Bioprocess Biosyst Eng DOI 10.1007/s00449-014-1124-6

ORIGINAL PAPER

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity Palanivel Velmurugan • Mahudunan Iydroose • Mohmed Hanifa Abdul Kader Mohideen • Thankiah Selva Mohan • Min Cho • Byung-Taek Oh

Received: 28 December 2013 / Accepted: 7 January 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract This study highlights the ability of nitratereducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag? ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag? ion and 720 min. The UV–Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM– EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible Electronic supplementary material The online version of this article (doi:10.1007/s00449-014-1124-6) contains supplementary material, which is available to authorized users. P. Velmurugan  M. Cho  B.-T. Oh (&) Division of Biotechnology, Advanced Institute of Environment and Bioscience, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan, Jeonbuk 570-752, South Korea e-mail: [email protected] M. Iydroose Department of Environmental Sciences, Bharathiar University, Coimbatore, Tamil Nadu 641-046, India M. H. A. K. Mohideen Department of Microbiology, Sadakathullah Appa College, Tirunelveli, Tamil Nadu 627002, India T. S. Mohan Department of Zoology, Rani Anna Govt. Arts and Science College, Tirunelveli, Tamil Nadu 627008, India

for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria. Keywords Bacillus subtilis  Bioreduction  Silver nanoparticles  MIC and MBC

Introduction Bionanotechnology is a new branch of nanotechnology and biotechnology combined together for enhancing environmentally benign technology for the synthesis of nanomaterials with specific functions. The diverse application of these materials as catalysts, sensors and in medicine depends critically on the size and composition of the nanomaterials [1]. The chemical method of synthesis leads to the presence of toxic chemical species absorbed on their surface which may have adverse effects in industrial application [2–4]. The use of toxic chemicals on the surface of nanoparticles and non-polar solvents in the synthesis procedure limits their applications in clinical fields [5]. Hence, the development of clean biocompatible, non-toxic and environmental-friendly methods for nanoparticles synthesis deserves merit. Since biological methods are regarded as safe, cost-effective, sustainable and environment friendly processes [6]. Researchers have focused their attention on biological systems in the past several years for the low-cost, energy-efficient, and non-toxic production of metallic nanoparticles [4,

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7]. Microorganisms such as bacteria, actinomycetes, and fungi are investigated in metal nanoparticles synthesis [2, 3, 8]. Green synthesis of nanoparticles has thus 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 [1, 9]. Extracellular production of metal nanoparticles has more commercial applications in various fields. Since polydispersity is the major concern, it is important to optimize the conditions for monodispersity in a biological process [10]. In case of intracellular production, the accumulated particles are of particular dimension and with less polydispersity [8]. Among the milieu of natural resources, prokaryotic bacteria have been most extensively researched for the synthesis of metallic nanoparticles [7]. Earlier several studies have reported the production of AgNPs by Bacillus subtilis 168 [1], Bacillus sp. [11], Bacillus stearothermophilus [11, 12] and Bacillus licheniformis [13, 14, 16]. Although it has been reported that metal silver is resistant to most microbial cells and can be used as a biocide or antimicrobial agent [1, 14], reports reveal that metallic silver is resistant to several bacterial strains [1, 13] and accumulates silver in the cell wall up to 25 % dry weight of the biomass, showing the possibility of separation of silver from its ore. In this study, we have made an attempt to investigate the microbial reduction of metallic silver into crystalline silver using nitrate-reducing B. subtilis EWB-46 isolated from municipal waste and the antimicrobial activity was assessed.

Materials and methods The bacteria culture, B. subtilis EWB-46, was obtained from municipal wastes (waste dumped outside of the city), Pollachi, TamilNadu, India. Isolation of total heterotrophic bacteria was done employing serial dilution followed by spread plate technique in nutrient agar. The isolated strains were inoculated in nutrient broth and incubated at 37 °C for 24 h on an orbital shaker at 220 rpm. After incubation the cultures were used for nitrate reductase assay (Nitrite/ Nitrate Assay Kit, colorimetric), and cell-free extract was separated by centrifugation at 9,060g for 10 min and assessed for the synthesis of AgNPs. The potent nitratereducing isolate was subject to reduction of Ag? ion (AgNO3); about 1 mM of Ag? ion (40 ml) was mixed with the obtained cell-free extract (10 ml). To obtain maximum AgNPs production, various process parameters were optimized: pH (5, 6, 7, 8, 9, 10,11 and 12), temperature (20, 30, 40, 50, 60, 70, 80 and 90 °C), metal ion concentrations (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and 5.0 mM) and time in min (60, 120, 240, 300, 360, 420, 480, 540, 600, 660, 720 and 780). Synthesis of AgNPs was monitored by visual inspection for color change from a clear light to

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brown with yellow shade and by measuring the absorbance maximum at 424 nm in UV–Vis spectra by drawing 3 ml (UV-1800 UV–Vis spectrophotometer, Shimadzu, Japan). All the chemicals, media, and analytical reagents used in this present work were purchased from Hi-Media Laboratories Pvt. Ltd. (Mumbai, India). Characterization and identification of the isolate The morphology and physiological characterization of the obtained isolate was performed according to the method described in Bergey’s manual of determinative bacteriology [15, 16]. The genomic DNA of the isolate was extracted by methods described in earlier reports [17]. In brief, the 16S ribosomal RNA gene was amplified by using the PCR method with Taq DNA polymerase and with suitable primers. The PCR product obtained was sequenced by an automated sequencer ABI PRISM (Model 3700). The sequences were compared using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/) SDS gel electrophoresis The SDS gel electrophoresis was performed according to Paulkumar et al. [17]. In brief, About 100 ml of cell-free extract was chilled and saturated using ammonium sulfate with continuous stirring for 1 h, kept overnight at 4 °C and then centrifuged at 9,060g for 15 min to obtain the precipitate. The precipitated protein was dialyzed using dialysis membrane after dissolving with 1 M phosphate buffer (pH 7.0). After dialysis, the obtained desalted protein was concentrated against crystals of sucrose and kept in the refrigerator at 4 °C for further assay. The protein was investigated by running the samples in SDS-PAGE as per standard procedure. After running of protein sample in the gel, the gel was stained with Coomassie brilliant blue and observed under gel documentation system. Characterization of silver nanoparticles The synthesized nanoparticles were further characterized by UV–Vis spectrum, SEM–EDS, FT-IR, TEM, AFM, XRD and NMR analysis. Energy-dispersive (EDS) spectroscopy was performed on a scanning electron microscope (LEO 1450VP, ZEISS, UK). The FT-IR spectrum of the silver nanoparticles was recorded on IRAffinity-1, Shimadzu, Japan, using KBr pellets and the spectrum was collected at a resolution of 4 cm-1 in the wave number region of 400–4,000 cm-1. TEM images were acquired on JEOL JEM 2100, Japan. The size and the surface topography of the drop-coated film of the silver nanoparticles were investigated with an atomic force microscope (AFM) (NTMDT Ntegra AFM system, The Netherlands). The powder

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X-ray diffraction (XRD) pattern of silver nanoparticles was recorded by PAN analytical X-ray diffractometer (XPERT PRO, The Netherlands). 1H NMR and 13C NMR spectra were recorded on Bruker AV 500 [500 MHz (1H) and 125 MHz (13C)] spectrometer using tetramethylsilane (TMS) as an internal reference. The chemical shifts are expressed in parts per million (ppm). Minimal inhibitory concentration/minimum bactericidal concentrations Two gram class of bacteria, P. fluorescens (gram negative bacteria) and S. aureus (gram positive bacteria), were used. These were obtained from the Postgraduate Practical Laboratory, Selvam College, Tamil Nadu, India, to examine the MIC and MBC of the obtained AgNPs. The MIC of the AgNPs was determined by applying the MTT assay using a 96-well microtiter plate [18]. 0.5 ml of the filtered (syringe filter) cell-free extract was used as negative control in all experiments. 5 ml of Luria-Bertani broth containing 10– 100 lg/ml of AgNPs was prepared with appropriate dilutions. For the determination of MIC, a single isolate from the Luria-Bertani agar plates was suspended and inoculated into 50 ll of Luria-Bertani broth. After 24 h of incubation, suspensions were diluted with Milli-Q Ultrapure water to obtain final inoculums of 5 9 105–5 9 106 Cfu/ml. Purity examination of the isolates were performed by gram staining throughout the study. Twofold serial dilutions of AgNPs solution was prepared in Luria-Bertani broth in 98-well plates starting from a stock solution of 10-2 M. A microtiter plate containing 0.05 ml of the serial compound dilution was filled with an equal volume of each bacterial inoculum. After incubation for 18–24 h at 35 °C, MIC was determined with POLARstar OPTIMA microplate reader (BMG LABTECH GmbH, Germany). The absorbance was compared with the positive control wells (broth containing inoculum with AgNPs) negative control wells (broth containing inoculum without AgNPs) [19–22]. To perform MBC, 10 ml of LB broth was added to a 50 ml sterile flask. The obtained AgNPs (0.05 g) were dispersed into the culture-containing flask and shaken for 24 h at 37 °C. Then, 100 ll of the culture from the flask was spread on nutrient agar medium (Difco TM LB Agar) and incubated. After incubation the colony-forming units (CFU) was counted with suitable dilution and reported as CFU/ml. The percentage of activity of bacterial reduction was calculated and expressed [23] as follows: 

Antibacterial activity ð%Þ ¼

1

Results and discussion A total of 56 isolates, B. subtilis EWP-46 were identified as potent nitrate reducers and was used for further study. The isolate (EWP-46) was named according to our convenience for further processing of isolates from EWP 1–56. When aqueous Ag? ion was added to the cell-free extract, the color change was noticed from clear to brown (Fig. 1 and inset) and indicated by the surface plasmon resonance (SPR) of metallic AgNPs at 420 nm. Through evaluating different pH values for the biosynthesis of AgNPs, it was found that pH 10 showed the maximum absorbance; the absorbance increased when pH increased from 5 to 10 (Fig. 2a) and decreased with increasing pH. This shows that the synthesis of silver nanoparticles is favored by an alkaline environment. This result was corroborated by Gurunathan et al. [24], who showed that maximum AgNPs production was obtained at pH 10 using the culture supernatant of E. Coli. Earlier, Gurunathan et al. [24] and Sanghi and Verma [25] reported that the reduction of metallic ions was sensitive to hydrogen ion concentrations and the morphology of the product. The proteins involved in synthesis may bind with silver at the thiol regions (–SH) forming a –S–Ag bond, a clear indication of what aids the conversion of Ag? to Ag0. In addition, the alkaline ion (–OH) is very much required for the reduction of metal ions. Additionally in alkaline condition, the enzymatic activity is high for the reduction of metal ions [25]. We identified a suitable temperature for maximum AgNPs production, and 60 °C favored the maximum absorbance. When the temperature increased from 2 to 60 °C, the absorbance of the reaction mixture increased (Fig. 2b). The result suggested that increase in temperature decreased the reduction process. This could be due to the inactivation or

 CFU =ml ðsampleÞ  100: CFU /ml ðcontrolÞ

The mean values of three independent replicates were expressed. Each experiment was performed in triplicate and the mean ± standard deviation (n = 3) recorded.

Fig. 1 UV–visible spectra of the Bacillus subtilis strain EWP-46 cell-free extract with Ag? recorded as a function of reaction time. Inset: reduction of silver ions as evidenced by color change

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Fig. 2 a Effect of pH, b temperature, c Ag? ion concentration, and d time for AgNPs production

degradation of biomolecules responsible for the reduction of silver. While evaluating the effect of different concentrations of Ag? ion in the reaction mixture, it was found that 1.0 mM Ag? ion showed the highest optical density (Fig. 2c). The maximum absorbance was recorded in 720 min at 420 nm and showed maximum AgNPs production (Fig. 2d); with increase in incubation time, the production increased. EDS analysis (Fig. 3) shows the elemental composition profile of the synthesized nanoparticles, which suggests silver to be the constituent element. The optical absorption peak near 3 keV indicates the presence of nano-sized metallic silver in pure form [19]. The TEM image of AgNPs is shown in Fig. 4a and AFM images are shown in Fig. 4b. AgNPs obtained from the optimized reaction parameters were used for TEM analysis. The synthesized AgNPs were more or less uniformly shaped and sized, but few were found to have irregular shapes (Fig. 4a). The average particle size was found to be between 10 and 20 nm. The irregular particle size could be due to different phases of growth [26]. Various shapes and size of nanoparticles obtaining through biological route are common, however it depends on the concentration ratio of the reducing agent used [27]. However, comparison of this

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difference is very scanty in other reports on biological synthesis of AgNPs [26, 27]. The surface topology of the formulated silver nanoparticles was studied by atomic force microscopy (AFM) analysis (Fig. 4b). Fourier transform infrared (FT-IR) analysis was performed to identify the possible biomolecules responsible for the reduction and capping of the reduced AgNPs synthesized using B. subtilis EWP-46 cell-free extract. Figure 5a shows the FT-IR spectra recorded of bacterial cell-free extract. The FT-IR spectrum of biosynthesized nanoparticles is shown in Fig. 5b, which demonstrates the band in the region of 1,520–1,629 cm-1 for the carbonyl group and 3,275– 3,800 cm-1 for the organic functional group such as hydroxyl or –NH groups. The peak at 2,460–2,784 cm-1 region of the spectrum corresponds to the P–H group. The peak at 3,275 cm-1 may be due to alcohols, phenols or carboxylic acids. Peaks between 1,400 and 1,800 cm-1 are a signal of C=O stretch, which was probably due to ketones [1, 28]. The peak at 1,590 cm-1 indicates the coupling of C–O and C–C stretches and 1,629 cm-1 indicates C=O stretch. A peak between 3,555 and 3,140 cm-1 shows the N–H stretch. The 1H NMR spectrum of the cell-free extract of B. subtilis EWP-46 (13C and 1H NMR data are attached to electronic supplementary material) displayed signals of

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0–70 ppm indicating the presence of alkane (CH3), the peaks from 70 to 160 indicated the presence of aromatic C and C=C, and peaks at 160–210 indicated aldehydes and ketone carbonyl group (C=0). 1H NMR carbon protons at 2.5–4.5 ppm and 6.5–8.0 ppm signals indicate (electronegative) N, O, and Cl. NMR peak interpretation was done with this website http://www.chem.ucalgary.ca; http:// www.chemwiki.ucdavis.edu. 1H NMR peak interpretation was taken from the website:. In conclusion, FT-IR and NMR data show that O–H, C=O, C–O and N–H groups

were present in the organic compounds, which act as a capping agent in biosynthesized silver nanoparticles. The proteins from B. subtilis EWP-46 cell-free extract were extracted by ammonium precipitation method and the molecular weight of the protein identified by SDS-PAGE (Fig. 6). In the figure, lane 1 contains the marker protein and lane 2 loaded with purified protein exhibits an intense band with a molecular weight of 43 kDa which might be the nitrate reductase enzyme responsible for the synthesis of silver nanoparticles [29]. The X-ray diffraction pattern of the AgNPs synthesized by B. subtilis EWP-46 cell-free extract is shown in (Fig. 7). A number of strong Bragg reflections can be seen which correspond to the (111), (200), (220), and (311) reflections of face-centered cubic (fcc) silver. Four peaks at 2h values of 37.26°, 48.86°, 57.82°, and 58.94° corresponding to (111), (200), (220), and (311) planes of silver were observed and compared with the standard powder diffraction card of the Joint Committee on Powder Diffraction Standards (JCPDS), silver file No. 04–0783. The XRD study confirms the presence of the obtained AgNPs in the crystalline form [1]. Antibacterial assay

Fig. 3 EDS spectrum of AgNPs

Fig. 4 a TEM image of the silver nanoparticles synthesized using Bacillus subtilis strain EWP-46 culture supernatant. Scale bar corresponds to 20 nm. b AFM image corresponds to silver

The MIC of synthesized AgNPs is determined in MTT assay using 96-well microtiter plate against P. fluorescens and S. aureus. The AgNPs exhibited the lowest MIC against both the bacteria (P. fluorescens 129 ± 10.8 lg/ml and S. aureus 116 ± 12.6 lg/ml), suggesting good antibacterial potential against both gram classes of bacteria studied. The metal silver is having antimicrobial properties in its own state and it have been using as a antimicrobial agent from last century. The other possible mechanism for

nanoparticles synthesized by Bacillus subtilis EWP-46 showing the nanoparticles with diameter of approximately up to 20 nm

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Bioprocess Biosyst Eng Fig. 5 FT-IR spectrum: a supernatant alone and b silver nanoparticles synthesized by Bacillus subtilis EWP-46 culture supernatant

antimicrobial activity by AgNPs was explained by Li et al. [5]; Kumar et al. [19] as a process of penetration into cell damage the cell membrane and release the cell content. For MBC, the colony count of control was 31 200 CFU ml-1, which decreased to (60 %) 140 CFU ml-1 for P. fluorescens and (40 %) 220 CFU ml-1 for S. aureus by AgNPs treatment. The nitrate reductase enzyme present in B. subtilis EWP-46 is possibly involved in the synthesis of AgNPs by inducing nitrate ions and reducing silver ions to metallic silver [16, 29]. Electron shuttle enzymatic metal reduction process might be a possible mechanism involved in the reduction of silver ions. Previous studies by Kalimuthu et al. [16] have indicated that NADH and NADH-dependent nitrate reductase enzyme are important factors in the biosynthesis of metal nanoparticles. Bacillus sp. is known to secrete the cofactor NADH and NADH-dependent enzymes, and especially nitrate reductase might be responsible for the bioreduction of Ag? to Ag0 and the subsequent formation of AgNPs [30, 31]. Kalimuthu et al.

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[16] explain that the same phenomenon has been hypothesized for magnetotactic bacteria. A similar phenomenon could happen in B. subtilis through the nitrate reductase enzyme present in it. Two unique nitrate reductase enzymes are present in B. subtilis. One is used for nitrate nitrogen assimilation and the other for nitrate respiration. The involvement of nitrate reductase in the production of AgNPs in vitro was demonstrated [12, 16], in which nitrate reductase were purified and used for the synthesis of nanoparticles. The reduction may occur by means of electrons from NADH, where NADH-dependent reductase can act as a carrier. It is concluded that nitrate-reducing bacterial strain has the capacity to reduce silver as well as nitrate. Cell-free extract of B. subtilis isolate EWP-46 was used for green synthesis of AgNPs with Ag? at room temperature. This is an economical, efficient, eco-friendly, and simple process. The nitrate reductase assay and the protein profiling explain that the bacteria have the nitrate reductase enzyme. The optimal parameters pH 10 and temperature 60 °C are suitable for converting bulk metal

Bioprocess Biosyst Eng Fig. 6 SDS-PAGE of protein. Lane 1 molecular weight marker; lane 2 contains extracellular protein of Bacillus subtilis EWP-46 with molecular weight of 43 kDa

arise from the phenotypic biochemical property of the culture as explained by Kumar et al. [19]. The predicted NMR data support that possibly biomolecules may be present in the cell-free extract. AgNPs have a broadspectrum antimicrobial activity against various gramPOSITIVE and gram-negative bacteria. Acknowledgments This research was supported by the Korean National Research Foundation (Korean Ministry of Education, Science and Technology, Award NRF-2011-35B-D00020). The preparation of the manuscript was supported by research funds from the Chonbuk National University in 2013. This work was also partially supported by Korea Ministry of Environment as ‘‘The GAIA Project (No. 2012000550021)’’.

References

Fig. 7 XRD pattern of silver nanoparticles synthesized using Bacillus subtilis strain EWP-46 culture supernatant

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Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity.

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) ...
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