www.ietdl.org Published in IET Nanobiotechnology Received on 11th March 2014 Revised on 4th May 2014 Accepted on 23rd May 2014 doi: 10.1049/iet-nbt.2014.0005

ISSN 1751-8741

Biosynthesis and characterisation of silver nanoparticles using Sphingomonas paucimobilis sp. BDS1 Yujun Gou1, Feng Zhang1, Xiaoyan Zhu1, Xiangqian Li1,2 1

Faculty of Life Science and Chemical Engineering, HuaiYin Institute of Technology, Huaian 223003, The People’s Republic of China 2 Enzyme and Biomaterials Center, Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaian, 223003, The People’s Republic of China E-mail: [email protected]

Abstract: Sphingomonas is a novel and abundant microbial resource for biodegradation of aromatic compounds. It has great potential in environment protection and industrial production. The use of microorganisms for the synthesis of nanoparticles is in the limelight of modern nanotechnology, since it is cost effective, non-toxic and friendly to the ever-overwhelmed environment. In this paper, the biosynthesis of silver nanoparticles (AgNPs) using Sphingomonas paucimobilis sp. BDS1 under ambient conditions was investigated for the first time. Biosynthesised AgNPs were characterised with powder ultraviolet–visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy and energy dispersive X-ray spectroscopy. The overall results revealed that well-dispersed face centred cubic spherical AgNPs in the range of 50–80 nm were produced on the surface of Sphingomonas paucimobilis sp. BDS1, after challenging pure wet biomass with silver nitrate solution. This suggests that the capture of silver ions may be a complex process of physical and chemical adsorption and the proteins on the surface of the bacteria may play the role of reduction and stabilising agent with regard to the result of FTIR.

1

Introduction

Nanoparticles are defined as particles having one dimension smaller than 100 nm [1, 2]. There is an ever-growing interest in the synthesis of nanomaterials because of their unique magnetic, electrical, catalytic, optical, piezoelectric, pyroelectric and photoconducting properties [3–6]. Synthesis of nanoparticles via chemical and physical methods has been employed in nanotechnology because of their affordability and ease of modulation in functional behaviour of nanostructures [7]. However, nanoparticles produced by a biogenic enzymatic process are far superior, in several ways, to those particles produced by the chemical and physical methods. Although the latter two methods are able to produce large quantities of nanoparticles with a defined size and shape in a relatively short time, they are complicated, outdated, costly and produce hazardous toxic wastes that are harmful, not only to the environment but also to human health [8, 9]. It has been emphasised earlier that the biological synthesis of nanomaterials is a quite promising area of modern nanobiotechnology, because this synthetic approach does not require hazardous toxic and expensive chemicals, besides the fact that it is not very energy intensive [10]. There is an increasing need to produce bio-nanoparticles through eco-friendly processes that are highly stable for large scale production [11–13]. IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 53–57 doi: 10.1049/iet-nbt.2014.0005

Silver nanoparticles (AgNPs) are the most widely used engineered nanomaterials [14]. They are commonly incorporated into a wide variety of commercial goods (e.g. personal care products, food containers, laundry additives, clothing, paintings and home appliances [15, 16]) and are also used for water treatment, drug and gene delivery, bone prostheses, dental work, burn wounds treatment, implantable materials, biosensors and bioimaging devices [17, 18]. The size, shape and surface morphology of AgNPs play a vital role in controlling the physical and chemical properties [19]. The synthesis of AgNPs by chemical reduction method was often performed in the presence of stabilising agent to prevent the unwanted agglomeration of colloids. Furthermore, the synthesis of AgNPs over a range of sizes and high monodispersity is one of the most challenging issues in current nanotechnology through chemical reduction. Consequently, biological approaches for size-controlled and well-dispersed AgNPs synthesis have been suggested as valuable alternatives to chemical methods [20]. Sphingomonas degrades dyes, pesticide, herbicide and especially polycyclic aromatic hydrocarbons (PAHs) [21– 24], which are toxicologically important substances that are widely distributed in the environment. It is also able to synthesise a variety of polymers such as gellan gum and xanthan gum [25]. Owing to its strong metabolic capability 53

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www.ietdl.org and various physiological functions, it attracts more and more researchers’ attention and gains increasing importance in the field of biotechnology in recent years. Here we report, for the first time, a green synthesis procedure of well-dispersed and size-controlled AgNPs using Sphingomonas paucimobilis sp. BDS1 and their characterisation.

2 2.1

Experimental details Sample collection and isolation

The samples for isolation of Sphingomonas paucimobilis sp. BDS1 were collected from a sewage treatment plant in Huai An in a sterile container and transported to the laboratory for further processing. The isolation procedure was carried out by standard serial dilution method and spread plate was performed on NO.1 medium (1% peptone, 0.3% beef extract, 0.5% NaNO3, 1% agar and 50 mg/l silver nitrate). The colonies that appeared were further purified by repeatedly streaking on NO.1 medium. 2.2

Molecular characterisation of the isolate

The genomic DNA of Sphingomonas paucimobilis sp. BDS1 was extracted using standard protocol. The 16S rRNA gene was amplified with the primers 27F (5-AGA GTT TGA TCC TGG CTC AG-3) and 1492R (5-GGC TAC CTT GTT ACG ACTT-3). For polymerase chain reaction (PCR), the conditions (30 cycles of 3 min at 94°C, 1 min at 55°C, 1 min at 72°C and a final total cycle for 10 min at 72°C) were performed. The purified PCR product was sequenced by Shanghai Majorbio Bio-pharm Technology Co., Ltd. The phylogenetic position of the isolated strain was assessed by performing a nucleotide sequence database search using the BLAST program from NCBI GenBank.

2.5

After the biosynthesis, samples of washed pellets were freeze-dried and used for characterisation. The localised surface plasmon resonance of AgNPs was characterised by power ultraviolet–visible (UV–vis) spectrophotometer (Shimadzu UV-VIS-NIR UV-3600, Japan) at a resolution of 1 nm in a wavelength range between 250 and 600 nm. The X-ray diffraction (XRD) spectra were recorded in Bruker D8 Advance (Germany) X-ray diffractometer and the X-ray diffracted intensities were recorded from 20° to 80° of 2θ angles. Further characterisation involved Fourier transform infrared (FTIR) spectroscopy analysis of the freeze-dried powder of AgNPs which was extracted and purified by ultrasonic dispersion, by scanning it in the range 400–4000 cm−1 at a resolution of 4 cm−1. The pellets were ultrasonic dispersed with ethanol, dropped on slides, air-dried and examined under field emission scanning electron microscopy (FESEM) (Quanta 250, USA) at 10 KV. In addition, the presence of silver metal in the sample was analysed by energy dispersive X-ray analysis (EDX) combined with FESEM. The numerical data of powder UV–vis, XRD, FTIR and EDX were processed by a software package Origin Pro 8.5.

3 3.1

Preparation of the biomass

For inoculum preparation, a loop of spore suspension of the bacterial culture was transferred from the agar slant into 100 ml sterile medium containing final concentrations of 0.3% NaNO3, 0.13% K2HPO4, 0.05% MgSO4·7H2O, 0.05% KCl, 0.003% FeSO4·7H2O and 3% sucrose in 250 ml Erlenmeyer flask. The flask was incubated at 37°C for 18 h on a rotary shaker at 200 rpm. Then several millilitres of late-log phase culture were inoculated into 500 ml Erlenmeyer flasks containing 200 ml medium. The inoculated flasks were incubated at 37°C, shaken at 200 rpm again for 18 h and then the cells were harvested by centrifugation (10 000 rpm, 10 min at 4°C) and washed three times with Millipore water. Finally, the wet biomass was collected. 2.4

Strain isolation and molecular characterisation

UV–visible spectroscopy

It is well known that AgNPs exhibit a yellowish-brown colour in water; this colour arises because of the excitation of surface plasmon vibrations in the metal nanoparticles [26]. After the wet biomass was exposed to 1 mM silver nitrate solution for 12 h, aqueous silver ions were reduced to AgNPs by sphingnomonas paucimobilis sp. BDS1. This was indicated by the obvious colour change from yellowish to yellowish-brown and verified by the surface plasmon resonance peak at 414 nm of AgNPs in powder UV–vis spectrum shown in Fig. 3.

Synthesis of AgNPs

Typically, 10 g of wet biomass was resuspended in 100 ml of aqueous solutions supplemented with 1 mM AgNO3 in 250 ml Erlenmeyer flasks and kept on rotary shaker (200 rpm) at 37°C in the dark for 12 h until an obvious colour change occurred. Then, the bacterial pellets were collected by centrifugation (10 000 rpm, 10 min at 4°C) and used for making samples for characterisation. 54

Results and discussion

The pure pale-yellow isolate (shown in Fig. 1a) obtained on NO.1 agar plate was identified as rod-shaped bacteria according to its morphological characterisation result of FESEM image (shown in Fig. 1b). It was further characterised as Sphingomonas paucimobilis sp. based on the molecular identification through 16S rRNA sequencing studies shown in Fig. 2. 3.2

2.3

Characterisation of AgNPs

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Fig. 1 Morphology of the isolate a Pure pale-yellow colonies of Sphingomonas paucimobilis sp. BDS1 isolated from sewage b FESEM image of rod-shaped Sphingomonas paucimobilis sp. BDS1 IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 53–57 doi: 10.1049/iet-nbt.2014.0005

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Fig. 2 Phylogenetic relationship based on 16S rRNA gene nucleotide sequences between Sphingomonas paucimobilis sp. BDS1 and reference sequence retrieved from NCBI Gen Bank constructed through neighbour joining method

The homogeneous spherical AgNPs are known to produce the surface Plasmon resonance band at 413 nm [27]. Surrounding conditions have a great influence on the characterisation of nanoparticles [28]. As shown in Fig. 3,

the active pellets which contain microbial biomass and AgNPs showed a peak at 414 nm in powder UV–vis spectrum, whereas the negative control did not, which confirms the synthesis of AgNPs. The band was relatively broad with an absorption tail in the longer wavelengths, which could in principle be because of the size distribution of the particles [29]. The insert photo of Fig. 3 indicated that the Ag+ had been reduced to Ag0 after in contact with the biomass of Sphingnomonas paucimobilis sp. BDS1 for 12 h. 3.3

Fig. 3 Powder UV–vis spectrum of dried powder samples before (negative) and after (active) the 12 h incubation of 1 mM AgNO3, which only the active showed a peak at 414 nm that conforming the formation of AgNPs Insert: active plots of maximum absorbance at 414 nm as a function of time

X-ray diffraction spectroscopy

The formations of the Ag nanoparticles were further confirmed by the XRD analysis. The XRD patterns of Ag nanoparticles using Sphingnomonas paucimobilis sp. BDS1 is shown in Fig. 4a and Fig. 4b shows that of negative control. In Fig. 4a, the characteristic diffraction peaks at 2θ values are 38.30°, 44.46°, 64.67° and 77.49°, which are assigned to the (1 1 1), (2 0 0), (2 2 0), and (3 1 1) planes of crystallographic face centered cubic (fcc) AgNPs, respectively. Their corresponding d-spacing values are 2.359, 2.044, 1.445, 1.231 and 1.180 Å of the AgNPs [30]. The high-intensity diffraction peak was observed at 38.30°, corresponding to the crystalline Ag. The broadening of the peaks was observed because of the effect of nanosized silver particles, which was confirmed by further indexing (Joint Committee on Powder Diffraction Standards file no. 04-0783).

Fig. 4 XRD patterns of biomass with or without AgNPs a XRD pattern of the freeze-dried biomass of Sphingnomonas paucimobilis sp. BDS1 with fcc crystalline AgNPs b XRD pattern of the freeze-dried biomass of Sphingnomonas paucimobilis sp. BDS1, no peaks was observed IET Nanobiotechnol., 2015, Vol. 9, Iss. 2, pp. 53–57 doi: 10.1049/iet-nbt.2014.0005

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www.ietdl.org approximate size of 50 to 80 nm. In our previous research, no AgNPs were found in the solution after treating the biomass with silver nitrate. As a result, these AgNPs appeared outside the biomass due to ultrasonic dispersion when FESEM samples were made. Thus, the result indicates that the reduction process is held in the surface. Consequently, these AgNPs could be easily dispersed outside the cell wall and then extracted and purified. Hence, it was an effective green procedure taking into consideration their controlled size, effective dispersion and the convenience of extracting and purification. The EDX (Fig. 6b) recorded in the spot-profile mode shows signals of Ag apart from C, O, Na and Mg that are likely because of the biomass background. The elements of Ca, Si were from the glass slide and Au was from the coating background. Fig. 5 FTIR spectrum of AgNPs synthesised by Sphingomonas paucimobilis sp. BDS1

3.4

Fourier transform infrared spectroscopy

Fig. 5 exhibited the FTIR spectrum of the samples. The bands seen at 3373 and 2928 cm−1 were assigned to the stretching vibrations of primary and secondary amines, respectively, while their corresponding bending vibrations were seen at 1663 and 1545 cm−1, respectively. The two bands observed at 1394 and 1073 cm−1 can be assigned to the C–N stretching vibrations of aromatic and aliphatic amines, respectively. Nevertheless, the overall observation confirms the presence of protein in the sample. In previous research, the capture of silver ions of microbe was confirmed to be a complex process of physical and chemical adsorption [31]. Additionally, it is reported that proteins can bind to nanoparticles either through free amine groups or cysteine residues in the proteins and via the electrostatic attraction of negatively charged carboxylate groups in proteins present in the cell wall of microbes [32]. Therefore, a complex process of physical and chemical adsorption of silver ions by Sphingomonas paucimobilis sp. BDS1, followed by reducing and stabilising the AgNPs by proteins on the surface of cell wall, may have occurred in this biosynthesis process. 3.5 Field emission scanning electron microscopy and energy dispersive X-ray spectroscopy The FESEM examination gave the electron micrograph (Fig. 6a) of the AgNPs on the surface of the Sphingomonas paucimobilis sp. BDS1 and in the surroundings with

Fig. 6 FESEM and EDX examination a FESEM image of AgNPs on the surface of the biomass and in the surroundings, which were 50–80 nm in size and well dispersed b EDX spectrum of samples recorded in the spot-profile mode, which showed the signals of Ag clearly 56

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4

Conclusion

In conclusion, we have reported the simple and green biological method for synthesising AgNPs using Sphingomonas paucimobilis sp. BDS1 for the first time. When challenging the biomass with silver nitrate, it reduced toxic silver ions to AgNPs with obvious colour change. These biosynthesised AgNPs were further characterised by powder UV–vis, XRD, FTIR, FESEM and EDX. All characterisation results indicated the existence of AgNPs, which were 50–80 nm in size, well-dispersed and with a fcc crystal structure.

5

Acknowledgment

This work was supported by Chinese National Science Foundation (Grant No. 20971050).

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