effects in the samples. Survey scans were recorded using a 600 µm spot size and a fixed pass energy of 160 eV, whereas narrow scan spectra were recorded using a 300 µm X-ray spot size and a fixed pass energy of 50 eV. Integrated photoelectron intensities were corrected for the Scofield cross-sections and the inelastic mean free path for kinetic energy. Spin-orbit doublet components for S 2p3/2 and S 2p1/2 are separated by 1.18 eV with a peak-area ratio of 2:1, and those for Pt 4f7/2 and Pt 4f5/2 are separated by 3.33 eV with a peak-area ratio of ∼1.3:1.28,29 Reference binding energies for Pt 4f and S 2p species were taken from Briggs and Seah28 and Knipe and Fleet,30 respectively.

Results Cyanobacterial and Abiotic Experiments. The results of cyanobacterial experiments using PtCl4° are shown in Figure 1. Upon addition of PtCl4° to the cyanobacterial culture, the pH and Eh values changed from ∼5 to 2.2-2.5 and from 0.45 to 0.750.83 V, respectively. All cyanobacteria were killed within several hours at all temperatures investigated (25-180 °C), probably because of the low pH and high temperatures. The optimal temperature for cyanobacteria growth is 29 °C; therefore, they would not survive the elevated temperatures presently investigated (60-180 °C). The soluble platinum concentrations significantly decreased within 7 days at 25-100 °C, and then slightly decreased by about ∼30 ppm after 7 days (Figure 1). At 180 °C, the soluble platinum was completely precipitated from solutions within 1 day (Figure 1). A significant increase in the precipitation of platinum occurred over the interval from 25 to 180 °C. There was no significant variation between duplicate experiments over a total of 68 individual experiments. The dark gray color of the reaction system was macroscopic evidence of platinum precipitation. The pH was slightly decreased about 0.3 pH units at 25-180 °C (Figure 1). Eh values generally decreased with time from 0.75 to ∼0.65-0.55 V for all cyanobacterial experiments. In abiotic experiments using PtCl4°, the total soluble platinum concentrations were relatively constant at 25 and 60 °C, but decreased with time at 80-180 °C (Figure 1). The precipitation of platinum was observed as a dark gray solid. Eh values showed (28) Briggs, D.; Seah, M. P. Practical Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, 2nd ed.; Wiley: Chichester, U. K., 1996. (29) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-ray Photoelectron Spectroscopy; Chastain, J., Ed.; Perkin-Elmer Corporation: Norwalk, CT, 1992. (30) Knipe, S. W.; Fleet, M. E. Can. Mineral. 1997, 35, 1485-1495.

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Figure 1. Variation of total soluble Pt, pH, and Eh with time for cyanobacterial and abiotic experiments with PtCl4° solution at 25-180 °C; C and A represent cyanobacterial and abiotic, respectively.

Figure 2. Micrographs of whole mounts of platinum particles cultured from cyanobacteria-PtCl4° at an incubation temperature of 25 °C and 28 days: (A) TEM of amorphous spherical platinumbearing nanoparticles; (B) SEM of spherical platinum-bearing particles; (C) TEM-SAED diffraction of amorphous platinumbearing nanoparticles; (D) TEM-EDS spectrum for panel A (the Cu signal is from the supporting grid). Scale bars in panels A and B are 0.5 µm.

no significant change between 25 and 80 °C; however, Eh decreased about 0.1-0.2 eV at 100-180 °C. TEM and SEM. TEM and SEM observations on products of cyanobacteria-PtCl4° experiments at 25-180 °C are presented in Figures 2-5. At 25 °C, the addition of PtCl4° to the cyanobacteria caused the precipitation of spherical platinumbearing nanoparticles with diameters ranging from 30 nm to 0.3 µm (Figure 2A,B). The nanoparticles were coated by organic materials from cyanobacteria. TEM-SAED of the particles in the 28-day experiment revealed that the spherical nanoparticles were amorphous to electron diffraction (Figure 2C), and TEM-

EDS showed the occurrence of platinum with sulfur, iron, and chloride (Figure 2D). Sulfur and iron were from the cyanobacteria cells, whereas chloride was trace of the platinum solution used (PtCl4°). At 60 and 80 °C, spherical nanoparticles of platinumorganics with diameters ranging from 30 nm to 0.2 µm were precipitated and connected into long beadlike chains by organic material (Figure 3). TEM-SAED of these particles revealed them to be amorphous to electron diffraction (data not shown), and TEM-EDS showed the occurrence of platinum with traces of sulfur and chloride (Figure 3E). At 100 °C, the long chains of platinum-bearing nanoparticles were still observed, but these particles were partly covered by very fine-grained platinum metal, formed by recrystallization (Figure 4A,B). TEM-SAED of the nanoparticles revealed powder ring diffraction patterns consistent with nanocrystalline platinum metal (Figure 4C). TEM-EDS showed the occurrence of platinum with sulfur, chloride, and iron (Figure 4D). At 180 °C, platinum nanoparticles were formed in a dendritic habit; that is, a branching “treelike” growth of crystals (Figure 5A,B). TEM-SAED of the nanoparticles revealed powder ring diffraction patterns consistent with nanocrystalline platinum metal (Figure 5C). TEM-EDS showed the occurrence of platinum with traces of sulfur and chloride (Figure 5D). The size of platinum nanoparticles was not studied systematically because the variation in size with temperature was much less than the variation within a single product. FIB-SEM-EDS of a longitudinally sectioned cyanobacterium reacted with PtCl4° at 25 °C for 28 days revealed that significant and fairly uniform amounts of platinum were deposited within the cell and were positively correlated with sulfur and chloride (Figure 6). In abiotic experiments, PtCl4° was stable in aqueous solution at 25-60 °C for 28 days, whereas nanoparticles and scattered platy nanocrystals of an unidentified platinum phase were precipitated at 80-180 °C (Figure 7A,B). TEM-SAED patterns of platinum nanoparticles were consistent with crystalline platinum metal (Figure 7D). TEM-EDS showed the occurrence of platinum with traces of chloride from the reagent used (Figure 7E).

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Figure 5. (A,B) TEM micrographs of whole mounts of nanoparticles of platinum metal assembled as dendrites in the cyanobacteriaPtCl4° systems at 180 °C and 1 day; (C) TEM-SAED diffraction powder-ring pattern consistent with nanoparticles of platinum metal; d-spacings of 0.224, 0.194, 0.137, and 0.117 nm correspond to reflections 111, 002, 022, and 113, respectively; (D) TEM-EDS spectrum for panel B. Scale bars in panels A and B are 0.5 and 0.1 µm, respectively. Figure 3. TEM micrographs of whole mounts of amorphous spherical nanoparticles of platinum, forming long beadlike chains in cyanobacteria-PtCl4° systems: (A) 60 °C and 14 days; (B,C) 80 °C and 21 days; (D) SEM micrograph of spherical platinum-bearing nanoparticles at 80 °C and 28 days; (E) TEM-EDS spectrum for panel B. Scale bars in panels A, B, C, and D are 0.25, 0.5, 0.1, and 1 µm, respectively.

Figure 4. (A,B) TEM micrographs of whole mounts of partially recrystallized spherical nanoparticles of platinum in cyanobacteriaPtCl4° systems at 100 °C and 28 days; (C) TEM-SAED diffraction powder-ring pattern consistent with very fine-grained platinum metal; d-spacings of 0.224, 0.194, 0.137, and 0.117 nm correspond to reflections 111, 002, 022, and 113, respectively; (D) TEM-EDS spectrum for panel B. Scale bars in panels A and B are 0.1 µm.

XPS. XPS spectra for reaction products of the cyanobacterial and abiotic experiments with PtCl4° solutions at 25-100 °C are presented in Figures 8 and 9, respectively. Semiquantitative

Figure 6. SEM micrographs of a longitudinal section of an individual cyanobacteria cell, and corresponding FIB-SEM-EDS elemental images for Pt, S, Cl, C, and O from cyanobacteria-PtCl4° experiments at 25 °C and 28 days. The bright region in the analyzed area, indicated by the arrow, was likely caused by a charging effect. Scale bars are 1 µm.

compositional analysis of the surface layers (∼15 Å depth) by broadscan XPS indicated Pt/S ratios of (1.3-3.3):1 and Pt/P ratios of (1.3-1.8):1 for the near-surface regions in cyanobacterial experiments. However, the analysis of the deeper near-surface region by TEM-EDS indicated Pt/S ratios close to 35.9:1 for a whole sample mount, whereas Pt/P ratios could not be determined because of the overlapping Pt and P peaks. Narrow region XPS spectra were acquired for Pt 4f, S 2p, and C 1s

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Figure 8. Narrow-region Pt 4f XPS spectra from the reaction products of cyanobacteria-PtCl4° experiments after 28 days: (A) 25 °C, showing subordinate Pt(IV) and dominant Pt(II); (B) 80 °C, showing Pt(II) (see Figure 3C,D); (C) 100 °C, showing broad peaks for Pt(II) and subordinate Pt(0). Figure 7. TEM micrographs of whole mounts of platinum precipitates in the abiotic system at 180 °C and 1 day: (A) euhedral crystals of unidentified platinum phase; (B) nanoparticles of platinum metal; (C) TEM-SAED diffraction pattern of panel A; (D) TEMSAED diffraction powder-ring pattern of panel B, consistent with nanoparticles of platinum metal; d-spacings of 0.224, 0.194, 0.137, 0.117, and 0.097 nm correspond to reflections 111, 002, 022, 113, and 004, respectively; (E) TEM-EDS for panel B. Scale bars in panels A and B are 0.1 µm.

spectral regions for the cyanobacterial experiments, but only the results for Pt are presented here (Figures 8 and 9). For the cyanobacterial experiments with the addition of PtCl4° at 25-100 °C, the spectra for the sedimented platinum particles were dominated by Pt(II) at ∼73.2-73.4 eV (with fwhm ∼ 1.53.0 eV). At 25 °C, the presence of a second component at ∼75.4 eV was assigned to Pt(IV), which was trace of the platinum reagent used (Figure 8), whereas, at 100 °C, minor peaks for Pt(0) were observed. Sulfur 2p spectra at 25-100 °C were dominated by sulfate, and minor peaks for reduced sulfur species were only observed at 80-100 °C. For abiotic experiments, the XPS spectra were dominated by a single peak for Pt° at 71.7 eV (and a spin-orbit doublet peak at +3.3 eV) with a fwhm of 1.1 eV, with a second component at 72.9 eV, which was assigned to Pt(II) (Figure 9). The Pt 4f7/2 peaks were shifted positively (+0.5 eV) in binding energy relative to that of Pt° at 71.2 eV (and a spin-orbit doublet peak at +3.3 eV) (Figure 9). Positive chemical shifts of the Pt 4f7/2 peaks beyond 71.2 eV have been attributed to several factors, including small particle size, platinum cluster compounds, and chemically bonded platinum complexes. In this study, the positive shift was likely due to platinum nanoparticle formation, as observed by TEM.

Discussion The interaction of cyanobacteria with PtCl4° solutions at 25180 °C results in a distinctive morphology for platinum-bearing

Figure 9. Narrow-region Pt 4f XPS spectra from the fluid/solid products of the abiotic PtCl4° experiments at 100 °C and 28 days, showing dominant Pt(0) and subordinate Pt(II).

nanoparticles and platinum metal. In cyanobacterial experiments using PtCl4°, platinum-bearing nanoparticles were precipitated both intracellularly and extracellularly (e0.3 µm). The presence of dispersed intracellular platinum suggests that platinum entered the cells as complexed PtCl4° [Pt(IV)] and was reduced to Pt(II) and then to Pt(0) by cyanobacteria. The cyanobacteria were immediately killed by either the platinum(IV)-chloride reagent or the acidic pH and the elevated temperatures (60-180 °C), and the resulting release of organics caused further precipitation of platinum. In the cyanobacteria systems at 25 °C, the growth of amorphous spherical Pt(II)-bearing nanoparticles (e0.3 µm) was observed in solutions, and, at 60-80 °C, smaller spherical Pt(II)-bearing nanoparticles (e0.2 nm) were glued together by organic material, forming long beadlike chains. At 100 °C, the nanoparticles of Pt(II)-organics were recrystallized, forming very fine-grained dark particles of platinum metal, and, at 180 °C, the recrystallized nanoparticles of platinum metal were branched, forming a dendritic morphology. The broadscan XPS results indicated the association of sulfur, phosphorus, and nitrogen with platinum(II), whereas SEM and TEM-EDS indicated the association of sulfur with platinum in the cyanobacteria-PtCl4° systems at 25-180 °C, suggesting the involvement of organic sulfur and possibly organic phosphorus in the

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reduction and complexation of PtCl4°. The stepwise reduction of PtCl4° solution by cyanobacteria is deduced to be as follows:

Pt(VI) [PtCl4°] f Pt(II) [Pt(II)-organics] f Pt(0) (1) where Pt(II) forms organometallic complexes. In contrast, in abiotic PtCl4° experiments at 80-180 °C, platinum metal was precipitated as nanoparticles by a reaction of the type

PtCl4 + 2H2O f Pt + 4H+ + 4Cl- + O2

(2)

which is promoted by heating. The ability of microorganisms to accumulate heavy metal ions has been reported previously,19-22 although the exact mechanism is still poorly understood. The presence of carboxyl groups, polyphosphate, and amino acids in algae is considered to be responsible for capturing metals,19,23,31 and similar clusters are presumably responsible for metal binding in cyanobacteria. The presence of polysaccharides in the cyanobacterial cell envelope and around the cells as water-soluble polymer could possess a large number of binding sites for metal ions.32 Also, most cyanobacterial polysaccharides have abundant uronic acid subunits, which, through their carboxyl groups, could efficiently bind metal ions. Another possible metal binding site in cyanobacteria is through the formation of metallothioneins or metalbinding proteins.19,33 Metallothioneins are proteins or polypeptides that bind metal ions as metal-thiolate, and often have a characteristic pattern of sulfur-containing amino acids.33,34 Although the exact mechanisms for platinum binding in (31) Mohamed, Z. A. Water Res. 2001, 35, 4405-4409. (32) Philippis, R. D.; Sili, C.; Paperi, R.; Vincenzini, M. J. Appl. Phycol. 2001, 13, 293-299. (33) Gardea-Torresdey, J. L.; Arenas, J. L.; Webb, R.; Fransisco, N. M. C.; Tiemann, K. J. J. Hazard. Subst. Res. 1997, 3, 1-18. (34) Turner, J. S.; Robinson, N. J. J. Ind. Microbiol. 1995, 14, 119-125.

cyanobacteria are yet to be determined, these studies show the importance of various clusters in the growth of platinum nanoparticles by cyanobacteria.

Conclusions In this study, synthesis of nanoparticles of platinum(II)organics and platinum metal by filamentous cyanobacteria from platinum(IV)-chloride (PtCl4°) complex has been demonstrated at 25-180 °C. The reaction of aqueous PtCl4° with cyanobacteria caused the precipitation of dispersed Pt(II)-organics within the bacterial cells, and the release of organic materials from cyanobacteria during their death. Further reaction of organic materials with PtCl4° in aqueous solution caused a reduction of Pt(IV) to Pt(II) and formed amorphous spherical Pt(II)-organic nanoparticles (e0.3 µm). With increase in temperature, the Pt(II)-organic nanoparticles were recrystallized and formed nanoparticles of crystalline platinum metal. At 180 °C, a dendritic habit of platinum metal was observed. In the abiotic experiments, spherical Pt(II)-bearing nanoparticles were not observed under similar conditions of temperature, pH, and reaction time. This study offers the first viable alternative method to standard abiotic chemical methods for the synthesis of spherical platinumbearing nanoparticles from platinum solutions. The use of cyanobacteria offers a means of developing “green nanofactories” for platinum catalysts, thereby reducing waste products and pollution by unnecessary chemical reagents. Acknowledgment. We thank Ronald Smith, Mark Biesinger, and Todd Simpson for their help during TEM, XPS, and SEMFIB analysis. The authors thank Tejal A. Desai for handling this manuscript and two anonymous reviewers for their constructive comments. This research was supported in part by the National Science Foundation LExEn Program and the Natural Sciences and Engineering Research Council of Canada Discovery Grants. LA060873S