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Extracellular Biosynthesis of Monodisperse Gold Nanoparticles by a Novel Extremophilic Actinomycete, Thermomonospora sp. Absar Ahmad,*,† Satyajyoti Senapati,‡ M. Islam Khan,† Rajiv Kumar,‡ and Murali Sastry*,§ Biochemical Sciences, Catalysis, and Materials Chemistry Divisions, National Chemical Laboratory, Pune - 411 008, India Received October 30, 2002. In Final Form: February 10, 2003
Introduction An important area of research in nanotechnology is the synthesis of nanoparticles of different chemical compositions, sizes, shapes, and controlled dispersities. Currently, there is a growing need to develop environmentally benign nanoparticle synthesis processes that do not use toxic chemicals in the synthesis protocol. As a result, researchers in the field of nanoparticle synthesis and assembly have turned to biological systems for inspiration. This is not surprising given that many organisms, both unicellular and multicellular, are known to produce inorganic materials either intra- or extracellularly.1 Some well-known examples of bio-organisms synthesizing inorganic materials include magnetotactic bacteria (which synthesize magnetite nanoparticles),2 diatoms (which synthesize siliceous materials),3 and S-layer bacteria (which produce gypsum and calcium carbonate layers).4 The secrets gleaned from nature have lead to the development of biomimetic approaches for the growth of advanced nanomaterials. Even though many biotechnological applications such as remediation of toxic metals employ microorganisms such as bacteria5 and yeast6 (the detoxification often occurring via reduction of the metal ions/formation of metal sulfides), it is only relatively recently that materials scientists have been viewing with interest such microorganisms as possible eco-friendly nanofactories.7,8 Bev* Authors for correspondence. E-mail:
[email protected];
[email protected]. † Biochemical Sciences. ‡ Catalysis. § Materials Chemistry Divisions. (1) (a) Simkiss, K.; Wilbur, K. M. Biomineralization; Academic Press: New York, 1989. (b) Biomimetic Materials Chemistry; Mann, S., Ed.; VCH Publishers: 1996. (2) (a) Loveley, D. R.; Stolz, J. F.; Nord, G. L.; Phillips, E. J. P. Nature 1987, 330, 252. (b) Spring, H.; Schleifer, K. H. Syst. Appl. Microbiol. 1995, 18, 147. (c) Dickson, D. P. E. J. Magn. Magn. Mater. 1999, 203, 46. (3) (a) Mann, S. Nature 1993, 365, 499. (b) Oliver, S.; Kupermann, A.; Coombs, N.; Lough, A.; Ozin, G. A. Nature 1995, 378, 47. (c) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129. (4) (a) Pum, D.; Sleytr, U. B. Trends Biotechnol. 1999, 17, 8. (b) Sleytr, U. B.; Messner, P.; Pum, D.; Sara, M. Angew. Chem., Int. Ed. Engl. 1999, 38, 1034. (5) Stephen, J. R.; Maenaughton, S. J. Curr. Opin. Biotechnol. 1999, 10, 230. (6) Mehra, R. K.; Winge, D. R. J. Cell. Biochem. 1991, 45, 30. (7) (a) Southam, G.; Beveridge, T. J. Geochim. Cosmochim. Acta 1996, 60, 4369. (b) Beveridge, T. J.; Murray, R. G. E. J. Bacteriol. 1980, 141, 876. (c) Fortin, D.; Beveridge, T. J. In Biomineralization. From Biology to Biotechnology and Medical Applications; Baeuerien, E., Ed.; WileyVCH: Weinheim, 2000; p 7. (8) (a) Klaus, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Proc. Natl. Acad. Sci. 1999, 96, 13611. (b) Klaus-Joerger, T.; Joerger, R.; Olsson, E.; Granqvist, C. G. Trends Biotechnol. 2001, 19, 15. (c) Joerger, R.; Klaus, T.; Granqvist, C. G. Adv. Mater. 2000, 12, 407.
eridge and co-workers have demonstrated that gold particles of nanoscale dimensions may be readily precipitated within bacterial cells by incubation of the cells with Au3+ ions.7 Klaus-Joerger and co-workers have shown that the bacteria Pseudomonas stutzeri AG259 isolated from a silver mine when placed in a concentrated aqueous solution of AgNO3 resulted in the reduction of the Ag+ ions and formation of silver nanoparticles of well-defined size and distinct morphology within the periplasmic space of the bacteria.8 Nair and Pradeep have synthesized nanocrystals of gold, silver, and their alloys by reaction of the corresponding metal ions within cells of lactic acid bacteria present in buttermilk.9 Very recently, JoseYacaman and co-workers have shown that gold nanoparticles may be synthesized in live alfalfa plants by gold uptake from solid media.10 In a break from tradition, which has hitherto relied on the use of prokaryotes such as bacteria in the intracellular synthesis of nanoparticles, we have recently shown that eukaryotic organisms such as fungi may be used to grow nanoparticles of different chemical compositions and sizes. A number of different genera of fungi have been investigated in this effort, and it has been shown that fungi are extremely good candidates in the synthesis of gold,11,12 silver,13 and, indeed, quantum dots of the technologically important CdS by enzymatic processes.14 The use of fungi is potentially exciting, since they secrete large amounts of enzymes and are more simple to deal with in the laboratory. However, one possible drawback to this approach could be that genetic manipulation of eukaryotic organisms as a means of overexpressing specific enzymes identified in nanomaterials synthesis would be much more difficult than that in prokaryotes. As can be seen from the above, the use of biological organisms in the deliberate and controlled synthesis of nanoparticles is a relatively new and exciting area of research with considerable potential for development. Actinomycetes are microorganisms that share important characteristics of fungi and prokaryotes such as bacteria.15 Even though they are classified as prokaryotes due to their close affinity with mycobacteria and the coryneforms (and thus amenable to genetic manipulation by modern recombinant DNA techniques), they were originally designated as “Ray Fungi” (Strahlenpilze). Focus on actinomycetes has primarily centered on their phenomenal ability to produce secondary metabolites such as antibiotics.16 For the nanotechnology related reasons mentioned above, we have recently enlarged the scope of our studies and have identified the alkalothermophilic (9) Nair, B.; Pradeep, T. Cryst. Growth Des. 2002, 2, 293. (10) Gardea-Torresdey, J. L.; Parsons, J. G.; Gomez, E.; PeraltaVidea, J.; Troiani, H. E.; Santiago, P.; Jose-Yacaman, M. Nano Lett. 2002, 2, 397. (11) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Ramani, R.; Parischa, R.; Ajaykumar, P. V.; Alam, M.; Sastry, M.; Kumar, R. Angew. Chem., Int. Ed. 2001, 40, 3585. (12) Mukherjee, P.; Senapati, S.; Mandal, D.; Ahmad, A.; Khan, M. I.; Kumar, R.; Sastry, M. ChemBioChem 2002, 3, 461. (13) Mukherjee, P.; Ahmad, A.; Mandal, D.; Senapati, S.; Sainkar, S. R.; Khan, M. I.; Parischa, R.; Ajayakumar, P. V.; Alam, M.; Kumar, R.; Sastry, M. Nano Lett. 2001, 1, 515. (14) Ahmad, A.; Mukherjee, P.; Mandal, D.; Senapati, S.; Khan, M. I.; Kumar, R.; Sastry, M. J. Am. Chem. Soc. 2002, 124, 12108. (15) Biology of Actinomycetes 88; Okami, Y., Beppu, T., Ogawara, H., Eds.; Japan Scientific Societies Press: Tokyo, 1988; p 508. (16) Sasaki, T.; Yoshida, J.; Itoh, M.; Gomi, S.; Shomura, T.; Sezaki, M. J. Antibiot. 1988, 41, 835.
10.1021/la026772l CCC: $25.00 © 2003 American Chemical Society Published on Web 03/11/2003
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Figure 1. (A) UV-vis spectra recorded as a function of time of reaction of a 10-4 M aqueous solution of HAuCl4 with Thermomonospora sp. biomass. The inset shows a test tube of the nanoparticle solution formed at the end of the reaction (120 h). (B) FTIR spectrum recorded from drop-cast films of the chloroauric acid solution after reaction with Thermomonospora sp. biomass for 120 h. The amide I and II bands are identified in the figure.
(extremophilic)17 actinomycete Thermomonospora sp. as an exciting candidate for the extracellular synthesis of gold nanoparticles by chemical reaction of the biomass with aqueous chloroaurate ions. We observe the efficient formation of a high concentration of gold nanoparticles of 8 nm average size and good monodispersity. To the best of our knowledge, this is the first report on the extracellular synthesis of metal nanoparticles using a prokaryotic microorganism. Presented below are details of the investigation. Experimental Details A novel alkalothermophilic (extremophilic) actinomycete, Thermomonospora sp., having optimum growth at pH 9 and 50 °C, was isolated from self-heating compost from the Barabanki district of Uttar Pradesh, India. This actinomycete was maintained on MGYP (malt extract, glucose, yeast extract, and peptone) agar slants. Stock cultures were maintained by subculturing at monthly intervals. After growing the actinomycete at pH 9 and 50 °C for 4 days, the slants were preserved at 15 °C. From an actively growing stock culture, subcultures were made on fresh slants and, after 4 days incubation at pH 9 and 50 °C, were used as the starting material for fermentation experiments. For the synthesis of the gold nanoparticles, the actinomycete was grown in 250 mL Erlenmeyer flasks containing 50 mL of MGYP medium which is composed of malt extract (0.3%), glucose (1%), yeast extract (0.3%), and peptone (0.5%). Sterile 10% sodium carbonate was used to adjust the pH of the medium to 9. After the pH of the medium was adjusted, the culture was grown with continuous shaking on a rotary shaker (200 rpm) at 50 °C for 96 h. After 96 h of fermentation, mycelia (cells) were 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 mycelial mass (10 g of wet mycelia) was then resuspended in 50 mL of 10-3 M aqueous AuCl4 solution in 250 mL Erlenmeyer flasks at pH 9. The whole mixture was put into a shaker at 50 °C (200 rpm) and maintained in the dark. The bioreduction of the AuCl4- ions in solution was monitored by periodic sampling of aliquots (2 mL) of the aqueous component and measuring the UV-vis spectra of the solution. UV-vis spectra of these aliquots were monitored as a function of time of reaction on a HewlettPackard diode array spectrophotometer (model HP-8452) operated at a resolution of 2 nm. Samples for transmission electron microscopy (TEM) analysis were prepared on carbon-coated copper TEM grids. The films on the TEM grids were allowed to stand for 2 min, following which the extra solution was removed using a blotting paper and the grid was allowed to dry prior to
measurement. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage at 120 kV. Fourier transform infrared (FTIR) spectroscopy measurements of drop-coated films of the chloroauric acid solution after 120 h of reaction with the biomass on Si (111) substrates were carried out on a Shimadzu 8201-PC instrument in the diffuse reflectance mode at a resolution of 4 cm-1. To identify the number of proteins secreted by the actinomycete and their molecular weights, the actinomycete biomass [10.0 g of wet mycelia (cells)] was resuspended in 100 mL of sterile distilled water for a period of 4 days. The mycelia were then removed by centrifugation, and the aqueous supernatant thus obtained was concentrated by ultrafiltration using a YM3 (molecular weight cutoff ) 3K) membrane and then dialyzed thoroughly against distilled water using a 3K cutoff dialysis bag. This concentrated aqueous extract containing protein was analyzed by PAGE (polyacrylamide gel electrophoresis) carried out at pH 8.3 according to the procedure published by Laemmli.18
Results and Discussion Reduction of the aqueous chloroaurate ions during exposure to the Thermomonospora sp. biomass may be easily followed by UV-vis spectroscopy. It is well-known that gold nanoparticles exhibit lovely pink-ruby red colors, arising due to excitation of surface plasmon vibrations in the gold nanoparticles.19 Figure 1A shows the UV-vis spectra recorded from the aqueous chloroauric acid-actinomycete reaction medium as a function of time of reaction. It is observed that the gold surface plasmon band occurs at ∼520 nm and steadily increases in intensity as a function of time of reaction. Complete reduction of the AuCl4- ions occurs after nearly 120 h of reaction, indicating that it is an extremely slow process. A picture of the chloroauric acid solution after completion of the reaction is shown in the inset of Figure 1A. An intense red (17) Extremophiles are microorganisms which thrive under conditions which are lethal to human beings, such as extremes of temperature [from -14 °C (psychrophiles) to 45 °C (thermophiles) to 110 °C (hyperthermophiles)]; extremes of pH [from 1 (acidophiles) to 9 (alkalophiles)]; very high barostatic pressure (barophiles); nonaqueous environment containing 100% organic solvents; excess heavy metal concentration; etc. These microorganisms have developed numerous special adaptations to survive in such extreme habitats, which include new mechanisms of energy transduction, regulating intracellular environment and metabolism, maintaining the structure and functioning of membrane and enzymes, etc. (18) Laemmli, U. K. Nature 1970, 227, 680. (19) Mulvaney, P. Langmuir 1996, 12, 788.
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Figure 2. (A and B) TEM micrographs recorded from drop-cast films of the gold nanoparticle solution formed by the reaction of chloroauric acid solution with Thermomonospora sp. biomass for 120 h at different magnifications. (C) Particle size distribution histogram determined from the TEM micrograph shown in part B. (D) Selected area diffraction pattern recorded from the gold nanoparticles shown in Figure 2B.
color is observed, showing the formation of gold nanoparticles. As indicated in the Experimental Section, the bioreduction was carried out in the dark and, clearly, the formation of gold nanoparticles is due to the actinomycete biomass. The fact that the surface plasmon band in the gold nanoparticle solution remains close to 520 nm throughout the reaction period indicates that the particles are dispersed in the aqueous solution with no evidence for aggregation. After completion of the reaction, the gold nanoparticle solution was separated from the actinomycete biomass and tested for stability. It was observed that the nanoparticle solution was stable for more than 6 months with little signs of aggregation (as determined by UV-vis spectroscopy measurements) even at the end of this period. The particles are thus stabilized in solution by a capping agent that is likely to be proteins secreted by the biomass (see FTIR results below). The biomass was colorless, indicating that the reduction of the gold ions took place extracellularly. Figure 1B shows the FTIR spectrum recorded from the chloroauric acid solution after reaction with Thermomonospora sp. for 120 h. The presence of two bands at 1660 and 1530 cm-1 is seen in the figure. The 1660 and 1530 cm-1 bands may be assigned to the amide I and II bands of
proteins, respectively.20 It is well-known that proteins can bind to gold nanoparticles through either free amine groups or cysteine residues in the proteins21 and, therefore, stabilization of the gold nanoparticles by surface-bound proteins is a possibility. The exact mechanism leading to the reduction of the metal ions is yet to be elucidated for this microorganism. As a first step in this direction, we have analyzed the proteins released into water by the actinomycete in terms of the number of different proteins secreted and their molecular weights. Preliminary gel electrophoresis measurements (Figure S1, Supporting Information) indicate that the actinomycete secretes four distinct proteins ranging in weight from 80 to 10 kDa. We believe that one or more of these proteins may be enzymes that reduce chloroaurate ions and cap the gold nanoparticles formed by the reduction process. It is also possible that the capping and stabilization of the gold nanoparticles are effected by a different protein. We are currently separating and concentrating the different proteins re(20) Caruso, F.; Furlong, D. N.; Ariga, K.; Ichinose, I.; Kunitake, T. Langmuir 1998, 14, 4559. (21) Gole, A.; Dash, C. V.; Ramachandran, V.; Mandale, A. B.; Sainkar, S. R.; Rao, M.; Sastry, M. Langmuir 2001, 17, 1674.
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leased by the actinomycete Thermomonospora sp. to test and identify the ones active in the above processes. In an earlier study, we had demonstrated that the fungus Fusarium oxysporum, when exposed to an aqueous solution of chloroauric acid, resulted in extracellular formation of gold nanoparticles.11 The reduction was faster than that observed in this study using Thermomonospora sp. (48 vs 120 h).11 However, the gold nanoparticles synthesized using the fungus were extremely polydisperse and ranged in size from 8 to 40 nm. Furthermore, the nanoparticles formed large aggregates in solution.11 If biosynthesis of gold nanoparticles using microorganisms is to be a viable alternative to chemical methods currently in vogue, then greater control over particle size and polydispersity would need to be established. This was one of our goals in screening different species of fungi and, now, actinomycetes. Figure 2A and B shows TEM pictures recorded from drop-coated films of the gold nanoparticles synthesized using Thermomonospora sp. after reacting chloroauric acid with the biomass for 120 h. The low magnification TEM image clearly shows dense assembly of uniformly sized gold nanoparticles (Figure 2A). Indeed, the whole surface of the grid was evenly covered with gold nanoparticles, as shown in this image. At slightly higher magnification, a better idea of the morphology and size of the particles may be had (Figure 2B). The particles are essentially spherical and appear to be reasonably monodisperse. The particle size histogram derived from the particles shown in this image and other similar images is shown in Figure 2C. It can be seen that the average particle size is ∼8 nm with some particles of 9-10 nm size and a very small percentage having diameters 7 and 12 nm. Thus, a significant improvement in the monodispersity has been achieved using actinomycetes. Whether the size and dispersity control is a consequence of larger amounts of proteins/enzymes secreted by Thermomonospora sp. in comparison with Fusarium oxysporum remains to be established. Another distinct possibility is that the nature and strength of interaction of different proteins with different crystallographic faces of gold nanocrystals may varysthis may lead to complex morphologies and size control. It is well-known that the complex morphology of calcium carbonate crystals in red
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abalone shells is modulated by insoluble proteins present in the organism,22 and a similar mechanism may be operative in our study on gold nanoparticles. It is also to be noted that the synthesis conditions are different in both cases. In the case of actinomycetes, the reaction is carried out under alkaline conditions and at slightly elevated temperatures. Under these extreme conditions, fungi such as Fusarium oxysporum would not survive. The use of extreme biological conditions in the synthesis could also be a contributory factor in the size and monodispersity control observed using actinomycetes. Figure 2D shows the selected area electron diffraction (SAED) pattern obtained from the gold nanoparticles shown Figure 2B. The Scherrer ring pattern characteristic of face centered cubic (fcc) gold is clearly observed, showing that the structures seen in the TEM images are nanocrystalline in nature. In conclusion, the efficient synthesis of fairly monodisperse gold nanoparticles by reaction of aqueous chloroaurate ions with the alkalothermophilic actinomycete Thermomonospora sp. has been described. The reduction of the metal ions and stabilization of the gold nanoparticles are believed to occur by an enzymatic process. From the nanotechnology point of view, identification of prokaryotic microorganisms such as actinomycetes in the extracellular synthesis of metal nanoparticles should offer greater scope for development via genetic manipulation through modern methods of recombinant DNA technology and protein engineering. Acknowledgment. S.S. thanks the Council of Scientific and Industrial Research (CSIR), Government of India, for a research fellowship. The TEM assistance of Ms. Renu Pasricha, Physical Chemistry Division, NCL Pune, is gratefully acknowledged. Supporting Information Available: Polyacrylamide gel electrophoresis picture of the protein extract from the actinomycete, Thermomonospora sp. (S1). This material is available free of charge via the Internet at http://pubs.acs.org. LA026772L (22) Su, X.; Belcher, A. M.; Zaremba, C. M.; Morse, D. E.; Stucky, G. D.; Heuer, A. H. Chem. Mater. 2002, 14, 3106.
