Biotechnology and Bioprocess Engineering 17: 835-840 (2012) DOI 10.1007/s12257-011-0582-9
RESEARCH PAPER
Extracellular Biosynthesis of Iron Oxide Nanoparticles by Bacillus subtilis Strains Isolated from Rhizosphere Soil P. Alagu Sundaram, Robin Augustine, and M. Kannan
Received: 13 November 2011 / Revised: 4 April 2012 / Accepted: 6 April 2012 © The Korean Society for Biotechnology and Bioengineering and Springer 2012
Abstract The biological synthesis of nanoparticles is emerging as a potential method for nanoparticle synthesis due to its non-toxicity and simplicity. We report the ability of Bacillus subtilis strains isolated from rhizosphere soil to produce iron oxide nanoparticles. B. subtilis strains having the potential for the extracellular biosynthesis of Fe3O4 nanoparticles were isolated from rhizosphere soil, identified and characterized. A bactericidal protein subtilin was isolated from all the isolates of B. subtilis, which is a characteristic for the species. The isolated subtilin was tested against the bacterial strain, E. coli. The supernatant of the bacterial culture was used for the synthesis of Fe3O4 nanoparticles. The formation of nanoparticles was assessed by using UV-Visible spectrophotometer. FTIR and SEM analysis were used in order to confirm the formation and size of the nanoparticles. Further, the effect of incubation time, pH, and temperature on the formation of Fe3O4 nanoparticles was studied. The successful synthesis of stabilized Fe3O4 nanoparticles, which was capped by the organic group, indicates the applicability of the isolated B. subtilis strain for the bulk synthesis of iron oxide nanoparticles. Keywords: biosynthesis, Fe3O4 nanoparticles, reduction, organic capping
P. Alagu Sundaram, Robin Augustine Department of Bioengineering, VHNSN College, Tamilnadu 626-001, India M. Kannan Department of Microbiology, VHNSN College, Tamilnadu 626-001, India Robin Augustine* Centre for Nanoscience & Nanotechnology, Mahatma Gandhi University, Kerala 686-560, India Tel: +91-956-2204140; Fax: +91-481-2730003 E-mail:
[email protected]
1. Introduction Iron oxide nanoparticles are technologically important because of their interesting magnetic and electrical properties. They are used in magnetic inks and magnetic fluids, as well as for the fabrication of magnetic cores regarding read/write heads for high-speed digital tapes or disc recording [1]. Even though synthesis methods including the hydrothermal reaction method, the sol-gel process, and the chemical co-precipitation can be used for the synthesis of iron oxide nanoparticles, the uniformity for the size of the particle with regards to these nanomaterial’s are generally poor [2]. Bacterial synthesis of magnetic nanoparticles is a wellknown approach due to its low cost, fine control over magnetic and thermal properties of the particles, control of particle sizes from 10 to 100 nm, and its higher saturation magnetization compared to typical chemically synthesized materials [3]. However, the fact that the biological nanoparticle synthesis generates nanoparticles at a much slower rate is one major drawbacks of the biological synthesis [4]. Thus, new strains of bacteria, which can synthesize nanoparticles in a much faster rate, should be isolated. Some extracellular enzymes show excellent redox properties and they can act as an electron shuttle in the reduction of metal. It is also evident that electron shuttles or other reducing agents like hydroquinones released by microorganisms are capable of reducing ions to nanoparticles [5]. Some bacteria like Mycobacterium paratuberculosis [6], Shewanella oneidensi [7], Geothrix fermentans [8] reduce Fe3+ oxides by producing and secreting small, diffusible redox compounds that can serve as an electron shuttle between the microbe and the insoluble iron substrate [9]. The bacteria B. subtilis has been used for the synthesis
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of silver nanoparticles by both extracellular and intracellular means [10]. Studies reveal that B. subtilis is able to reduce Au3+ ions in order to produce octahedral gold particles of nanoscale dimensions within the bacterial cells by incubating the cells with a gold chloride solution [11-13]. However, the ability of this bacterium to reduce Fe2O3 is yet to be demonstrated. Therefore, the present study aimed at developing a biological synthetic route for the cost effective, non-toxic production of iron oxide nanoparticles through the use of a new isolated B. subtilis is believed to be highly promising.
2. Materials and Methods 2.1. Isolation, screening, and identification of a Bacillus subtilis strain from rhizosphere soil Soil isolates of B. subtilis were obtained by screening rhizosphere soil samples. Three different soil samples were used for the isolation of the organism. Soil samples were processed according to Beima Aslim et al. [14]. Each gram of soil samples was suspended in 99 mL of sterile distilled water and shaken vigorously for 2 min. The samples were heated at 60oC for 60 min in a water bath and then serially diluted and streaked onto BHI (Brain Heart Infusion) Agar (Aldrich, USA). Suspected colonies were Gram stained and confirmed through biochemical tests according to Garrity et al. [15]. The candidate culture of Bacillus subtilis was supplemented with egg yolk and then incubated for 24 ~ 40 h at a temperature of 35 ± 2oC. 2.2. Collection of supernatant B. subtilis stock cultures were renewed by sub culturing every month. A Luria Broth (LB) medium was prepared by the addition of 1% Bacto-tryptone [Difco], 0.5% yeast extract [Difco], and 1% NaCl (pH 7.5). After autoclaving, LB was inoculated with the new isolates and incubated at 37ºC and 150 rpm for 36 h. The freshly grown cells were removed by centrifugation at 5,000 rpm for 10 minutes and the supernatant was used for the synthesis of nanoparticles. 2.3. Extracellular biosynthesis of iron nanoparticles using the culture supernatant of Bacillus subtilis An aqueous solution of 2 mM Fe2O3 (50 mL) was treated with 50 mL of B. subtilis supernatant solution in a 250 mL Erlenmeyer flask (pH adjusted to 8.5). The whole mixture was incubated at 35oC (200 rpm) for 5 days under a dark condition. A control without bacterial supernatant was also maintained under similar conditions. For the preliminary determination of iron oxide nanoparticles, Ultraviolet-
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Visible Spectroscopy (UV-Vis) in the range 200 ~ 1,100 nm was performed in a Perkin-Elmer Lambda 2 Spectrophotometer. The same experiment was performed at different time interval, pH and temperature. 2.4. Effect of temperature on the formation of nanoparticles To find out the effect regarding temperature on the nanoparticle formation, the reaction was carried out at three different temperatures (28, 36, and 45oC). The pH of the reaction system was chosen as 9, the reaction time period kept as 24 h and, the rotation rate of the reaction system was chosen as 200 rpm. Furthermore, other conditions were kept the same as the preliminary experiment. Afterwards, the particular incubation time absorbance at 250 nm was taken as mentioned earlier. 2.5. Effect of pH on the formation of nanoparticles In this experiment the temperature of the reaction system was taken as 36oC, the reaction time period kept as 24 h, and the rotation rate of the reaction system was chosen as 200 rpm. In addition, other conditions were kept the same as the preliminary experiment. Afterwards, the particular incubation time absorbance at 250 nm was taken as mentioned earlier. 2.6. Effect of incubation time on the formation of nanoparticles To understand the effect of incubation time on the formation of nanoparticles, all the parameters, except incubation time, were kept constant as in the previous experiments. Optical Density was measured after each 20 h incubation period up to 120 h. 2.7. Characterization of the synthesized nanoparticles The studies for size, morphology, and composition of the nanoparticles were performed by means of scanning electron microscopy (HITACHI, S-3000H). Histograms of size distribution were calculated from the SEM images by measuring the diameters of at least 50 particles. The FT-IR (Perkin-Elmer Spectrum RX1) analysis was used for the characterization of iron oxide nanoparticles. FTIR absorption spectra before and after the reduction of Fe2O3 were taken in the range 400 ~ 4,000 cm-1. XRD analysis was used to find out the structure and the size of the synthesized nanoparticles. The grain size was determined by using Sherrer’s equation, D = kλ/ B cosθ where D is the mean grain size, k is a geometric factor (=0.89), λ is the X ray wavelength, B is the full width at half maximum (FWHM) of the diffraction peak and θ is the diffraction angle.
Extracellular Biosynthesis of Iron Oxide Nanoparticles by Bacillus subtilis Strains Isolated from Rhizosphere Soil
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Table 1. Results of biochemical tests for the isolated strains of Bacillus subtilis Isolates
Citrate test
B.subtilis 1 B.subtilis 2 B.subtilis 3
– – –
Vogous proscer test Methyl red test – – –
Fig. 1. Bacterial growth inhibition on E.coli plate by subtilin isolated from three B.subtilis isolates.
+ + +
Indole test
Oxidase test
Catalase test
+ + +
+ + +
+ + +
Fig. 2. Visible colour change before (A) and after (B) the formation of Fe3O4 nanoparticle.
3. Results and Discussion 3.1. Isolation, screening, and identification of Bacillus subtilis strain from rhizosphere soil Three soil samples containing B. subtilis were collected, which was confirmed by their biochemical characters. Centrally located spores of Gram positive bacilli were analyzed by Gram staining (Data not shown). Biochemical characterization of the three isolates is shown in Table 1. A bactericidal protein subtilin was isolated from all three isolates of B. subtilis tested against the bacterial strain, E. coli. Fig. 1 depicts the antibacterial activity of isolated subtilin on E. coli. 3.2. Extra cellular biosynthesis of iron nanoparticles using the culture supernatant of Bacillus Subtilis The ferric oxide solution was mixed with the culture supernatant of B. subtilis. The formation of biosynthesized iron nanoparticles was observed by the color changes (Fig. 2). Unreduced Fe2O3 was brick red in color whereas Fe3O4 nanoparticles were dark brown in color due to its specific surface plasmon resonance property. The UV- Visible spectrum of biosynthesized nanoparicles (Fig. 3), clearly indicate that the iron oxide surface plasmon band occurs at around 250 ~ 350 nm, suggesting that the particles are well dispersed in the aqueous solution.
Fig. 3. Absorption spectra of Fe3O4 nanoparticles.
The iron oxide nanoparticle solution was also tested at the end of the reaction period for stability. The particles are thus stabilized in solution by the capping agent, which is likely to be proteins secreted by the B. subtilis. 3.3. Effect of temperature on the formation of nanoparticles From Fig. 4, it is clear that the rate of formation for Fe3O4 nanoparticles reduced with the increase of reaction
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Fig. 4. Effect of incubation temperature on the formation of nanoparticles.
temperature. When the reaction temperature is higher than 50oC, there is no observable formation of nanoparticles within a time period of 24 h. This may be due to the inactivation or degradation of biomolecules responsible for the reduction of Fe2O3. This is controversial in regards to the chemical synthesis of nanoparticles whereby increasing the reaction temperature enhanced the rate of formation for Fe3O4 nanoparticles. 3.4. Effect of pH on the formation of nanoparticles From Fig. 5, the rate of formation for Fe3O4 nanoparticles increases as the solution pH increases up to 9 and there was a sudden decrease afterwards. When the pH is higher than 11, the rate of formation of nanoparticle is very low and remained as such up to the maximum pH. It might be due to the inactivation or degradation of biomolecules responsible
Fig. 5. Effect of solution pH on the formation of nanoparticles.
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Fig. 6. Effect of incubation time on the formation of nanoparticles.
for the reduction. Further, when the pH of the reaction system increases, Fe(OH)3 was generated in the first step, which was owing to the hydrolysis of Fe3+. Then, Fe(OH)2 was generated as the pH of the reaction system increased, which was owing to the hydrolysis of Fe2+. Finally, Fe3O4 alone can be formed as the solution pH increases. It reveals that the nucleation of the Fe3O4 nucleus happens more easily when the solution pH is lower than 11, while the growth of the Fe3O4 nucleus occurs more easily when the solution pH is higher than 11. 3.5. Effect of incubation time on the formation of nanoparticles As incubation time increases, the yield of nanoparticle formation also increased up to a certain limit. From Fig. 6, it is clear that the highest formation for nanoparticles is around 80 h of incubation. Afterwards, a sudden decline in the yield of nanoparticles was observed. This might be due to the completion of nanoparticle formation in the solution or the unavailability of Fe2O3 for further reaction. 3.6. Scanning electron microscopy The SEM image of iron nanoparticles is shown in Fig. 7. The SEM image shows the distribution of individual iron oxide nanoparticles as well as a few aggregates. From the SEM image it is clear that the biosynthesized nanoparticles are spherical in morphology and each of the iron oxide nanoparticles has a size in the range of 60 ~ 80 nm. The nanoparticles were not in direct contact even within the aggregates, indicating stabilization of the nanoparticles by a capping agent. As discussed earlier, biosynthesized nanoparticles are exceptionally stable, which is likely to be due to capping with proteins or secondary metabolites secreted by the bacteria.
Extracellular Biosynthesis of Iron Oxide Nanoparticles by Bacillus subtilis Strains Isolated from Rhizosphere Soil
Fig. 7. SEM image of Fe3O4 nanoparticles.
3.7. FT–IR spectra The FT-IR spectrum of biosynthesized nanoparticles is shown in Fig. 8, which demonstrates the band in the region of 850 and 400 cm-1 for iron oxide, 1,630 ~ 1,780 cm-1 for the carbonyl group, 3,200 ~ 3,600 cm-1 for the organic functional group such as hydroxyl or –NH groups. 2,440 ~ 2,275 cm-1 region of the spectrum corresponds to P-H group. FT-IR spectrum of Fe2O3 was given for the comparison (Fig. 9). Strong bands between 850 and 400 cm-1 are due to the stretching vibration of Fe-O bonds in Fe3O4 [16]. The
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bands at 460 and 570 cm-1 are assigned to specific vibrations of Fe-O bonds [17]. The intensity of these bands is very weak in the case of biosynthesized Fe3O4 in comparison with neat Fe2O3. This may be due to the capping effect of the functional groups. There is a peak at 3,285.77 cm-1 and this may be due to phenols, alcohols or carboxylic acids. Peaks in between 1,730 and 1,625 cm-1 are an indication of C=O stretch and there is one at 1,656.31 cm-1, which was probably due to ketones. Presence of both O-H stretch and C=O stretch may be due to the capping of carboxylic acid moieties on the iron oxide nanoparticles. Further, the presence of C-O Stretch in between 1,300 and 1,000 cm-1 may be due to the covalent linking of ester or ether groups to the nanoparticle. Esters demonstrate their carbonyl C=O stretch at 1,750 ~ 1,735 cm-1, but also exhibit their characteristic absorption at 1,300 ~ 1,000 cm-1 from the couplings of C-O and C-C stretches. Thus, the peak at 1,063.75 cm-1 indicates the coupling of C-O and C-C stretches and 1,656.31 cm-1 indicates C=O stretch. Hence, there may be an ester link with iron oxide nanoparticle. A single N-H stretch in between 3,500 and 3,100 cm-1 along with an N-H bend in between 1,550 and 1,450 may due to the presence of secondary amine. The phosphorus-hydrogen linkage was also identified; the P-H absorption band in an infrared spectrum is known and appears in the range of 2,440 ~ 2,275 cm-1 [18]. In our tests, the peak indicating the presence of the phosphorus-hydrogen linkage occurred at approximately 2,348.48 cm-1. The overall result of FT-IR indicates that certain organic compounds contain O-H, C=O, C-O, N-H groups, which act as a capping agent in biosynthesized iron oxide nanoparticles compared to free ferric oxide. 3.8. X-ray diffraction patterns Fig. 10 shows X-ray diffraction patterns with regards to the
Fig. 8. FT-IR spectrum of Fe2O3.
Fig. 9. FT-IR spectrum of Fe3O4 nanoparticles.
Fig. 10. X-Ray diffraction pattern of iron oxide nanoparticles.
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biosynthesized Fe3O4 nanoparticles. The XRD peaks of the Fe3O4 are compared with standard ones in JCPDS. A series of characteristic peaks at 2θ = 30.17o, 35.46o, 43.38o, 53.69o, 57.23o, and 62.77o, which corresponds to (220), (311), (400), (422), (511), and (440) Bragg reflection, respectively and is also comparable with standard magnetite XRD patterns. This implies that the synthesized Fe3O4 nanoparticles are cubic spinel in structure [19]. The average diameter of Fe3O4 nanoparticles was determined according to Sherrer’s equation, D = kλ/ B cosθ [20]. The estimated average size of the Fe3O4 nanoparticles is about 70 nm.
4. Conclusion The B. subtilis strain with enhanced potential for the biosynthesis of iron oxide nanoparticles was isolated from rhizosphere soil, which was identified by several biochemical tests. This organism has the ability to synthesize metal nanoparticles extracellularly at a much faster rate. The synthesis of iron oxide nanoparticles is mediated by the extracellular secretion of enzymes or secondary metabolites, which is advantageous for obtaining stabilized nanoparticles capped by protein and/or other organic compounds from B. subtilis. The SEM analysis shows that the nanoparticles are of 60 ~ 80 nm in size. The FT-IR results depict the presence of O-H, C=O, C-O, N-H, P-H groups on the nanoparticles, which act as stabilizers as well as a link to other molecules like therapeutic agents onto these functional moieties. The XRD pattern also confirms the efficacy of B. sutilis to synthesize Fe3O4 nanoparticles extracellularly and the lattice structure is found to be cubic spinel. The size calculated from XRD data also supports the SEM data and is in the range of 70 nm.
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