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Bacterial synthesis of silicon/silica nanocomposites† Sanjay Singh,a Umananda M. Bhatta,b P. V. Satyam,b Alok Dhawan,c Murali Sastry‡a and B. L. V. Prasad*a
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Received 18th December 2007, Accepted 18th March 2008 First published as an Advance Article on the web 8th April 2008 DOI: 10.1039/b719528a The synthesis of silicon/silica nanoparticle composites by the bacterium Actinobacter sp. is demonstrated. More specifically, the formation of silicon/silica nanocomposite is shown to occur when the bacterium is exposed to K2SiF6 precursor under ambient conditions. Based on the earlier reports where this bacterium has been shown to synthesize iron oxide and iron sulfide nanoparticles, it is hypothesized that this bacterium secretes reductases and oxidising enzymes, which lead to the Si/SiO2 nanocomposite synthesis. The particles obtained by bacterial synthesis were thoroughly characterized. The cytotoxicity studies revealed that the particles do not display any cytotoxicity to human skin cells. The synthesis of silica nanoparticles by bacteria in the present study demonstrates the versatility of the organism, and the formation of elemental silicon by this environmentally friendly process expands further the scope of microorganism based nanomaterial synthesis.
Introduction Natural systems depict a rich repository of nanoparticle synthesis. Consequently researchers in this newly exploding field are pursuing them with great vigour. Many organisms, both unicellular and multicellular, are known to produce inorganic materials either intracellularly1 or extracellularly2 with wellknown examples including magnetotactic bacteria (which synthesize magnetic nanoparticles),3–5 diatoms (which synthesize siliceous materials)6–8 and S-layer bacteria (which produce gypsum and calcium carbonate layers).9,10 Silica, one of the most important and common chemical substances in nature,11 occupies a prominent position in scientific research, because of its wide use in various industrial applications, such as pigments, pharmaceuticals, electronic and thin film substrates, electronic and thermal insulators, and humidity sensors.12 Silica nanocrystals also play an important role in materials such as resins, catalysts and molecular sieves.13 The chemical syntheses of silica and silica-based materials, while being very effective on one hand,14 are relatively expensive and eco-hazardous, often requiring extremes of temperature, pressure, and pH. Thus, there has been increasing demand to develop more environmentally friendly processes15 to make these materials. Therefore, taking a cue from biotechnological processes such as toxic metal remediation that use microorganisms such as bacteria16 and yeast17 (the detoxification either involves reduction of the metal ions to metals or formation of compounds such as sulfides and a Materials Chemistry Division, National Chemical Laboratory, Pashan Road, Pune, 411008, India b Institute of Physics, Bhubaneswar, 751 005, India c Developmental Toxicology Division, Industrial Toxicology Research Centre, Lucknow, 226 001, India. E-mail:
[email protected]; Fax: +91 20 25902636; Tel: +91 20 25902013 † Electronic supplementary information (ESI) available: FTIR spectra of Si/SiO2 nanoparticle composites and images depicting the morphology of cell lines before and after exposure to these composites. See DOI: 10.1039/b719528a ‡ Current Address: Tata Chemicals Innovation Centre, Anmol Pride, Baner, Pune-411 045, India.
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carbonates etc.), materials scientists have been of late viewing these organisms as possible ecofriendly nanofactories.18–23 It all probably started with the interesting report by Klaus and co-workers21–23 showing that the bacterium Pseudomonas stutzeri AG259, isolated from a silver mine, resulted in the reduction of Ag+ ions and the formation of silver nanoparticles of welldefined size and distinct morphology within the periplasmic space of the bacteria when exposed to an aqueous solution of AgNO3. Subsequent results include a report by Nair and Pradeep24 where nanoparticles of gold, silver, and their alloys have been shown to form upon the reaction of their corresponding metal ions within cells of lactic acid bacteria present in buttermilk. Mandal et al.,25 have reported that the bacterium Clostridium thermoaceticum and Klebsiella aerogenes have been used for the synthesis of CdS nanoparticles and sphalerite nanoparticles from sulfate reducing bacteria under anaerobic conditions. There are indeed several other such reports and for brevity all have not been included here.26 In continuation of the synthesis of metal nanoparticles, there are reports regarding the synthesis of metal oxide nanoparticles using bacteria. Bharde et al.27 have isolated, as a contaminant, a bacterium Actinobacter sp. from a potassium ferrocyanide and potassium ferricyanide containing flask kept open for one week. Subsequently when a cultured part of this bacterium was challenged with a mixture of potassium ferrocyanide and potassium ferricyanide, the formation of crystalline iron oxide nanoparticles was observed. While it is clear that this bacterium must have developed resistance to the Fe3+/Fe2+ ion mixture in which it grew and hence the formation of Fe3O4 is expected to some extent, its response to other metal/non metal containing precursors would be an interesting aspect to investigate. The impetus for such investigations comes from earlier reports where it is clearly shown that challenging the microorganisms by metal ion precursors that they have never encountered in their life cycle could lead to interesting products exemplifying the great potential of these methods.28 In the present case we found that Actinobacter sp. not only survived the exposure of SiF62 ions but led to the concurrent hydrolysis and reduction of this precursor finally J. Mater. Chem., 2008, 18, 2601–2606 | 2601
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forming silicon/silica (Si/SiO2) nanocomposites. The synthesis is quite rapid (48 h) compared to other biosynthetic processes and is accomplished at room temperature, without use of any extreme situation and at relatively neutral pH. It is hypothesized that addition of the precursor (K2SiF6) to Actinobacter sp. culture causes a stressful environment (ionic stress) and in order to nullify this stressed situation the bacteria secrete some enzymes that hydrolyze the metal precursor to form Si/SiO2 nanocomposites. There are several interesting aspects to the results presented here. Firstly, unlike many celebrated chemical sol–gel syntheses,14 the present one results in crystalline nanoparticles. Secondly, some of the Si(IV) ions seem to have been reduced all the way to elemental silicon by the enzymes secreted leading to the formation of Si/SiO2 nanocomposites. As far as we are aware this is probably the first report of the formation of elemental silicon by any microorganism and could have great ramifications for further research. Presented below are the details of investigation.
Experimental details Synthesis of Si/SiO2 nanocomposites The aerobic bacterium Actinobacter sp. was isolated as a contaminant from a flask containing potassium ferrocynide and potassium ferricynide.27 The bacterial cell colony was inoculated in a 500 mL Erlenmeyer flask containing 100 mL of Luria broth medium and incubated for 24 h under shaking conditions (200 rpm) at 27 C. After incubation, the bacterial cells were harvested and thoroughly washed with autoclaved water under sterile conditions. The harvested bacterial biomass (~1 gm of wet weight) was then resuspended in 100 mL of an aqueous solution of 1 103 M K2SiF6 separately in a 500 mL Erlenmeyer flask and kept on a shaker (200 rpm) at 27 C. The reaction between the bacterial biomass and the SiF62 was carried out for a period of 48 h. The bio-transformed reaction products were collected under sterile conditions after separating the bacterial biomass from the reaction medium through centrifugation at 5000 rpm for 10 min.
(DMEM) supplemented with 10% foetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM glutamine, 50 U mL1 penicillin, 50 mg mL1 streptomycin and 100 mM non-essential amino acids. DMEM was purchased from GIBCO. Cells were cultured for 3–4 d (80% confluency) before the assay. For MTT assay, the cells were seeded in 96 well plates at a density of 10 000–15 000 cells per well in 100 mL of complete medium. The cells were incubated for 24 h in a humidified incubator at 37 C and in an atmosphere of 5% CO2–95% air. After incubation, the complete medium was discarded and 100 mL of freshly prepared Si/SiO2 nanocomposites (different concentration) in DMEM were added to each well. MTT assay MTT [3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide], a pale yellow dye is converted into formazan, a violet compound, by the activity of succinate dehydrogenase of mitochondria. Since the conversion takes place in living cells, the amount of formazan produced is directly correlated with the number of viable cells present. The MTT assay was done following the method of Mosmann29 with slight modification. In brief, cells (10 000–15 000 cells per well in 100 ml of medium) were seeded in a 96 well plate and allowed to adhere for 24 h at 37 C in an atmosphere of 5% CO2–95% air. The original medium was replaced with serum free medium containing Si/SiO2 nanocomposites ranging from 10 mM to 50 mM and incubated for 3 h at 37 C. The treatment concentrations were discarded and 100 mL serum free medium and 10 mL MTT (5mg mL1) in PBS was added to each well and re-incubated for another 1 h at 37 C. The reaction mixture was carefully taken out and 200 mL of DMSO was added to each well and mixed thoroughly. After 10 min, the color was read at 530 nm, using a Multiwell Microplate Reader (Biotek, USA). The untreated sets were also run parallel under identical conditions and served as controls. The relative cell viability as a percentage was calculated as (A530 of treated samples/A530 of untreated samples) 100. The data presented are the mean SD from three independent experiments. Morphological analysis
Control experiments In control experiments, the bacterial biomass was resuspended in autoclaved deionized water in the absence of K2SiF6 and the filtrate obtained thereafter was characterized for the presence of Si/SiO2 nanocomposite. As expected this reaction did not result in the formation of Si/SiO2 nanocomposite. In another control experiment, the hydrolysis of K2SiF6 in autoclaved deionized water in the absence of bacterial biomass was studied by TEM and FTIR. This control experiment was also negative and no Si/ SiO2 nanocomposite could be detected. Cell culture The A431 cell line (human epithelial cell line) was used for the cytotoxicity study of Si/SiO2 nanocomposites. The A431 cell line (ATCC No. CRL-1555) was initially procured from the National Centre for Cell Sciences, Pune, India and has been maintained at the Industrial Toxicology Research Centre, Lucknow, India. The cells were maintained in Dulbecco’s Modified Eagle Medium 2602 | J. Mater. Chem., 2008, 18, 2601–2606
Morphology of cells before and after the Si/SiO2 nanocomposite treatment was examined under a phase-contrast inverted microscope (Leica Germany). The changes in the cells were quantified using automatic image analysis software Leica Q Win 500, hooked up to the inverted phase-contrast microscope. Sample preparation for MTT A stock solution at 10 mM was prepared by mixing 6 mg of Si/ SiO2 nanocomposite in 10 mL of DMEM. This mixture was sonicated thrice for one minute each time with an interval of 10 s at 15 W. Sample characterization The purified and dried silica powder was crushed with KBr, pelleted and the FTIR spectra were recorded on a Perkin-Elmer Spectrum One instrument at a resolution of 4 cm1. Samples for the transmission electron microscopy (TEM) were prepared by This journal is ª The Royal Society of Chemistry 2008
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drop coating the isolated and resuspended solution on carboncoated copper grids. Selected area electron diffraction (SAED) as well as high resolution transmission electron microscopy (HRTEM) measurements were also carried out from the same TEM grid. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage of 120 kV. HRTEM measurements were carried out on a JEOLJEM-2010 UHR instrument operated at a lattice image resolution of 0.14 nm. X-Ray diffraction (XRD) measurements of drop coated films of the silica on glass substrates were carried out on a Philips PW 1830 instrument operated at a voltage of 40 kV and a current of 30 mA with Cu Ka radiation. X-Ray photo-emission spectroscopy (XPS) measurements of films of silica nanoparticles cast on to Cu substrates were carried out on a VG MicroTech ESCA 3000 instrument at a pressure greater than 1 109 Torr. The general scan and C 1s, Si 2p, N 1s, O 1s and F 1s core level spectra were recorded with un-monochromatized Mg Ka radiation (photon energy ¼ 1253.6 eV) at a pass energy of 50 eV and electron takeoff angle (angle between electron emission direction and surface plane) of 60 . The overall resolution was ~1 eV for the XPS measurements. The core level spectra were background corrected using the Shirley algorithm30 and the chemically distinct species were resolved using a nonlinear least squares fitting procedure. The core level binding energies (BEs) were aligned with the adventitious carbon binding energy of 285 eV. For the energy dispersive analysis of X-rays (EDAX) the sample was prepared on copper substrate by drop coating of purified Si/SiO2 nanocomposites. EDAX measurements were performed on a Leica Stereoscan-440 scanning electron microscope (SEM) instrument equipped with a Phoenix EDAX attachment.
Results and discussion The reaction product obtained from the Actinobacter sp.–K2SiF6 reaction mixture was analyzed by TEM. Fig. 1A and B shows the representative TEM images recorded from the extracellular product obtained by the reaction of Actinobacter sp. with K2SiF6 for 48 h. The particles embedded in the biomolecular matrix are quasi-spherical in morphology having a size of around 10 nm. In Fig. 1B (inset), selected area electron diffraction (SAED) analysis of the as-prepared silica nanoparticles clearly shows the presence of crystalline nature in these particles. These diffraction spots could be indexed to the tridynite polymorph of SiO2 wherein the ˚ , 2.85 A ˚ , and 1.91 A ˚ ) match reasonably d values obtained (3.12 A ˚ ˚ and 1.90 A ˚ ) for the well with the reported d values (3.11 A, 2.84 A 2 1 4, 3 0 2 and 3 1 13 planes respectively for tridynite SiO2.31 Fig. 1C, shows the TEM image recorded from the calcined sample of Si/SiO2 nanocomposite. Fig. 1C (inset) shows the SAED pattern from a calcined sample. In Fig. 1D, EDAX measurements of drop coated films of as-prepared silica nanoparticles on Cu substrates clearly reveal the presence of Si and O suggesting the presence of silica nanoparticles. Also, signals for K, C, N and O were obtained, which could confirm the side products of the Si precursor used and the capping proteins present on the nanoparticle surface. In accordance with the SAED patterns (Fig. 1B and C, insets), the crystalline nature of silica particles is again confirmed by XRD patterns (Fig. 2A) obtained from thin films of Si/SiO2 This journal is ª The Royal Society of Chemistry 2008
Fig. 1 TEM micrographs at different magnifications of Si/SiO2 nanocomposites synthesized by the exposure of K2SiF6 to the Actinobacter sp. before (A and B) and after (C) calcination at 400 C for 4 h. The insets in B and C are the SAED patterns recorded from representative Si/SiO2 nanocomposites. In D the EDAX spectrum of the as-prepared sample can be seen.
nanocomposites from as synthesized as well as calcined samples. Drop coated films of as-prepared as well as calcined Si/SiO2 nanocomposites show well-defined Bragg reflections indicating that the particles are crystalline. The most striking results from XRD (Fig. 2A) patterns are the presence of peaks corresponding to elemental silicon (denoted by +) along with silica (denoted by :).31 The silica peaks could be assigned to a mixed phase of tridynite (indicated by : and T) and crystoballite (indicated by :) polymorphs (Fig. 2A). The Bragg reflections for silicon become more intense after calcination (1 1 1 and 3 1 1 crystal planes), this could be due to the improvement in crystallinity upon heat-treatment as well as the loss of amorphous protein coating on the particles. As far as we are aware the formation of elemental silicon by a bio-organism is the first of its kind and we further confirmed it by other studies as described in the following.
Fig. 2 (A) XRD pattern recorded from the as-prepared (curve 1) and calcined (curve 2) samples of biosynthesized Si/SiO2 nanocomposites. (B) Absorbance (curve 1) and emission spectra (curve 2) of the as-prepared sample. The emission spectra are recorded with an excitation of 280 nm.
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Fig. 2B presents the optical absorbance as well as the emission spectra (lex ¼ 280 nm) of the as-prepared sample. The absorbance monotonously increases with decreasing wavelength and is generally featureless except for a small change in the slope at 425 nm. The two emission bands one at l ¼ 352 nm and the other at l ¼ 442 nm are noteworthy. As the excitation is at 280 nm, the emission at ~352 nm can be attributed to the tryptophan and tyrosine residues of the protein capping on the nanoparticles. However, the longer wavelength emission is very interesting. This could be ascribed to the elemental silicon present in the sample. Credence to this hypothesis comes from well established reports that polycrystalline silicon emits at l ¼ 450 nm.32 This phenomenon of photoluminescence is known to occur when there is an interface between Si nanocrystals in the SiO2 matrix in the nanoscale size regime.33 The formation of silica and silicon in the present study is more prominently evidenced by X-ray photoelectron spectroscopy (XPS), a very sensitive technique (Fig. 3) for investigations involving different oxidation states of the elements. Fig. 3A shows the C 1s core level spectrum that could be decomposed into three chemically distinct components centered at 281.9 eV, 285.0 eV and 287.19 eV. The low binding energy peak at 281.9 eV is attributed to the presence of aromatic carbon present in amino acids from proteins bound to the surface of silica nanoparticles.34 The high binding energy peak at 287.19 eV is attributed to electron emission from carbons in carbonyl groups present in proteins bound to the nanoparticles’ surface.35,36 The C 1s component centered at 285.0 eV is due to electron emission from adventitious carbon present in the sample. In Fig. 3B, the Si 2p spectrum could be resolved in to two spin–orbit pairs (spin–orbit splitting z 0.6 eV)37 with 2p3/2 binding energies (BEs) of 103.3 eV38,39 and 98.8 eV.39 The high BE components at 103.3 eV (Si 2p3/2) and 102.7 eV (Si 2p1/2) agree excellently with the reported values for SiO2, while low BE components present at 98.84 eV (Si 2p3/2) and 98.20 eV (Si 2p1/2) agree with the reported values for silicon.39 The absence of any peak from F 2p levels (inset Fig. 3C) unambiguously supports the almost complete hydrolysis of
Fig. 3 XPS spectra recorded from the as-prepared samples of biosynthesized Si/SiO2 nanocomposites. Panels A, B, C and D show the curve fitting for C 1s, Si/SiO2, N 1s and O 1s, respectively. In panel C (inset) the signal for F 1s is shown.
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SiF62 ions in the nanocomposite samples. Fig. 3C shows the N 1s core level spectra that can be decomposed in to two chemically distinct components centered at 399.6 eV and 402.2 eV. The high binding energy and lower binding energy components can be attributed to the presence of protonated amines and free amines present in the capping proteins40 respectively. Higher binding energy components can also be attributed to N atoms present in amide bonds. In addition to C 1s, N 1s and Si 2p, the sample was also scanned for O 1s signals (Fig. 3D) that show two components having BE of 532.2 eV and 529.8 eV. Oxygen in the Si–O–Si environment is known to show an O 1s BE component at 532.5 eV37 while the low binding energy component at 529.8 eV could be due to oxygen present in capping proteins. As a final confirmation for the presence of silicon along with silica in the as-prepared sample, HRTEM investigations were undertaken. Fig. 4A, B and C show the HRTEM images of the as-prepared sample. Clear lattice planes (indicated by +) having ˚ (Fig. 4B), 2.73 A ˚ (Fig. 4B) and 3.10 A ˚ separations of 3.34 A (Fig. 4C) are observed, that match reasonably well with reported ˚ , 2.69 A ˚ and 3.13 A ˚ ) for (200), (211) and (111) values (3.29 A 31 planes respectively for Si. Further, along with the presence of lattice planes for silicon, HRTEM images reveal lattice planes ˚ (Fig. 4A and (indicated by :) corresponding to silica at 3.24 A ˚ ˚ B), 3.02 A (Fig. 4A) and 2.59 A (Fig. 4A) matching reasonably well with reported values of silica.31 Therefore, the HRTEM analysis undoubtedly establishes that silicon and silica are both present in the sample. The biological synthesis of Si/SiO2 nanocomposites by Actinobacter sp. could be a very complex phenomenon. As has been mentioned previously this bacterium grew as a contaminant in a flask containing Fe2+/Fe3+ ions.27a It has now been established that this bacterium can secrete iron reductase and membrane bound iron oxidase27b when exposed to Fe3+/Fe2+ ions and therefore the synthesis of magnetite (Fe3O4) or maghaemite (g-Fe2O3) does not come as a surprise. The synthesis of Si/SiO2 as seen in the present case, though unexpected to certain extent, is not surprising either. It is well established now that challenging microorganisms with unfamiliar metal ion precursors induces the secretion of new proteins that can effect chemical transformations such as reduction or hydroxylation so as to nullify the
Fig. 4 HRTEM images (A, B and C) from different regions showing the presence of lattice spacing matching with Si (+) as well as SiO2 (:). (D) Schematic representation of the synthesis mechanism of Si/SiO2 nanocomposites.
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stress.28 We hypothesize that the synthesis of Si/SiO2 nanocomposites by Actinobacter sp. follows a similar pathway and leads to the release and cumulative action of certain non-specific reductases and oxidizing enzymes (Fig. 4D). Some of these enzymes are seen capping the nanoparticle surface as confirmed by FTIR spectra (ESI Fig. S1†) that clearly reveal the amide I and amide II bands along with the antisymmetric Si–O–Si stretching mode41 at 1100 cm1. The reduction of Si(IV) to elemental Si could be attributed to the secreted reductase enzyme. The silicon surface is very reactive and is always covered with its native oxide. Here also the elemental Si in the nanocrystalline particles is prone to oxidation when exposed to the ambient conditions under which the experiments are carried out. The action of an oxidizing enzyme could expedite the process finally leading formation of Si/ SiO2 nanocomposites. Currently efforts are underway to isolate, purify and characterize these enzymes. Silica nanoparticles are being used in several applications that have direct contact with the skin.42 Thus it is important that we undertake a systematic cytotoxicity study of any new material prepared before their application is realized. The cytotoxicity of Si/SiO2 nanocomposites in the present case was determined by MTT assay (Fig. 5). A431 cell lines were treated with different concentrations (10 mM, 5 mM, 1 mM, 500 mM, and 50 mM) of Si/ SiO2 nanocomposites for 3 h. Very high concentrations (10 mM and 5 mM) of nanocomposites showed significant cytotoxicity as only 14.74% and 37.5% mitochondrial activity was recorded (Fig. 5). While in the case of lower concentrations (1 mM to 50 mM) of nanocomposites, mitochondrial activity was found to be more than 67.8% (Fig. 5). Further, the cell morphology (ESI Fig. S2B and C†) study also reveals that exposing skin cells to higher concentrations of Si/SiO2 nanocomposites (10 mM and 5 mM respectively) causes drastic change in their morphology leading to cell death, supporting to MTT results. On the other hand at a 1mM nanocomposite concentration (ESI Fig. S2D†), though some changes in the cell morphology can be seen, no significant cytotoxicity was observed as 67.8% of mitochondrial activity was recorded. Compared with the cell morphology of the control (ESI Fig. 2A†), no change in morphology was seen when the cells were treated with 500 mM and 50 mM (ESI Fig. 2E and F,† respectively) concentrations of Si/SiO2 nanocomposites, supporting the MTT results.
Fig. 5 Effect of different concentrations of Si/SiO2 nanocomposites on cell viabilities (% mitochondrial activity) of human epithelial (A431) cell line (MTT assay) after 3 h of exposure. Values are mean SD from three independent experiments.
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Conclusion A potentially rewarding bacterium mediated synthesis of Si/SiO2 nanocomposite is disclosed for the first time. The samples are thoroughly characterized with different techniques including emission, HRTEM, XRD and XPS spectra. Plausible proteins responsible for such formation are identified. With a view to establishing the compatibility of these nanocomposites with human skin, their cytotoxicity with A431 cell lines was investigated. These studies revealed that cell lines survive the contact with these nanocomposites upto 1 mM concentrations.
Acknowledgements SS thanks CSIR for a fellowship. We thank Dr Mahima Bajpayee and Ms Virginia D’Britto for the help with MTT experiments. The authors gratefully acknowledge several useful discussions with Dr Atul Bharde. The authors acknowledge financial assistance through the DST funded Unit on Nanoscience and Technology (DST-UNANST) at NCL and a joint grant to NCL and ITRC.
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