J Nanopart Res (2009) 11:1811–1815 DOI 10.1007/s11051-009-9621-2
BRIEF COMMUNICATION
Microbial synthesis of silver nanoparticles by Bacillus sp. Nalenthiran Pugazhenthiran Æ Sambandam Anandan Æ Govindarajan Kathiravan Æ Nyayiru Kannaian Udaya Prakash Æ Simon Crawford Æ Muthupandian Ashokkumar
Received: 31 October 2008 / Accepted: 2 March 2009 / Published online: 13 March 2009 Ó Springer Science+Business Media B.V. 2009
Abstract A silver resistant Bacillus sp. was isolated through exposure of an aqueous AgNO3 solution to the atmosphere. Silver nanoparticles were synthesized using these airborne bacteria (Bacillus sp.). Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analyses confirmed that silver nanoparticles of 5–15 nm in size were deposited in the periplasmic space of the bacterial cells; a preferable cell surface location for the easy recovery of biogenic nanoparticles. Keywords Bacteria Nanoparticles Bacillus sp. Silver Nanobiotechnology
N. Pugazhenthiran S. Anandan (&) Nanomaterials & Solar Energy Conversion Lab, Department of Chemistry, National Institute of Technology, Trichy 620015, India e-mail:
[email protected] G. Kathiravan N. K. Udaya Prakash Marina Labs, Choolaimedu, Chennai 600094, India S. Crawford School of Botany, University of Melbourne, Melbourne, Victoria 3010, Australia M. Ashokkumar (&) School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia e-mail:
[email protected]
Introduction Nanomaterials are at the leading edge of the rapidly developing field of nanotechnology. The synthesis of nanomaterials of specific composition and size is a burgeoning area of materials science research. The properties of these materials in applications as diverse as catalysis, sensors and medicine depend critically on the size and composition of the nanomaterials. New routes to the preparation of these materials extend the choice of properties that can be obtained. Several synthetic techniques that usually employ atomic, molecular, and particulate processing in a vacuum or in a liquid medium are in use (Daniel and Astruc 2004; Yonezawa and Toshima 1995; Link et al. 1999; Silvert et al. 1996; Mizukoshi et al. 1997; Vinodgopal et al. 2006; Anandan et al. 2008; Treguer et al. 1998; Hodak et al. 2000; Chen and Yeh 2001; Mandal et al. 2006). Most of the techniques are capital intensive, as well as inefficient in materials and energy use. Hence, there is an ever-growing need to develop clean, nontoxic, and environmentally benign synthetic procedures. Consequently, researchers have used biological synthesis, since this technique provides particles with good control over the size distribution. The main reason for this may be that the processes devised by nature for the synthesis of inorganic materials on nano- and micro- scales have contributed to the development of a relatively new and largely unexplored area of research based on the use of microbes in the biosynthesis of nanomaterials (Mandal et al. 2006;
123
1812
Sastry et al. 2004). Among the microorganisms, prokaryotic bacteria have received the most attention in the area of bio-synthesis of nanoparticles. Microbial resistance against heavy metal ions such as Fe, Co, Ni, Cu, Zn, As, Cd, Hg, Pb or U has been explored for bioleaching processes of ores (Dopson and Lindstrom 1999; Bacelar-Nicolau and Johnson 1999; Lundgren and Silver 1980) and for biological metal recovery systems (White et al. 1997, 1998; Misra 1992; Nies 1992; Jeong et al. 1997). Early studies reveal that Bacillus subtilis 168 is able to reduce Au3? ions to produce octahedral gold particles of nanoscale dimensions (5–25 nm) within the bacterial cells by incubation of the cells with gold chloride solution (Beveridge and Murray 1980; Southam and Beveridge 1994; Fortin and Beveridge 2000) under ambient conditions. Silver is highly toxic to most microbial cells and can be used as biocide or antimicrobial agent (Slawson et al. 1992a, b). Nevertheless, it has been reported that several bacterial strains are silver resistant (Pooley 1982; Slawson et al. 1992a, b) and may even accumulate silver at the cell wall to as much as 25% of the dry weight biomass, thus indicating their use for industrial recovery of silver from ore materials (Pooley 1982). The silver resistant bacterial strain Pseudomonas stutzeri AG259 accumulates silver nanoparticles in the size range, 35–46 nm, along with some silver sulphide, in the cell (Haefeli et al. 1984; Klaus et al. 1999; Silver 2003). The exact reaction mechanisms leading to the formation of silver nanoparticles by the silver resistant bacteria is yet to be elucidated. In this study, we have made an attempt to corroborate the microbial reduction of water soluble Ag? to Ag0 using an airborne bacteria (Bacillus sp.) present in the atmosphere.
J Nanopart Res (2009) 11:1811–1815
was inoculated with a loopful of bacteria and incubated for a period of 7 days in darkness at room temperature. The bacteria were then harvested by centrifugation (10,000 rpm) at room temperature. The harvested cells were analyzed by TEM. For comparison, petridishes containing only the culture supernatant without silver nitrate solution and only silver nitrate (without culture supernatant) were incubated under similar experimental conditions. Upon visual observation, the culture supernatant incubated in the presence of silver nitrate showed a colour change from yellow to brown whereas no colour change could be observed in culture supernatant without silver nitrate and silver nitrate solution without the culture. These control experiments indicate that the Ag? ion reduction is not just a thermal process. TEM, EDX and electron diffraction analyses Pellets of freshly harvested Ag-loaded bacteria were fixed in 2.5% (w/v) aqueous glutaraldehyde, centrifuged, re-suspended in 1.5 ml of 0.1 M phosphate buffer (pH-7.2) at 4 °C and post fixed in 1% Osmium tetraoxide at 4 °C in 0.1 M phosphate buffer (60 min) for TEM. Samples were dehydrated using a graded series of acetone. After two 15 min washes in acetone, cells were embedded in fresh araldite followed by polymerization at 60 °C for 24 h. Ultrathin sections (90 nm) were cut on a Leica Ultracut R microtome, and mounted on pioloform-coated Cu grids. The sections were stained with 2% aqueous uranyl acetate for 10 min and triple lead stain for 5 min. Micrographs were taken with a Philips CM120 transmission electron microscope at 120 kV using a Gatan Multiscan 600CW digital camera. Energy-dispersive X-ray (EDX) spectra were acquired using TECNAI G2 model transmission electron microscopy.
Experimental details Bacterial strain and growth conditions
Results and discussion
Atmospheric bacteria were grown on nutrient agar substrate containing 3.5 mM AgNO3 (Aldrich) under aerobic conditions at 30 °C. A single colony was isolated from a dozen petridishes exposed. The isolated bacteria were identified as Bacillus sp. through their morphology and biochemical studies (Holt et al. 1994). The isolated colony was sub-cultured into 50 ml of nutrient broth containing 3.5 mM AgNO3. The broth
To confirm silver precipitation, Bacillus sp. were observed using TEM after exposure to a 3.5 mM aqueous AgNO3 solution. Figure 1 (a–e) shows the TEM images of thin sections of Bacillus sp. after exposure to a 3.5 mM aqueous AgNO3 solution. As shown in low magnification TEM images (Fig. 1a–c) silver nanoparticles were formed in most bacterial cells.
123
J Nanopart Res (2009) 11:1811–1815
1813
Fig. 1 TEM images of Bacillus sp. reduced Ag nanoparticles [low resolution images (a–c) and high resolution images (d and e)]. Lattice fringes of silver nanoparticles were observed from HRTEM image (e). FFT pattern (f) confirms (110) plane of silver nanoparticles. SAED spectrum (g) confirms Bacillus sp. reduce Ag? to Ag0
123
1814
In a higher magnification TEM image (Fig. 1d–e), biogenic nanoparticles 10–15 nm in size were observed in the periplasmic space of the bacterial cells, which is between the outer and inner cell (plasma) membranes. However, in contrast to many other reports of metal-resistant bacteria, for which efflux of toxic ions is the main detoxification mechanism (Silver and Phung 1996; Gupta et al. 1999), the majority of the accumulated silver here is deposited as seed like particles between the outer membrane and the plasma membrane. The sizes of the deposits in our results is so small (5–15 nm) compared to previous studies using P. stutzeri Ag259 (35–46 nm), which may be caused by different cell growth and metal incubation conditions (Haefeli et al. 1984). The FFT pattern (Fig. 1f) confirms the plane (110) of silver nanoparticle and thus from the electron diffraction pattern it is evidenced that the silver nanoparticles are crystalline in nature. Also, the lattice fringes observed in micrograph (Fig 1f) supports the crystalline nature of the silver nanoparticles. The selected area electron diffraction (SAED) (Fig 1g) results related to the metallic silver nanoparticles indicates the reduction of Ag? to Ag0 by Bacillus sp. Therefore, it can be concluded that the resting cells of Bacillus sp. can reduce Ag? ions to elemental silver (Ag0) in their periplasmic space. In summary, a simple microbial reduction method has been developed for synthesizing Ag nanoparticles. This methodology could be used for synthesizing a number of metallic nanoparticles involving other metals with good size and shape morphology. Acknowledgement The research described herein was supported by the Department of Innovation, Industry, Science and Research, Australia and Department of Science and Technology, India.
References Anandan S, Grieser F, Ashokkumar M (2008) Sonochemical synthesis of Au-Ag core-shell bimetallic nanoparticles. J Phys Chem C 112:15102–15105. doi:10.1021/jp806960r Bacelar-Nicolau P, Johnson DB (1999) Leaching of pyrite by acidophilic heterotrophic iron-oxidizing bacteria in pure and mixed cultures. Appl Environ Microbiol 65:585–590 Beveridge TJ, Murray RGE (1980) Sites of metal deposition in the cell wall of Bacillus subtilis. J Bacteriol 141:876–877 Chen YH, Yeh CS (2001) A new approach for the formation of alloy nanoparticles: laser synthesis of gold–silver alloy
123
J Nanopart Res (2009) 11:1811–1815 from gold–silver colloidal mixtures. Chem Commun (Camb) 371–372. doi:10.1039/b009854j Daniel MC, Astruc D (2004) Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology. Chem Rev 104:293–346. doi:10.1021/ cr030698? Dopson M, Lindstrom EB (1999) Potential role of thiobacillus caldus in arsenopyrite bioleaching. Appl Environ Microbiol 65:36–40 Fortin D, Beveridge TJ (2000) From biology to biotechnology and medical applications. In: Baeuerien E (ed) Biomineralization. Wiley-VCH, Weinheim, pp 7–22 Gupta A, Matsui K, Lo JF, Silver S (1999) Molecular basis for resistance to silver cations in Salmonella. Nat Med 5:183– 188. doi:10.1038/5545 Haefeli C, Franklin C, Hardy K (1984) Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine. J Bacteriol 158:389–392 Hodak JH, Henglein A, Giersig M, Hartland GV (2000) Laserinduced inter-diffusion in AuAg core-shell nanoparticles. J Phys Chem B 104:11708–11718. doi:10.1021/jp002438r Holt et al (1994) Bergey’s manual of determination bacteriology. William & Wilkins, Baltimore Jeong BC, Hawes C, Bonthrone KM, Macaskie LE (1997) Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme. Microbiology 143:2497–2501 Klaus T, Joerger R, Olsson E, Granqvist CG (1999) Silverbased crystalline nanoparticles, microbially fabricated. Proc Natl Acad Sci USA 968:13611–13614. doi:10.1073/ pnas.96.24.13611 Link S, Wang ZL, El-Sayed MA (1999) Alloy formation of gold-silver nanoparticles and the dependence of the plasmon absorption on their composition. J Phys Chem B 103:3529–3533. doi:10.1021/jp990387w Lundgren DG, Silver M (1980) Ore leaching by bacteria. Annu Rev Microbiol 34:263–283. doi:10.1146/annurev.mi.34. 100180.001403 Mandal D, Bolander ME, Mukhopadhyay D, Sarkar G, Mukherjee P (2006) The use of microorganisms for the formation of metal nanoparticles and their application. Appl Microbiol Biotechnol 69:485–492. doi:10.1007/ s00253-005-0179-3 Misra TK (1992) Bacterial resistances to inorganic mercury salts and organomercurials. Plasmid 27:4–16. doi:10.1016/ 0147-619X(92)90002-R Mizukoshi Y, Okitsu K, Maeda Y, Yamamoto TA, Oshima R, Nagata Y (1997) Sonochemical preparation of bimetallic nanoparticles of gold/palladium in aqueous solution. J Phys Chem B 101:7033–7037. doi:10.1021/jp9638090 Nies DH (1992) Resistance to cadmium, cobalt, zinc, and nickel in microbes. Plasmid 27:17–28. doi:10.1016/0147619X(92)90003-S Pooley FD (1982) Bacteria accumulate silver during leaching of sulphide ore minerals. Nature 296:642–643. doi:10. 1038/296642a0 Sastry M, Ahmad A, Khan MI, Kumar R (2004) Microbial nanoparticle production. In: Niemeyer CM, Mirkin CA (eds)
J Nanopart Res (2009) 11:1811–1815 Nanobiotechnology. Wiley-VCH, Weinheim, Germany, pp 126–135 Silver S (2003) Bacterial silver resistance: molecular biology and uses and misuses of silver compounds. FEMS Microbiol Rev 27:341–353. doi:10.1016/S0168-6445(03) 00047-0 Silver S, Phung LT (1996) Bacterial heavy metal resistance: new surprises. Annu Rev Microbiol 50:753–789. doi:10.1146/ annurev.micro.50.1.753 Silvert PY, Vijayakrishnan V, Vibert P, Herrara-Urbina R, Elhsissen KT (1996) Synthesis and characterization of nanoscale Ag-Pd alloy particles. Nanostruct Mater 7:611– 618. doi:10.1016/0965-9773(96)00039-6 Slawson RM, Trevors JT, Lee H (1992a) Silver accumulation and resistance in Pseudomonas stutzeri. Arch Microbiol 158:398–404. doi:10.1007/BF00276299 Slawson RM, Van Dyke MI, Lee H, Trevors JT (1992b) Germanium and silver resistance, accumulation, and toxicity in microorganisms. Plasmid 27:72–79. doi:10.1016/ 0147-619X(92)90008-X Southam G, Beveridge TJ (1994) The in vitro formation of placer gold by bacteria. Geochim Cosmochim Acta 58:4527–4530. doi:10.1016/0016-7037(94)90355-7
1815 Treguer M, de Cointet C, Remita H, Khatouri J, Mostafavi M, Amblard J, Mostafavi M, Amblard J, Belloni J, de Keyzer R (1998) Dose rate effects on radiolytic synthesis of goldsilver bimetallic clusters in solution. J Phys Chem B 102:4310–4321. doi:10.1021/jp981467n Vinodgopal K, He Y, Ashokkumar M, Grieser F (2006) Sonochemically prepared platinum-ruthenium bimetallic nanoparticles. J Phys Chem B 110:3849–3852. doi: 10.1021/jp060203v White C, Sayer JA, Gadd GM (1997) Microbial solubilization and immobilization of toxic metals: key biogeochemical processes for treatment of contamination. FEMS Microbiol Rev 20:503–516. doi:10.1111/j.1574-6976.1997. tb00333.x White C, Sharman AK, Gadd GM (1998) An integrated microbial process for the bioremediation of soil contaminated with toxic metals. Nat Biotechnol 16:572–575. doi: 10.1038/nbt0698-572 Yonezawa T, Toshima N (1995) Mechanistic consideration of formation of polymer-protected nanoscopic bimetallic clusters. J Chem Soc Faraday Trans 91:4111–4119. doi: 10.1039/ft9959104111
123
