Biosynthesis of Silver Nanoparticles by Filamentous Cyanobacteria

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Biosynthesis of Silver Nanoparticles by Filamentous Cyanobacteria from a Silver(I) Nitrate Complex Maggy F. Lengke,* Michael E. Fleet, and Gordon Southam Department of Earth Sciences, UniVersity of Western Ontario, London, Ontario N6A 5B7, Canada ReceiVed May 9, 2006. In Final Form: NoVember 23, 2006 The biosynthesis of silver nanoparticles has been successfully conducted using Plectonema boryanum UTEX 485, a filamentous cyanobacterium, reacted with aqueous AgNO3 solutions (∼560 mg/L Ag) at 25-100 °C for up to 28 days. The interaction of cyanobacteria with aqueous AgNO3 promoted the precipitation of spherical silver nanoparticles and octahedral (111) silver platelets (of up to 200 nm) in solutions. The mechanisms of silver nanoparticles via cyanobacteria could involve metabolic processes from the utilization of nitrate at 25 °C and also organics released from the dead cyanobacteria at 25-100 °C.

Introduction The synthesis of silver nanoparticles has attracted much attention because their unique shape-dependent optical, electrical, and chemical properties have potential applications in nanotechnology. Silver nanoparticles are used in photographic reactions,1 catalysis,2,3 and chemical analysis.4 Silver has been known to exhibit strong toxicity to a wide range of microorganisms and has been used extensively in many antibacterial applications.5 Therefore, the development of silver nanoparticles is expected to open new avenues to fight and prevent disease.5 Biological processes have recently been considered as possible methods for the synthesis of nanoparticles, especially the development of “green” synthetic approaches for nanoparticles. The biosynthesis of silver nanoparticles of different sizes, ranging from 1 to 70 nm, and shapes, including spherical, triangular, and hexagonal, has been conducted using bacteria, fungi, plants, and plant extract.6-14 The formation of extracellular and intracellular silver nanoparticles by bacteria (Pseudomonas stutzeri AG259; Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, Salmonella typhus, Phoma sp. 3.2883, and Staphylococcus aureus) has been demonstrated by reacting the cells with silver-

(I) nitrate (AgNO3).5-7,12 The suggested mechanisms for the bioreduction of silver by bacteria involve deoxyribonucleic acid (DNA)7 or sulfur-containing proteins.5 The reaction of fungi (Verticillium sp., Aspergillus fumigatus, and Fusarium oxyporum) with silver(I) nitrate caused the reduction of silver through a nitrate-dependent reductase for Fusarium oxyporum,13 whereas for other fungi the mechanism was speculated to involve carboxylate groups from the cell wall.8,10,14 The formation of spherical silver nanoparticles (2-20 nm) inside live alfalfa plants occurs through silver metal uptake from agar media.11 The reaction of silver(I) nitrate with germanium leaf extract caused the formation of spherical-dominated silver nanoparticles: the suggested mechanism of silver reduction here involved reaction with terpenoids.9 In this study, the formation of silver nanoparticles was investigated using silver(I) nitrate in the presence of the filamentous cyanobacterium, Plectonema boryanum (strain UTEX 485). This organism was chosen in this study because cyanobacteria form one of the largest and most important groups of photoautotrophic bacteria on earth and are known to have a high-affinity transport system for nitrate.15 Experimental Section

* Corresponding author. E-mail: [email protected] or [email protected]. Present address: Geomega Inc., 2995 Baseline Road, Suite 202, Boulder, Colorado 80303. (1) Mostafavi, M.; Marignier, J. L.; Amblard, J.; Belloni, J. Radiat. Phys. Chem. 1989, 34, 605-617. (2) Verykios, X. E.; Stein, F. P.; Coughlin, R. W. Catal. ReV. Sci. Eng. 1980, 34, 197-234. (3) Claus, P.; Hofmeister, H. J. Phys. Chem. B 1999, 103, 2766-2775. (4) Pal, T. J. Chem. Educ. 1994, 71, 679-681. (5) Morones, J. R.; Elechiguerra, J. L.; Camacho, A.; Holt, K.; Kouri, J. B.; Ramirez, J. T.; Yacaman, M. J. Nanotechnology 2005, 16, 2346-2353. (6) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C-G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 13611-13614. (7) Feng, Q. L.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. J. Biomed. Mater. Res. 2000, 52, 662-668. (8) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parishcha, R.; Ajaykumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Nano Lett. 2001, 1, 515-519. (9) Shankar, S. S.; Ahmad, A.; Murali, S. Biotechnol. Prog. 2003, 19, 16271631. (10) Sastry, M.; Ahmad, A.; Khan, M. I.; Kumar, R. Curr. Sci. 2003, 85, 162-170. (11) Gardea-Torresdey, J. L.; Gomez, E.; Peralta-Videa, J. R.; Parsons, J. G.; Troiani, H.; Jose-Yacaman, M. Langmuir 2003, 19, 1357-1361. (12) Chen, J. C.; Lin, Z. H.; Ma, X. X. Lett. Appl. Microbiol. 2003, 37, 105108. (13) Duran, N.; Marcato, P. D.; Alves, O. L.; De Souza, G. I. H.; Esposito, E. J. Nanobiotechnol. 2005, 3, 1-7. (14) Bhainsa, K. C.; D’Souza, S. F. Colloids Surf., B 2005, 47, 160-164.

Cyanobacteria Cultures. The filamentous cyanobacterium, Plectonema boryanum strain UTEX 485 (obtained from the culture collection at the University of Texas at Austin, Texas), was grown in batch cultures in BG-11 medium16 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 8.0 using a 1 M NaOH solution. Before experimentation, the culture was transferred (20% (v/v) of inoculum), and grown for approximately 6-8 weeks to reach a stationary “growth” phase. It was then centrifuged and washed three times with sterile distilled, deionized water to remove salts and trace metals from the medium before being used for the cyanobacterial experiments. Cyanobacterial and Abiotic Experiments. The cyanobacterial experiments were conducted to examine the role of cyanobacteria in the synthesis of silver nanoparticles from aqueous solutions of AgNO3 (AgNO3; Alfa Aesar Company, Ward Hill, MA). The experiments were conducted using washed cyanobacteria and without the presence of the BG-11 medium. To initiate the experiments, 5 mL of silver solution (∼560 mg/L) was added to 5 mL of washed (15) Suzuki, I.; Kikuchi, H.; Nakanishi, S.; Fujita, Y.; Sugiyama, T.; Omata, T. J. Bacteriol. 1995, 177, 6137-6143. (16) Rippka, R.; Deruelles, J.; Waterbury, J.; Herdman, M.; Stanier, R. Y. J. Gen. Microbiol. 1979, 111, 1-61.

10.1021/la0613124 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/18/2007

Biosynthesis of Ag Nanoparticles by Cyanobacteria cyanobacteria culture (∼10 mg dry weight). The experiments were conducted at 25, 60, and 100 °C for up to 28 days after the incubation period with silver solutions and maintained in the dark, and cyanobacteria populations were measured with time. For experiments at 60 and 100 °C, reactants were contained in sealed borosilicate vials in temperature-regulated laboratory ovens (Blue M Electric Company and Fisher Isotemp Oven, respectively). pH, Eh, total silver, and sulfur were measured with time. All experiments were conducted in duplicate. Abiotic control experiments consisting of ∼560 mg/L silver were conducted using AgNO3 solution without the presence of cyanobacteria. Bacterial Viability and Total Bacterial Counts. The effects of AgNO3 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, OR). 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/ORP/temperature meter. The pH electrode was calibrated using buffer solutions 4, 7, and 10 with analytical uncertainties in the measurement of pH of (0.05 pH unit. The Eh was measured using an ORP electrode and calibrated using ZoBell’s solution.17 Total silver concentrations were measured on the centrifuged samples over the course of the experiments with a Perkin-Elmer 3300-DV inductively coupled plasma optical emission spectrometer (ICP-OES). The uncertainty in measured silver is e5%, with a detection limit of 0.05 mg/L for silver. Transmission Electron Microscopy (TEM). Unstained whole sample mounts and thin sections of cyanobacteria and silver nanoparticles from the experiments were examined with a Phillips CM-10 transmission electron microscope (TEM) operated at 80 kV and a Phillips EM400T transmission electron microscope (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 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. Samples for ultrathin sections were fixed in 2% glutaraldehyde (electron microscopy grade; Electron Microscopy Sciences, Hatfield, PA), enrobed in Noble Agar (Difco), dehydrated with a 25, 50, 75, and 100%(aq) ethanol series (anhydrous ethanol; Commercial Alcohols Inc., Brampton, Ontario, Canada), and embedded in London Resin White (LR White; Electron Microscopy Sciences, Hatfield, PA) curing with an accelerator. The embedded samples were ultrathin sectioned on a Reichert-Jung Ultracut E ultramicrotome using a diamond knife to a thickness of 70 nm and collected on Formvar carbon-coated 200 mesh copper grids. X-ray Photoelectron Spectroscopy (XPS). The reaction products obtained from the cyanobacterial experiments using AgNO3 at 25 and 100 °C and day 28 were analyzed by XPS to investigate the oxidation state of silver and sulfur. All samples were dried in the vacuum chamber of the XPS. 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 368.30 ( 0.05 eV (with a peak fwhm of 1.0(0.05 eV) for the Ag 3d5/2 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 graphitelike C at 285.00 eV to compensate for charging effects in the samples. Survey scans were recorded (17) Nordstrom, D. K. Geochim. Cosmochim. Acta 1977, 41, 1835-1841.

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Figure 1. Variation of total soluble Ag, pH, and Eh values with time for cyanobacterial and abiotic experiments with AgNO3 solution at 25 to 100 °C. A and C represent abiotic and cyanobacterial experiments, respectively. 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,18 and the doublet components for Ag 3d5/2 and Ag 3d3/2 are separated by 6 eV.19 Reference binding energies for Ag 3d and S 2p species were taken from Moulder et al.19 and Knipe and Fleet,20 respectively.

Results Cyanobacterial and Abiotic Experiments. The results of cyanobacterial and abiotic experiments using AgNO3 are shown in Figure 1. On addition of AgNO3 to the cyanobacteria cultures, the soluble silver concentrations decreased by about 290 mg/L after 28 days at 25 °C. At 60 °C, the soluble silver decreased by about 330 mg/L after 14 days and then decreased very slightly (∼14 mg/L) until the end of experiments. At 100 °C, the soluble silver was completely precipitated from solutions within 28 days. A greyish-black silver precipitate on cyanobacteria was observed macroscopically. The pH was constant at 5.0 and 4.7 at 25 and 60 °C, respectively, but decreased from 4.7 to 3.3 at 100 °C (Figure 1). Eh values were relatively constant at ∼0.8 V after 28 days at 25 and 60 °C but decreased from 0.8 to 0.6 V at 100 °C. All cyanobacteria were killed within several hours at all temperatures investigated (25 to 100 °C). In abiotic experiments using AgNO3, total soluble silver concentrations were constant until the completion of experiments at all temperatures investigated. pH values were relatively constant at 4.7. Eh values showed no significant change at 25 and 100 °C but slightly decreased by about 0.08 V at 60 °C. (18) Briggs, D.; Seah, M. P. Practical Surface Analysis by Auger and X-ray Photoelectron Spectroscopy, 2nd ed.; Wiley: Chichester, U.K., 1996. (19) 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. (20) Knipe, S. W.; Fleet, M. E. Can. Mineral. 1997, 35, 1485-1495.

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Figure 2. TEM micrographs of whole mounts of cyanobacterial cells cultured in the presence of AgNO3 at 25 °C and 28 days. (A) Precipitation of silver nanoparticles on the cyanobacterial surface. (B) TEM micrograph of a thin section of cyanobacteria cells with nanoparticles of silver deposited inside the cell. (C, D) Spherical nanoparticles of silver precipitated in solution. (E) TEM-SAED diffraction powder-ring pattern consistent with crystalline nanoparticles of Ag with a possible trace of silver sulfide (*). d spacings of 0.235, 0.204, 0.144, and 0.123 nm corresponding to reflections 111, 200, 220, and 311, respectively. (F) TEM-EDS for area D. Scale bars in A, B, C, and D are 0.5 and 0.2 µm and 25 and 50 nm, respectively.

TEM. TEM observations on products of cyanobacteriaAgNO3 experiments at 25-100 °C are presented in Figures 2-4. At 25 °C, the addition of AgNO3 to the cyanobacteria caused the precipitation of silver nanoparticles at cell surfaces (Figure 2A) and within the cells (Figure 2B). Small spherical silver nanoparticles with size ranging from 1 to 15 nm were also precipitated

in solution (Figure 2C,D). TEM-SAED of the nanoparticles revealed powder ring diffraction patterns (Figure 2E) consistent with nanocrystalline silver with possible trace silver sulfide. TEMEDS showed the occurrence of silver with traces of phosphorus, sulfur, and iron (Figure 2F). At 60 °C, silver nanoparticles were deposited at cell surfaces (Figure 3A). In solution, spherical

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Figure 3. TEM micrographs of whole mounts of cyanobacterial cells cultured in the presence of AgNO3 at 60 °C and 28 days. (A) Precipitation of silver nanoparticles on the cyanobacterial surface. (B) Spherical nanoparticles of silver precipitated in solution. (C) TEM-SAED diffraction powder-ring pattern consistent with crystalline nanoparticles of Ag; d spacings of 0.235, 0.204, 0.144, and 0.123 nm correspond to reflections 111, 200, 220, and 311, respectively. (D) TEM-EDS for area B. Scale bars in A and B are 0.25 µm and 50 nm, respectively.

silver nanoparticles with size ranging from 1 to 40 nm were present (Figure 3B). TEM-SAED of the nanoparticles revealed powder ring diffraction patterns (Figure 3C) consistent with nanocrystalline silver. TEM-EDS showed the occurrence of silver with traces of phosphorus, sulfur, and iron (Figure 3D). At 100 °C, the cyanobacterial cells were encrusted with silver nanoparticles, and separation of some filaments into their constituent cells was observed (Figure 4A). The spherical silver nanoparticles were still observed and ultimately resulted in the precipitation of octahedral crystal platelets of silver within the cells and in solution (Figure 4B-D). These particles are equivalent to the nanotriangles or nanohexagons of silver in previous work.14 TEM-SAED of the nanoparticles revealed powder ring diffraction patterns (Figure 4E) consistent with nanocrystalline silver. TEMEDS showed the occurrence of silver with traces of phosphorus, sulfur, and iron (Figure 4F). The size of the octahedral silver particles ranged from 5 to 200 nm, all with nanometer-scale thickness. The solutions of AgNO3 were stable for 28 days at 25-100 °C in abiotic experiments; therefore, silver nanoparticles were not formed. XPS. XPS spectra for reaction products of the cyanobacterial experiments with AgNO3 solutions at 25 and 100 °C are presented in Figure 5. Narrow-region XPS spectra were acquired for Ag 3d, S 2p, and C 1s spectral regions, but only results for Ag are

presented here (Figure 5). Semiquantitative compositional analysis of the surface layers (∼15 Å depth) by broadscan XPS indicated Ag/S ratios of 2.3:1 and 1:1 and Ag/P ratios of 3.5:1 and 3:1 for the near-surface region in the cyanobacterial experiments at 25 and 100 °C, respectively. However, the analysis of the deeper near-surface by TEM-EDS had indicated Ag/S and Ag/P ratios close to 14:1 and 26:1, respectively, for a whole sample mount. For cyanobacterial experiments with solutions of AgNO3 at 25 °C, the XPS spectra were dominated by a single peak for Ag0 at 368.3 eV (and a spin-orbit doublet peak at +6.0 eV) with a fwhm of 1.5 eV (Figure 5A). The S spectra for the sediment show a main peak with a centroid at ∼168.1 eV (and a doublet peak at +1.18 eV) corresponding to the SO42- species and minor peaks for reduced sulfur species. For the cyanobacterial experiments with the addition of AgNO3 at 100 °C, the spectra for the sedimented silver particles were dominated by Ag0 at 368.3 eV (with a fmhm of ∼1.3 eV) (Figure 5B). Sulfur 2p spectra were dominated by SO42- species with minor polysulfide (163.3 eV) and one S-O species (166.2 eV), which is interpreted as thiosulfate.

Discussion The reaction of cyanobacteria with AgNO3 solutions results in the formation of silver nanoparticles from 25 to 100 °C. The

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Figure 4. TEM micrographs of whole mounts of cyanobacterial cells cultured in the presence of AgNO3 at 100 °C and 28 days. (A) Silver nanoparticles encrusted on cyanobacterial cells. (B) TEM micrograph of a thin section of cyanobacteria cells with nanoparticles of silver deposited inside the cell. (C) Octahedral, spherical, and anhedral nanoparticles of silver precipitated in solution. (D) Octahedral silver platelets. (E) TEM-SAED diffraction powder-ring pattern consistent with crystalline nanoparticles of Ag; d spacings of 0.235, 0.204, 0.144, and 0.123 nm correspond to reflections 111, 200, 220, and 311, respectively. (F) TEM-EDS for area B. Scale bars in A, B, C, and D are 1, 0.05, and 0.1 µm and 50 nm, respectively.

presence of spherical silver nanoparticles was observed in all experiments from 25 to 100 °C both intracellularly (