Biosynthesis of bismuth nanoparticles using Serratia

www.ietdl.org Published in IET Nanobiotechnology Received on 27th December 2010 Revised on 13th September 2011 doi: 10.1049/iet-nbt.2010.0043

ISSN 1751-8741

Biosynthesis of bismuth nanoparticles using Serratia marcescens isolated from the Caspian Sea and their characterisation P. Nazari1 M.A. Faramarzi1 Z. Sepehrizadeh1 M.R. Mofid 2 R.D. Bazaz 3 A.R. Shahverdi1 1

Department of Pharmaceutical Biotechnology and Biotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Biochemistry, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran 3 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran E-mail: [email protected]

Abstract: Today, synthesis of nanoparticles (NPs) using micro-organisms has been receiving increasing attention. In this investigation, a bismuth-reducing bacterium was isolated from the Caspian Sea in Northern Iran and was used for intracellular biosynthesis of elemental bismuth NPs. This isolate was identified as non-pigmented Serratia marcescens using conventional identification assays and the 16s rDNA fragment amplification method and used to prepare bismuth NPs. The biogenic bismuth NPs were released by liquid nitrogen and highly purified using an n-octanol water two-phase extraction system. Different characterisations of the purified NPs such as particle shapes, size and purity were carried out with different instruments. The energy-dispersive X-ray and X-ray diffraction (XRD) patterns demonstrated that the purified NPs consisted of only bismuth and are amorphous. In addition, the transmission electron micrograph showed that the small NPs formed larger aggregated NPs around ,150 nm. Although the chemical syntheses of elemental bismuth NPs have been reported in the literature, the biological synthesis of elemental bismuth NPs has not been published yet. This is the first report to demonstrate a biological method for synthesising bismuth NPs and their purification with a simple solvent partitioning method.

1

Introduction

An exciting strategy in nanotechnology has contributed to the synthesis of metal nanoparticles (NPs) with particular characteristics. Among the synthetic methods, synthesis of metal NPs and other nanomaterials by micro-organisms compete with other techniques because of the method’s environmental impact such as clean, non-toxic and ecofriendly features [1 – 4]. Today, there has been increasing interest in the use of metal NPs in different health materials or industrial products such as antimicrobials, catalysts, lubricants and microelectronics instruments [2, 5, 6]. Among the elements, semimetal bismuth has many applications. For example, it is used in electronics for its highly anisotropic behaviour and low effective mass [7]. Moreover, bismuth compounds, such as bismuth subsalicylate and bismuth subcitrate have been widely used in treating gastrointestinal disorders since the 18th century [8]. Recently, many reports have been published in the literature on the synthesis of bismuth NPs from bismuth ions using chemical and radiolytic reducing methods, electron irradiation and microwave treatment [9]. However, the synthesis of bismuth NPs by micro-organisms has not yet been investigated and should be understood. In this study, we screened different samples from the Caspian Sea to isolate a bismuth NP-producing micro-organism. The 58

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isolated strain was identified and employed for preparing bismuth NPs. Biogenic bismuth NPs were extracted and purified from cell debris using a liquid – liquid phases-partitioning method and characterised by different methods such as UV – visible (UV – Vis) spectroscopy, transmission electron microscopy (TEM), energy-dispersive spectroscopy, X-ray diffraction (XRD) analysis and laser light scattering technique for determining particle size.

2 2.1

Materials and methods Screening of bismuth NPs-producing bacteria

Freshwater samples from the Caspian Sea in northern Iran were collected in sterile bottles during the summer of 2008. Portions of collected sea samples water were aseptically transferred to fresh CASO broth media (Merck, Germany), and all inoculated media were incubated aerobically at 308C at 150 rpm for 24 h. Nutrient agar (NA) medium (1000 ml) (Merck, Germany) was prepared and sterilised at 1218C for 20 min. The sterile NA medium was then allowed to cool to 458C. Subsequently, 1000 mg of sterile bismuth subnitrate (Merck, Darmstadt, Germany) was aseptically weighted and added to the above NA medium. The prepared bismuth supplemented agar media (1 mg/ml) was dispensed in sterile Petri dishes, and a screening programme applied. IET Nanobiotechnol., 2012, Vol. 6, Iss. 2, pp. 58– 62 doi: 10.1049/iet-nbt.2010.0043

www.ietdl.org To isolate the bismuth-producing bacteria, several prepared NA plates containing bismuth subnitrate (1 mg/ml) were streaked with a loop full of the above-cultivated CASO broth. All plates were incubated aerobically for 48 h at 308C. Conversion of Bi+3 to elemental bismuth leads to form brownish colony and this colour was preliminary used as provisional marker for screening of Bi+3-reducing bacteria. In the next step, a single dark brown colony was selected and re-cultivated in the Bi+3 supplemented NA plates (1 mg/ml) at the described conditions to obtain a pure culture (48 h, 308C). The organism was maintained on the Bi+3-supplemented NA medium plates (1 mg/ml) at 48C for further experiments. 2.2

Identification of bacterial isolate

The following phenotypic and physiological characterisations of the isolate were carried out according to the methods described in determinative bacteriology textbooks [10, 11]: Gram staining, cell morphology, pigmentation, oxygen requirements by fluid thioglycollate medium, motility, oxidase test, lactose fermentation, indole test, urease test, lysine decarboxylase test, H2S production and growth at 4 and 408C. In addition, the identification procedures were further confirmed by the 16s rDNA fragment amplification method using polymerase chain reaction (PCR). A single colony was picked up and suspended in 50 ml of distilled water and lysed by heating at 958C for 10 min. Cell lysates, after centrifugation, were used for PCR amplification. Amplification of 16S rDNA was performed using the following forward and reverse primers: Forward 5′ CAAgTCgAgCggTAACACAg 3′ Reverse 5′ CgTATTCACCgTAgCATTCTg 3′ The reaction mixture was composed of 2 ml of lysed cell suspension, 1 ml of each forward and reverse primers, 12.5 ml PWO master (Roche, Germany) and distilled water up to 25 ml. The mixture was incubated at 948C for 2 min and then cycled 30 times as the following profile: 948C for 20 s, 568C for 30 s, 728C for 1.5 min, and then incubated for 7 min at 728C. The amplified DNA fragments were purified from agarose 1% gel using the QIAquick gel extraction kit (Qiagen, USA) according to the supplier’s instructions and was sent for automated sequencing using the above primers (Gen Fanavaran Co., Iran). The obtained 16S rDNA sequence was aligned with nucleotide databases using the BLAST program, and the sequence was submitted to GenBank. 2.3

liquid nitrogen, the pellets were frozen and then were disrupted with a pestle. The resulting slurry was centrifuged at 5000 g for 15 min to remove some soluble intracellular components. Subsequently, the pellets containing bismuth NPs and cell debris were suspended in a two-phase partitioning system of 1:2 n-octyl alcohol and distilled water. Then the mixture was shaken vigorously. The two mixed phases were completely separated by centrifuging at 5000 g for 10 min. After this process, the biogenic bismuth particles were observed at the bottom of the tubes. The supernatants were discarded, and the settled NPs were sequentially washed with chloroform, ethyl alcohol and distilled water. The cleaned bismuth NPs were resuspended in deionised water and subjected to further characterisation assays.

Synthesis of bismuth NPs using bacterium

Bismuth NPs were prepared by reducing the bismuth subnitrate using the bacterial isolate from the Caspian Sea. In the first step, sterile nutrient broth (Merck, Germany) was supplemented with the bismuth subnitrate (0.2% (w/v)) and inoculated with a loopful of the mentioned bacterial isolate (2 – 3 colonies of a fresh overnight culture). This inoculated liquid medium was incubated aerobically at 308C in a shaker incubator (150 rpm). After 48 h, bacterial cells were separated from the culture medium by the centrifugation process (8000 rpm, 4000 g, 10 min). The pellets were washed with NaCl solution (0.9% (w/v)) and centrifuged at the same condition. The washing process was repeated three times to ensure the removal of any undesirable materials. Cell pellets were transferred to a mortar, and by adding some IET Nanobiotechnol., 2012, Vol. 6, Iss. 2, pp. 58 –62 doi: 10.1049/iet-nbt.2010.0043

2.4

Characterisation of bismuth NPs

The UV – Vis spectra of the purified bismuth NPs suspension in the wavelength range 200 – 400 nm [12] were measured on a Labomed Model UVD-2950 UV – VIS double beam PC scanning spectrophotometer, operated at a resolution of 1 nm. These NPs were also characterised with TEM (model EM 208 Philips). Before characterisation, aqueous suspension containing the bismuth NPs was passed through the ultrasonication process, and a few drops of suspension were deposited on carbon-coated copper TEM grids and dried at room temperature. Micrographs were achieved using a TEM (model EM 208 Philips) operated at an accelerating voltage at 100 kV. To observe the NPs’ surface features and to determine the NPs’ elemental composition, a scanning electron microscope (SEM) equipped with energy-dispersive X-ray spectroscopy (EDX) was employed. For SEM observation, NPs were mounted on specimen stubs and coated with gold in a sputter coater device model SCD 005 (BAL-TEC, Switzerland). Samples were analysed with SEM (Philips XL30, Netherlands) operated at 16 kV, and EDX was recorded by focusing on a cluster of NPs. The crystalline structure of the bismuth NPs was investigated with the XRD technique using an X-ray diffractometer (Philips X’Pert Pro, Netherlands) ˚ ) over a scanning range of with CoKa radiation (l ¼ 1.78 A Bragg angles from 108 to 1108. The generator was operated at 40 kV with a 30 mA current.

3 3.1

Results and discussion Identification of the microorganism

Although different water samples were collected from the Caspian Sea, only one sample (Bis55) from the northern part of Ziba Kenar contained a bacterium with the ability to convert bismuth ions to elemental bismuth. Fig. 1 shows the colonies of this isolate on the plain NA medium (plate A) and bismuth-supplemented NA medium (plate B). In microbiological examinations, the strain had Gram-negative rods with no pigmentation, and showed good growth on sheep blood agar, MacConkey agar and eosin methylene blue agar media. The isolate was facultative and grown in a temperature range of 25– 408C. In addition, this strain was negative in the oxidase, lactose and urease tests. Furthermore, no H2S production and no indole fermentation were observed in the biochemical diagnostic tests for this isolate. In contrast, this isolate was motile and halotolerant up to 7% NaCl, and the lysine decarboxylase test was positive. Further study showed that this strain did not produce red pigments (prodigiosin) at different temperatures. 59


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Fig. 1 Photograph from NA with (plate B) and without (plate A) bismuth subnitrate streaked with isolated S. marcescens This isolate reduced bismuth ions to elemental bismuth and formed brownish colonies (plate B)

These results were in agreement with non-pigmented Serratia sp. based on the standard diagnostic tests for identifying bacteria [10, 11]. After the amplification of bacterial 16S rDNA with PCR, an amplified fragment of about 1250 bp was developed. It was aligned to sequences in GenBank, and the alignment further confirmed that the isolated bismuth ion-reducing bacterium was representative of Serratia marcescens. The sequence was submitted to GenBank and assigned the following accession number: GQ471957. Alignment results revealed 99% identity with the S. marcescens bacterium. 3.2

the extracted bismuth NPs at different concentrations (0.5, 1 and 2 mM) are shown in Fig. 3. The spectra obtained show minor absorption peaks at 263, 254 and 262 nm, which are supported by previous work reported in the literature [12, 13]. Fig. 4 shows the corresponding size distribution of bismuth NPs measured by laser light scattering after partitioning and purification by the liquid –liquid two-phase system. The purified bismuth NPs showed a bimodal size distribution pattern. In these bimodal size distributions, two peaks were observed; one was at an average size of 122 nm, and another was at an average size of 342 nm. It seems bismuth NPs may be formed as aggregated particles or may be aggregated during the separation process using the two-phase partitioning solvent system. Therefore purified bismuth NPs were further subjected to ultrasound treatment and further investigated using TEM. Fig. 5 shows the representative TEM images of purified bismuth NPs prepared by S. marcescens with different magnification. The lower TEM image with higher magnification shows small non-spherical-shaped bismuth NPs that aggregated and formed irregular particles whose size range was ,150 nm. The EDX analysis of the biogenic bismuth NPs confirmed

Bismuth NPs’ preparation and characterisation

Fig. 2 shows the test tubes containing the bismuth NPs enriched bacterial cell extract obtained with the disruption process by liquid nitrogen (tube A), and this slurry before (tube B) and after (tube C) separation by n-octyl alcohol. Cell debris materials dissolved in the n-octyl alcohol – water system or remained at the interface of the aqueous and alcoholic phases in the first run of the separation of the bismuth NPs (tube C). However, after the solvent extraction and centrifugation process, bismuth NPs was steeled in the bottom of the test tube (tube C). The UV – Vis spectrum of

Fig. 3 UV –Vis absorption spectra of biogenic bismuth NPs colloids measured at different concentrations

Fig. 2 Photograph from test tubes containing bismuth-enriched cell extract (tube A) before (tube B) and after the separation process (tube C) with the two-solvent phase-partitioning system 60


Fig. 4 Bismuth NPs’ size distribution pattern obtained by laser light scattering technique IET Nanobiotechnol., 2012, Vol. 6, Iss. 2, pp. 58– 62 doi: 10.1049/iet-nbt.2010.0043

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Fig. 5 Transmission electron micrographs of biogenic bismuth NPs purified by the two-solvent phase system recorded with different magnification

the presence of elemental bismuth signals (left-hand illustration in Fig. 6). EDX microanalysis of the purified bismuth NPs exhibited optical absorption bands with peaks at 2.4, 11.8 and 14 keV, which is typical of the absorption of metallic bismuth [14, 15]. The SEM image of the purified bismuth NPs showed the particles with irregular shapes created some aggregates with various lengths, and single spheres were rarely seen (right-hand image in Fig. 6). The XRD pattern of the bismuth NPs presented the presence of broad peaks without any clear crystalline structure. This pattern indicated the amorphous phase of bismuth NPs (Fig. 7).

In this study, the ability of a marine isolated bacterium identified as S. marcescens to form bismuth NPs was investigated. Many reports have been published in the literature on the bacterial synthesis of metal NPs. The biosynthesis of metal NPs is considered a green technology and not only carried out by bacteria but also can be performed by different fungi or higher organisms, such as plants [16, 17]. For example, research on the biosynthesis of silver NPs [2, 18, 19], titanium NPs [20], selenium NPs [4, 21] and gold NPs [22, 23] has been recently published by different authors. According to our literature survey, the biosynthesis of bismuth NPs has not yet been investigated. In addition, there are few reports published in the literature on the chemical synthesis of bismuth NPs in which different reducing agents such as sodium borohydride have been used to form bismuth NPs [5, 7, 24]. Bismuth is a non-essential semimetal ion for bacteria and has toxic effects against some species [8, 25]. The detoxification potential of different bacterial strains for metal oxyanions has previously been reported [21]. Bismuth as a toxic metal can also be accumulated by micro-organisms in their cytoplasm or in other intracellular compartments such as the cell wall [8, 25]. At present, the mechanism of accumulation by S. marcescens is not known and should be further investigated. To uptake to the cells, bismuth ions may compete with other ions [8, 25]. In this investigation, S. marcescens was observed to reduce the bismuth ions and form intracellular bismuth NPs. One of the most essential steps in the microbial synthesis of NPs is recovery of biogenic NPs. It has been proved that biphasic systems have great potential for efficient partitioning of bioproducts and NPs, and we have recently used this biphasic systems for successful recovery and purification of selenium NPs from Bacillus sp. MSh-1 [4]. Similarly, in the current study, based on the lipophilic nature of the cell debris and insolubility of bismuth NPs in the aqueous or organic phases, we used an organic-aqueous partitioning system. In addition, according to the EDX results (left illustration in Fig. 5), the NPs just composed the bismuth atoms. A minor absorption band in the UV–Vis spectrum was observed for the biogenic bismuth NPs samples prepared at different times (Fig. 3). Differences between the size distribution pattern measured by the laser light scattering technique and the TEM images are because of significant NP aggregation, which has been discussed as a reason for broadening of the UV–Vis spectrum and bimodal shape in size distributions pattern measured with the laser light scattering technique. However, TEM images of purified bismuth NPs showed small irregularly shaped bismuth NPs with considerable aggregation (Fig. 5). The significant aggregation between elemental

Fig. 6 Scan electron microscopy image (left) and EDX spectrum of biogenic bismuth NPs prepared by S. marcescens IET Nanobiotechnol., 2012, Vol. 6, Iss. 2, pp. 58 –62 doi: 10.1049/iet-nbt.2010.0043

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Fig. 7 XRD pattern of purified bismuth NPs prepared by S. marcescens

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bismuth NPs may be associated with strong diamagnetite or magnetite properties of bismuth at the nanoscale level. However, the reason for this aggregation has not been revealed during this study and should be investigated in future work.

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Conclusion

We screened and employed S. marcescens for biosynthesis of bismuth NPs as a green synthetic method. Prepared NPs were released and purified using a two-phase partitioning system. TEM images showed groups of small NPs aggregated and formed larger particles ranging in size ,150 nm. EDX spectrum and XRD analysis of the purified samples indicated that amorphous elemental bismuth was prepared using bacterial isolates from the Caspian Sea. To the best of our knowledge, based on the literature survey, this is the first report on the biosynthesis of bismuth NPs using a microorganism and its characterisation with different instrumental devices.

5

Acknowledgments

This work was supported by a grant (no. 7178) from Tehran University of Medical Sciences. We also thank K. Mollazadeh Moghaddam for his assistance in the collection of samples from northern Iran.

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