A novel bacterial isolate Stenotrophomonas maltophilia as living factory for synthesis of gold nanoparticles
© Nangia et al; licensee BioMed Central Ltd. 2009
Received: 27 March 2009
Accepted: 20 July 2009
Published: 20 July 2009
This article has been retracted. The retraction notice can be found here.
The synthesis of gold nanoparticles (GNPs) has received considerable attention with their potential applications in various life sciences related applications. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system, which has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. In the present study, we have demonstrated the synthesis of gold nanoparticles by a novel bacterial strain isolated from a site near the famous gold mines in India. A promising mechanism for the biosynthesis of GNPs by this strain and their stabilization via charge capping was investigated.
A bacterial isolate capable of gold nanoparticle synthesis was isolated and identified as a novel strain of Stenotrophomonas malophilia (AuRed02) based on its morphology and an analysis of its 16S rDNA gene sequence. After 8 hrs of incubation, monodisperse preparation of gold nanoparticles was obtained. Gold nanoparticles were characterized and found to be of ~40 nm size. Electrophoresis, Zeta potential and FTIR measurements confirmed that the particles are capped with negatively charged phosphate groups from NADP rendering them stable in aqueous medium.
The process of synthesis of well-dispersed nanoparticles using a novel microorganism isolated from the gold enriched soil sample has been reported in this study, leading to the development of an easy bioprocess for synthesis of GNPs. This is the first study in which an extensive characterization of the indigenous bacterium isolated from the actual gold enriched soil was conducted. Promising mechanism for the biosynthesis of GNPs by the strain and their stabilization via charge capping is suggested, which involves an NADPH-dependent reductase enzyme that reduces Au3+ to Au0 through electron shuttle enzymatic metal reduction process.
Synthesis of GNPs and subsequent linkage to biomolecules has contributed immensely in various life sciences related applications such as drug-delivery, gene transfer, bioprobes in cell and tissue analysis for visualization of micro- and nano-objects, and for observation of the biological processes at nano-scale etc. [1–5]. These nanoparticles, in general, are synthesized using a number of synthetic procedures in various polar and non-polar media [6–8]. Recently, there has been tremendous excitement in the study of nanoparticles synthesis by using some natural biological system. This has led to the development of various biomimetic approaches for the growth of advanced nanomaterials. Microorganisms, such as bacteria, yeast and fungi, are known to produce inorganic materials either intra- or extracellularly [9–12]. These microorganisms play an important role in remediation of metals through reduction of metal ions. Some of these microorganisms can survive and grow even at high metal ion concentrations. They are often exposed to extreme environmental conditions, forcing them to resort to specific defense mechanisms to quell such stresses, including the toxicity of foreign metal ions or metals . The toxicity of metal ions is reduced or eliminated by changing the redox state of the metal ions and/or precipitation of the metals intracellularly, thus, forming the basis of the synthesis of nanoparticles . However, the actual mechanism for the biosynthesis of GNPs by different microorganisms and their stabilization via charge capping is still not well understood. It was shown in one of the earlier studies that the possible mechanism of biosynthesis of silver nanoparticles might involve the reduction of silver ions due to the electron shuttle enzymatic metal reduction process. The enzyme involved in the synthesis of silver nanoparticles may be nitrate reductase present in microorganism, which may be induced by the nitrate ions and reduced silver ions to metallic silver . Duran et al also suggested the possible mechanism of biosynthesis of silver nanoparticles by Fusarium oxysporum strains (fungi). They proposed the involvement of enzymatic electron shuttle relationship for the formation of Ag+ ions and the subsequent formation of silver nanoparticle . Although there are reports on the biochemical steps involved in metallic nanoparticles synthesis by microorganisms [17–20], there are virtually no reports available which may elucidate enzymatic basis of gold nanoparticle synthesis by Stenotrophomonas maltophilia.
In this paper, we report the rapid synthesis of GNPs by Stenotrophomonas maltophilia (Acc No GQ220749), a novel bacterial strain isolated from soil samples from the Singhbhum gold mines (located in the Jharkhand state of India). A possible mechanism of biosynthesis of gold nanoparticles from gold chloride (HAuCl4) involves the role of specific NADPH-dependent reductase enzyme present in the organism that converts Au3+ to Au0 through electron shuttle enzymatic metal reduction.
Results and discussion
Isolation and characterization of strain capable of synthesisizing gold nanoparticles
Biochemical tests of the isolated strain Stenotrophomonas maltophila AuRed02
Glucose o/f test
Methyl red test
Carbohydrate utilization tests
Characterization of gold nanoparticles synthesized by Stenotrophomonas maltophilia
Promising mechanism aspects of biosynthesis by Stenotrophomonas maltophilia
The process of synthesis of well-dispersed nanoparticles using a highly efficient microorganism Stenotrophomonas maltophilia has been reported in this study leading to the development of an easy bioprocess for synthesis of GNPs of desired size and shape. The results presented demonstrate that a specific NADPH-dependent enzyme present in the isolated strain reduces Au3+ to Au0 through an electron shuttling mechanism leading to the synthesis of nearly monodispersed GNPs. This green route of biosynthesis of GNPs is a simple, economically viable and an eco-friendly process.
Isolation and characterization of bacteria from the gold enriched soil
Soil samples from the gold enriched sites near famous Singhbhum gold mines, Jharkhand state, India were used as inoculum, serially diluted and plated onto Nutrient Agar media (Himedia, India). The plates were incubated at 30°C for 24 hrs. The colonies obtained were further subcultured on Nutrient Agar supplemented with 1 mM HAuCl4 (Sigma, India) and incubated at 30°C for 24 hrs. In one of the isolates, the light yellow color of HAuCl4 changed to wine red indicating that the organism was utilizing HAuCl4 for the synthesis of GNPs. The morphological and physiological characterization of the selected isolate was carried out by biochemical tests using the Bergeys Manual of Determinative Bacteriology . The cell morphology was investigated using Zeiuss-EVO 40 scanning electron microscope (SEM). Further characterization of isolate was done by means of 16S rRNA gene analyses . The 16S rDNA sequencing was done by M/s Bangalore Genei, India.
To obtain stable gold nanoparticles, the conditions of the characterized isolate were optimized for different concentrations of gold chloride, production media, temperature, pH and time. The characterized isolate was inoculated into 50 ml sterile Nutrient Broth (NB) and subsequently 1 gm of wet biomass was harvested after 16 hrs of incubation. The biomass obtained was washed thrice with deionised water (pH 7.0) and added to a 100 ml Erlenmeyer flask containing 1 mM gold chloride solution prepared in de-ionized water spiked with 400 mg Yeast Extract. The Erlenmeyer flasks were incubated at 25°C for 8 hrs to isolate the nearly monodisperse spherical gold nanoparticles. To isolate the pure GNPs, cells were disrupted using 0.2% (v/v) Triton X-100. The disrupted samples were centrifuged at 3500 rpm for 15 min at 4°C and washed thrice with deionized water to remove cell-debris. The supernatant was used for the characterization of GNPs.
Biosynthesis of gold nanoparticles and their characterization
To determine the peak time-point of maximum gold nanoparticles synthesis, the absorption spectra of the supernatants were taken at different time intervals and recorded using a Hitachi U2800 spectrophotometer. TEM studies were carried out using Jeol 2100 microscope operating at 120 kV accelerating voltage. Samples were prepared by placing a drop of GNPs solutions on carbon-coated TEM grids. The films on the TEM grids were allowed to dry for 5 min at room temperature before analysis. Energy dispersive spectroscopy analysis (EDS) was carried out to confirm the synthesis of gold nanoparticles using FEI E-SEM Quanta 200. Particle size and charge distribution (zeta potential) was analyzed using dynamic light scattering system (Beckman Coulter, USA) by illuminating the colloidal gold solution with He-Ne Laser (633 nm) in a sample cell.
This work was funded by a grant from the joint Indo-Russia Integrated Long Term Programme (ILTP), which we gratefully acknowledge. Authors gratefully acknowledge Mr Kumar Rajesh for necessary technical support.
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