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Biogenic synthesis of floral-shaped gold nanoparticles using a novel strain, Talaromyces flavus

Biogenic synthesis of floral-shaped gold nanoparticles using a novel strain, Talaromyces flavus Ann Microbiol (2014) 64:1055–1063 DOI 10.1007/s13213-013-0744-4 ORIGINAL ARTICLE Biogenic synthesis of floral-shaped gold nanoparticles using anovel strain, Talaromyces flavus Eepsita Priyadarshini & Nilotpala Pradhan & Lala Behari Sukla & Prasanna Kumar Panda & Barada Kanta Mishra Received: 21 June 2013 /Accepted: 22 October 2013 /Published online: 19 November 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 . . Abstract A biogenic route was adopted towards the synthesis Keywords Biogenic synthesis Gold nanoparticle . . of gold nanoparticles using the extract of a novel strain, Talaromyces flavus Extracellular Nanobiotechnology Talaromyces flavus. Reduction of chloroauric acid by the fungal extract resulted in the production of gold nanoparticle, which was further confirmed by the concordant results obtain- Introduction ed from UV–visible spectroscopy, energy dispersive spectros- copy (EDS), and dynamic light scattering (DLS) analysis. The last decade has been witnessing a fascinating area of Morphology and the crystal nature of the synthesized nano- research termed nanotechnology: “the synthesis and fabrica- particles were characterized using transmission electron mi- tion of nanomaterials and their applicability” (Eustis and croscopy (TEM), X-ray diffraction (XRD) and selected area El-Sayed 2006). The European Union Commission defines electron diffraction (SAED). A direct correlation was observed nanoparticles as “particles having one or more dimensions in between nanoparticle formation and the concentration of re- the size range of 1−100 nm” and nanomaterials as “natural, ducing agent present in the fungal extract. The time-dependent incidental or manufactured material containing particles, in an kinetic study revealed that the bioreduction process follows an unbound state or as an aggregate or an agglomerate and autocatalytic reaction. Crystalline, irregular, and mostly flower- where, for 50 % or more of the particles in the number size shaped gold nanoparticles with a mean hydrodynamic radius of distribution, one or more external dimensions is in the size 38.54±10.34 nm were obtained. pH played a significant role on range 1–100 nm.” The increasing application of nanomaterials production of mono-dispersed nanoparticle. FTIR analysis par- concomitant with their growing demand has led to the devel- tially deciphered the involvement of –NH , −SH, and –CO 2 opment of a plethora of routes for their synthesis and size groups as the probable molecules in the bio-reduction and modification. Among the noble metals (silver, gold, and plat- stabilization process. Compared to the conventional methods, inum), gold has been of paramount importance since ancient a time-resolved, green, and economically viable method for times. However, gold nanoparticles (GNP) in comparison to floral-shaped nanoparticle synthesis was developed. bulk gold have attracted specific attention because of their interesting optical and catalytic properties (Sardar et al. 2009; Eustis and El-Sayed 2006). Electronic supplementary material The online version of this article GNP have found application in diverse fields like optics (doi:10.1007/s13213-013-0744-4) contains supplementary material, which is available to authorized users. (Hong and Hall 2012), electronics (Schmid and Simon 2005), electrochemistry (Huang et al. 2005), catalysis (Stratakis and E. Priyadarshini (*) N. Pradhan Academy of Scientific and Innovative Research, CSIR-Institute of Garcia 2012), sensing and detection agents (Scampicchio Minerals and Materials Technology, Bhubaneswar 751013, India et al. 2006), and in biomedical spheres (Dykman and e-mail: lisa.eepsita@gmail.com Khlebtsov 2012) because of their stability, resistance to oxi- : : dation, and biocompatibility. Recently, GNP are being widely N. Pradhan L. B. Sukla P. K. Panda Bioresources Engineering Department, CSIR-Institute of Minerals used for catalysis and SERS studies, which demand the use of and Materials Technology, Bhubaneswar 751013, India complex multi-shaped nanostructures like star-shape, urchin- like, and floral-shaped (Boca et al. 2011;Nehl etal. 2006). B. K. Mishra Such ashape enhances thetotal surfaceareaaswellas CSIR- Institute of Minerals and Materials Technology, Bhubaneswar 751013, India show an enhanced Raman scattering effect. Floral-shaped 1056 Ann Microbiol (2014) 64:1055–1063 GNP in comparison with spherical GNP is known to exhibit cotton blue stain using a phase contrast microscope (Nikon strong surface plasmon resonance along with a very high H550L). Molecular characterization was done by 28S rDNA electromagnetic field, localized at its protrusions (Nehl et al. gene sequencing. DNA was isolated and the fragment of 2006; Kim et al. 2009). However, the existing methods for gene was amplified using the universal primers DF: floral-shaped GNP synthesis involves the use of toxic chem- 5 ’ -ACCCCGCTGAACTTAAG C - 3 ’ an d D R: ical reagents and tedious multistep procedures (Wang et al. 5’-GGTCCGTGTTTCAAGACGG-3'. The obtained se- 2008) making the process hazardous and expensive. quence was then subjected to BLAST analysis and highly Several physicochemical methods such as laser ablation, homologous gene sequences were selected and represented sonoelectrochemistry, photochemical and chemical reduction in the form of phylogenetic tree. exist for GNP synthesis (Sylvestre et al. 2004;Serwas et al. 2008; Dong and Zhou 2007; Scampicchio et al. 2006). Biosynthesis of gold nanoparticles However, researchers are currently focusing on the biological route that utilizes an environmentally friendly solvent medium A loopful of fungal spore was aseptically inoculated in 100 ml and a nontoxic material for stabilization. Biological systems of sterile cornmeal medium (HIMEDIA) composed of corn- have proved to be natural nano-factories leading to the pro- meal (20 g/l), peptic digest of animal tissue (10 g/l), yeast duction of stably-capped GNPs (Sharma et al. 2009). Bacillus extract (4 g/l), and dextrose (10 g/l). The flasks were incubated subtilis was the first reported biological system for GNP at 35 °C for 72 h in an orbital shaker incubator at 150 rpm synthesis (Beveridge and Murray 1980). Later, eukaryotes (Kuhner, Switzerland). The biomass obtained was filtered like fungi, algae and plant extracts were also found to effec- using Whatman filter paper and washed thoroughly with tively reduce gold ions to GNP (Soni and Prakash 2012; sterile distilled water to remove all media components. The Luangpipat et al. 2011; Ghosh et al. 2012). Several fungi like washed biomass was further incubated in 100 ml of sterile Aspergillus niger, Aspergillus nidulans, Penicillum sp. and distilled water under similar conditions for 72 h. Incubation in Verticillium sp. have also been used for GNP synthesis (Soni sterile distilled water subjects it to a stress condition, which and Prakash 2012; Prusinkiewicz et al. 2012;Liangwei etal. allows the extracellular secretion of proteins and metabolites. 2011; Mukherjee et al. 2001). Fungi are greatly preferred for The filtrate obtained after incubation was termed the extracel- their capacity to secrete high amount of proteins, along with lular fluid (ECF). In another set of experiment, ECF was the advantage of easy handling, which make the downstream boiled for 30 min and was termed the boiled extracellular process simple (Sastry et al. 2003). fluid (BECF). In yet another set of experiment, the mycelia The current work describes the synthesis of extracellular along with the ECF obtained after 72 h were boiled for 30 min. floral-shaped GNP, by the reduction of gold ions using The resulting solution was filtered and termed the boiled the aqueous extract of the fungus Talaromyces flavus. biomass filtrate (BBF). All three filtrates were used for GNP Talaromyces flavus, a novel fungal strain, has been reported production. to inhibit various soil-borne plant pathogens such as Sclerotinia The ECF, BECF, and BBF were treated with chloroauric sclerotiorum, Rhizoctonia solani, and Verticillium dahlia. acid (HAuCl ) and incubated for GNP synthesis. The concen- (Boosalis 1956; Fravel et al. 1986; Mclaren et al. 1994). tration of HAuCl was maintained at 1 mM, barring a few However, to the best of our knowledge, its potential to reduce experiments. Three sets of control, comprising of cornmeal gold ions to GNP has not yet been reported in the literature. medium, ECF taken at 0 and 72 h of incubation were run This is the first report of a simple, green and rapid protocol for simultaneously. The first two sets of the control were treated floral-shape GNP production using a fungal extract. The effect with 1 mM HAuCl and the last set was run without HAuCl 4 4 of different parameters required for the optimization of yield, addition. controlled synthesis and minimization of the time for synthesis The effect of ionic strength on the stability of GNP synthe- were studied. sized by biological and chemical method was studied by treating the synthesized GNP colloidal suspension with NaCl. Chemical synthesis of GNP was carried out using Materials and methods sodium citrate as the reducing agent (Turkevitch et al. 1951). The critical coagulation concentration (CCC) represents the Fungal strain and its characterization NaCl concentration up to which the synthesized GNP remain stable without aggregation (Xie et al. 2003). In brief, CCC was The fungal strain used in the study was isolated from a mine- determined by treating the synthesized GNP (biological and contaminated site and subsequently maintained as pure chemical) with gradual increasing concentration of NaCl, culture on potato dextrose agar (HIMEDIA, India) slants. until a shift in the original SPR peak was observed. The The strain was designated as IF3. Morphological characteri- stability with respect to the SPR band was established by zation of the fungal strain was carried out by lactophenol recording the UV–visible absorbance of the mixture. Ann Microbiol (2014) 64:1055–1063 1057 Characterization of the synthesized GNP and analysis when resolved on agarose gel. The consensus sequence of of Extracellular fluid 611 bp of D2 region of the 28S rDNA gene generated was used to carry out BLAST. Based on the maximum identity Biosynthesis of GNP was visually observed by the change score, the first ten sequences were selected and the phyloge- in color of the reaction mixture (ECF treated with HAuCl ), netic tree was constructed using MEGA 4 software. On the andconcordantproductionofGNP wasconfirmedusing basis of nucleotide homology and phylogenetic analysis UV–vis Spectroscopic analysis (CECIL) in the range of (Fig. 1c), the isolate was identified to be Talaromyces flavus 190–1,100 nm. Transmission electron microscopy (TEM) strain NRRL 2098 (GenBank Accession No.: EU021596.1). analysis of synthesized GNP was carried out to determine their size and shape. TEM microscope (FEI, TECNAI-G2) Biosynthesis of gold nanoparticles using the fungal extracts 20-TWIN operating at 200 kVequipped with a GATAN CCD and its kinetic analysis camera was used for TEM-EDX analysis. The sample for TEM analysis was prepared by coating a drop of the colloidal On treating the ECF with HAuCl , a visible change in color gold solution onto a carbon-coated copper grid and allowing from colorless to burgundy was observed within 24 h of the solvent to evaporate under an IR lamp prior to analysis. To incubation. Change in color of the solution indicated the bio- investigate the possible functional groups involved in GNP reduction of chloroauric acid with the resultant formation of synthesis, Fourier transform infrared spectroscopy (FTIR, GNP.The formationofGNP wasconfirmedbyUV–vis Perkin-Elmer Model Spectrum 1) analysis was carried out. spectra with the appearance of a characteristic SPR peak in −1 The spectra were measured in the range of 4,000–400 cm at the range of 520–580 nm (Haiss et al. 2007). The control set −1 aresolutionof2cm . A few drops of the sample were layered comprising cornmeal medium showed GNP production indi- on top of a circular glass slide and allowed to dry, resulting in a cating that the media components were also capable of reduc- thin film that was then analyzed. The crystalline nature and ing gold ions. However, the second set of control (ECF taken phase analysis of the synthesized GNP were studied using at 0 h of incubation) when treated with HAuCl was not synchotron X-ray diffraction measurement. Diffraction capable of GNP production, indicating complete washout of pattern measurements were carried out on a Philips X’pert the medium components during biomass washing. The last set Pro, Panalytical X-ray powder diffractometer, having Cu-K of the control (ECF obtained after 72 h of incubation without (λ=1.54 Å) radiation, working at 40 kV/30 mA. The X-ray addition of HAuCl ) did not show any change in color; patterns were obtained in the 2θ range of 10–79°, using however, when treated with HAuCl , it showed GNP produc- 0.0050 step size. Dynamic light scattering (DLS) measure- tion. The results thus clearly indicate that the fungal biomol- ments were carried out to determine the hydrodynamic radius ecules secreted into the ECF during the 72 h of incubation of synthesized GNP using a Dawn Heleos II (Wyatt) system were solely responsible for the reduction of the gold ions to operating at a wavelength of 658 nm. GNP. Similarly, BECF and BBF were also treated with All the three filtrates obtained (ECF, BECF, and BBF) were HAuCl to study the role of denatured proteins on GNP 4, subjected to protein assay. Total protein content before and synthesis. Interestingly, characteristic SPR peaks for GNP after GNP production was determined by the Folin-Lowry were obtained in both cases (Fig. 2). In addition, GNP syn- method (Lowry et al. 1951). thesis in BBF was comparatively fast, i.e. within 5 h of HAuCl addition. This rapid production may be due to the additional release of intracellular reducing components into Result and discussion the extract when subjected to boiling. The kinetics of GNP biosynthesis was investigated to study Identification of the strain capable of GNP synthesis the effect of reaction time on gold ion reduction. For this, aliquots of the reaction mixture were withdrawn at regular The morphological and molecular identity of the fungus is time intervals and scanned to obtain the absorption spectra. depicted in Fig. 1. When grown on PDA plate, light yellow- The evolution of optical density (OD) or absorbance at colored mycelia with green spores and orange-yellow colora- 540 nm for GNP synthesized using ECF and BECF is pre- tion were observed behind the plate (Fig. 1a). Figure 1b shows sented in Fig. 3a. It is apparent that, initially, GNP synthesis the morphological feature of the fungal strain as observed using ECF and BECF was slow, up to 120 and 240 min, under a phase contrast microscope. respectively, after which the reaction was quite rapid for ECF, Molecular characterization of the fungal strain was done by which showed a continuous increase in absorbance up to 24 h 28S rDNA sequencing. The fragment of the D1/D2 region of beyond which there was a slow increase. Comparatively, a the LSU (large subunit 28S rDNA) gene obtained from the slower pace of reaction was observed for BECF which isolated plasmid DNA was amplified by PCR. The inset in showed a smaller but continuous formation of GNP even Fig. 1c shows the single discrete amplicon band of 650 bp beyond 48 h. Even at 48 h of incubation, reaction is not 1058 Ann Microbiol (2014) 64:1055–1063 a b c Penicillium marneffei AB363758.1 Penicillium marneffei AB219804.1 Penicillium marneffei AB363757.1 Penicillium marneffei (AB363756.1) Penicillium marneffei (AB363759.1) 700 bp Penicillium marneffei (AB363755.1) Acremonium cellulolyticus Y-94 (AB474751.1) Penicillium cf. verruculosum RS7PF (EU579531.1) Penicillium verruculosum (AF510496.1) IF3 Talaromyces flavus strain NRRL 2098 (EU021596.1) Fig. 1 a, b Morphological characteristics of Talaromyces flavus. c Phylogenetic tree depicting the evolutionary relationship of Talaromyces flavus.The inset in (c) shows the agarose gel image of PCR amplicon (Lane 1 D1/D2 region of LSU (large subunit 28S rDNA) amplicon band; lane 2 DNA marker) complete as Au ions were still available for reduction. The The observed rate constant (k ) is obtained from the slope of obs reaction process may be slower because of less proteins/native the plot (Fig. 3b). The observed rate constant (k )for ECF obs −1 proteins and/or reducing agent available in the BECF after and BECF was found to be 0.032 and 0.014 h , respectively. boiling. Secondly, the obtained curves were sigmoidal in shape suggesting an autocatalytic reaction. An autocatalytic reaction involves a continuous but slow nucleation process Optimization of conditions for gold nanoparticle biosynthesis followed by fast growth. For an autocatalytic reaction, the The effect of initial concentration of gold salt (HAuCl )on reaction rate is obtained by plotting ln[a/(1-a)] with respect to time (where a =OD(t)/OD(∞); OD(t) and OD(∞)are the GNP formation was studied by varying its concentration in the reaction mixture. Figure 4a shows the UV–vis spectra of GNP O.D. at times t and ∞, respectively) and ln[a/(1-a)] is expected to change linearly with progressing time (Huang et al. 1993). synthesized at different gold salt concentrations. At a low HAuCl concentration of 0.5 mM, two distinct peaks at 535 and 740 nm was observed. The appearance of a peak in the 2.5 longer wavelength region is because of longitudinal plasmon ECF only resonance implying the formation of anisotropic particles (Yu ECF + HAuCl4 et al. 1997). With the gradual increase in gold salt concentra- BECF + HAuCl4 BBF + HAuCl4 tion, a red shift in the transverse SPR peak was observed along 1.5 with the complete disappearance of the longitudinal plasmon resonance peak. At 1 mM concentration, the SPR peak was observed at around 540 nm with the highest absorbance intensity. At a higher concentration of 1.5 and 2 mM, a broad SPR peak at around 555 nm was observed. This indicates that 0.5 increasing the HAuCl concentration beyond a certain limit leads to the formation of large and aggregated GNP. The obtained kinetic data showed that the observed rate constant 240 440 640 840 −1 was maximum for 1 mM concentration (k =0.032 h ). The Wavelength (nm) obs rate constant was found to increase with concentration, up to Fig. 2 UV–vis absorption spectra of GNP synthesized using boiled fungal extracts (condition: 1 mM HAuCl ,24 h) 1 mM HAuCl , and thereafter decreased with further increase 4 4 Absorbance Ann Microbiol (2014) 64:1055–1063 1059 2.5 ECF BECF BECF ECF a b 1.6 -1 Kobs (ECF) = 0.032h 1.5 1.2 0.5 0.8 -1 Kobs (BECF) = 0.014h 0.4 -0.5 0 -1 0 500 1000 1500 2000 2500 3000 0 10203040506070 80 Time (mins) Time (hours) Fig. 3 a Evolution of absorbance intensity with time for GNP synthesized using ECF and BECF at 1 mM HAuCl concentration. b Autocatalytic kinetic plot of resulting optical data of GNP synthesized using ECF and BECF in concentration. The k was 0.030, 0.032, 0.022, and the formation of GNP (data not shown), which indicates obs −1 0.019 h for 0.5, 1, 1.5, and 2 mM HAuCl concentrations, the possible involvement of protein molecules as reduc- respectively. Figure 4b illustrates the effect of initial concen- ing or stabilizing agents in GNP synthesis. This study tration of HAuCl on the observed rate constant. The result further proves that the components present in the fungal thus indicates that the reduction rate depends on the concen- ECF carry out the bioreduction process and subsequent tration of initial gold salt. As 1 mM HAuCl concentration GNP production. showed the maximum intensity of GNP production, it was The influence of biomass amount on GNP production was consequently taken as the optimized concentration for further studied by varying the sucrose concentration in the growth experiments. (cornmeal) medium. High concentrations of carbon source are The concentration of ECF was varied to study the effect of known to enhance the growth of microorganisms leading to biomolecules on GNP biosynthesis. In brief, ECF was con- increased amounts of biomass, which in turn would enhance centrated by lyophilization and treated in increasing concen- the secretion of biomolecules. As hypothesized, a gradual trations (5, 10, 15, and 20 mg/ml) with 1 mM HAuCl The increase in sucrose concentration favored an increase in fungal UV–vis spectra presented in Fig. 5 reveals that, with the biomass, resulting in higher protein content in the ECF (and gradual increase in ECF concentration, the absorbance inten- may be other biomolecules responsible for GNP formation). sity of the synthesized GNP also increases, thereby Figure 6a presents the relationship between the sucrose con- confirming a direct relationship between the concentration of centration in the growth medium, the corresponding biomass fungal biomolecules and GNP synthesis. A decrease in protein weight, and protein concentration. At 2 % sucrose concentra- concentration in the reaction mixture was also observed after tion, a sharp SRP peak was observed at 545 nm that signified 0.04 ECF only ab 0.5mM 0.03 1mM 1.5 1.5mM 2mM 0.02 0.01 0.5 230 330 430 530 630 730 830 930 0.5 1 1.5 2 Wavelength ( nm) HAuCl4 Conc (mM) Fig. 4 a UV–vis absorption spectra of GNP synthesized at different concentrations of HAuCl b The effect of initial concentration of HAuCl on the 4 4 observed rate constant Absorbance (540 nm) Absorbance ln a/(1-a) -1 k h obs 1060 Ann Microbiol (2014) 64:1055–1063 5mg/ml 10mg/ml 1.2 15mg/ml 20mg/ml 0.8 0.4 300 500 700 900 56789 10 Wavelength (nm) pH Fig. 5 UV–vis absorption spectra of GNP synthesized at different con- Fig. 7 Effect of initial pH of ECF on absorption maxima (SPR) of centrations of ECF after 24 h of reaction synthesized GNP homogeneity and monodispersity of the synthesized nanopar- using dilute HCl and NaOH prior to HAuCl addition, keep- ticles. With a gradual increase in sucrose concentration, nar- ing other parameters constant. At different pH, the SPR peaks row peaks with enhanced intensity were observed. Even a were centered at different wavelength maxima. GNP synthe- decrease in reduction time was observed. With 5 % sucrose sized at inherent pH (pH 8) and pH 9 exhibited evident peaks concentration, visible GNP production was observed within at 540 nm. A peak with the maximum intensity was obtained 3 h of treating the ECF with HAuCl . Increasing the sucrose at the inherent pH. However, with a decrease in the pH of concentration in media thus helps in a rapid reduction the reaction mixture, the SPR peak shifted towards a and enhanced GNP production. Figure 6b shows the longer wavelength and a decrease in intensity was also absorbance intensity of synthesized GNP at different su- observed (Fig. 7). It has been reported that mostly crose concentrations. Higher sucrose concentration led to high amino, sulfydryl, and carboxylic acid groups are in- protein concentration which in turn resulted in enhanced volved in nanoparticle formation. They are positively GNP production. Thus, it can be inferred that proteins charged at low pH due to excessive protonation, altering the most likely play a role in the reduction and stabilization surface charge of the biomolecules and ultimately resulting in of GNP. mild reduction and heterogeneity of the synthesized nanopar- Literature survey emphasized that pH is yet another ticles (Ahmad et al. 2002). Therefore, acidic pH triggers the important factor in controlling the size of nanoparticles during formation of polydispersed nanoparticles. A gradual increase their synthesis (Nayak et al. 2010). With this in mind, the in pH leads to an increase in the reduction rate with controlled effect of the pH of the reaction mixture on GNP production nucleation, and results in the formation of monodispersed and was investigated. The initial pH of fungal ECF was varied homogenous nanoparticles. 60 14 2 Protein conccentration (mg/ml) a b Biomass weight (g/100 ml) 1.6 1.2 0.8 0.4 0 0 0 012345 012345 Sucrose (%) added to Medium Sucrose Conc. (%) Fig. 6 a Effect of increasing sucrose concentration in the growth medium on biomass weight and protein concentration of ECF. b Effect of different sucrose concentration on absorbance intensity of synthesized GNP Protein (mg/ml) Absorbance Biomass (g/100 ml) Absorbance maxima (SPR) Absorbance at 545 nm Ann Microbiol (2014) 64:1055–1063 1061 Fig. 8 UV–vis absorption 1.5 ab Original spectra depicting the effect of 10mM NaCl 1.2 ionic strength (NaCl 30mM NaCl concentration) on a biological 40mM NaCl 0.9 and b chemically synthesized GNP 0.6 0.9 0.3 300 400 500 600 700 Wavelength (nm) 0.6 Original 40mM NaCl 100mM NaCl 220mM NaCl 0.3 300 400 500 600 700 800 900 Wavelength (nm) Effect of ionic strength on stability of biological diffractogram (Supplementary Fig. 1). The biosynthesized and chemically synthesized GNP GNP were in an elemental form, Au , as established by the XRD analysis. The distinct Bragg reflection peaks at 2θ Nanoparticles undergo coagulation at high salt (ionic strength) values of 38.0, 44.08, and 64.4 closely matched the standard concentrations. Coagulation occurs due to the compression of XRD data for gold (JCPDS Card No: 00-001-1172) and the electrical double layer in the presence of high salt concen- corresponded to the crystal facets of (111), (200), and (220). trations (Chandran et al. 2012). In the present study, it was The formed GNPs were consistent with the face-centered observed that coagulation of chemically synthesized GNP cubic (fcc) structure of gold, and thus synthesized GNP were occurred at much lower concentrations of NaCl compared to undoubtedly crystalline in nature. An additional peak at 2θ biological GNP. The biological GNP were stable up to value of 27.69 corresponding to that of AuCl was also ob- 220 mM of NaCl concentration (Fig. 8a), whereas chemically served (JCPDS Card No: 00-018-0568), depicting its exis- synthesized GNP coagulated at 40 mM of NaCl (Fig. 8b). This tence as the dissolved precursor ion. suggests the high stability of biological GNP because of TEM images of GNP synthesized using ECF and BECF efficient capping that strongly shields the GNP, thereby after 24 h confirms the metal particles to be in nanoscale, but resisting coagulation. were irregularly shaped and of varying size (Fig. 9a, b). The GNP formed using ECF were of floral shape. They possess a gold core surrounded by asymmetrical protrusions. Synthetic Characterization of synthesized gold nanoparticle procedures undertaken for floral-shaped nanoparticle synthe- sis involves multiple steps and the use of hazardous The exact phase composition and crystalline structure of the chemicals, whereas, in the present case, floral-shaped GNP biosynthesized GNP were analyzed from the XRD a b c Fig. 9 Transmission electron micrographs of GNP synthesized using a ECF, b BECF, and c nucleation points of GNPs in ECF Absorbance Absorbance 1062 Ann Microbiol (2014) 64:1055–1063 synthesis involved a single step protocol and was rapid. Since Thus, FTIR spectra partially deciphered the involvement floral-shaped nanoparticles are known to exhibit a high sur- of –NH, –CO, and –SH groups as the possible capping face area and enhanced plasmon resonance, this single step agents. biogenic synthesis of floral GNPs using T. flavus can be a major breakthrough in catalysis as well as biomedical appli- cations. In the case of BECF, the synthesized GNP were Conclusion and future perspectives polydispersed with a mixture of hexagonal, spherical, and triangular-shaped nanoparticles. Figure 9c shows the presence In this paper, we report a rapid, ecofreindly, and feasible single of some minute particles (even smaller than 1 nm) along with step route for floral-shaped GNP biosynthesis using a novel large particles. The former indicates the points of nucleation fungal strain, Talaromyces flavus. The synthesized nanoparti- which fuse to form bigger particles. cles were of floral-shaped pattern with a mean hydrodynamic Energy dispersive spectroscopic (EDS) analysis radius of 38.54±10.34. nm The bioreduction of gold ions to (Supplementary Fig. 2) showed the presence of strong signals form GNP followed an autocatalytic reaction model, with an corresponding to elemental gold along with signals for C, O, −1 observed rate constant of 0.032 h . A direct relationship and Cu. Copper peaks were detected due to the use of copper between the concentration of fungal biomolecules and GNP grids in the TEM analysis, and the carbon and oxygen peaks biosynthesis was confirmed. FTIR analysis revealed the in- are due the fungal biomolecules. The SAED image exhibits volvement of –NH, –CO, and –SH groups in the stabilization several diffraction ring patterns and further confirms the crys- of synthesized GNPs. Thus, in addition to antimicrobial prop- talline nature of the synthesized GNP. The particles were erties of the Talaromyces flavus extract, its capacity to syn- arranged in different planes which is in agreement with the thesize floral-shaped GNPs is an additional breakthrough XRD spectra. benefit in the fields of catalysis, SERS study, and biomedi- The sizes of the GNP synthesized using ECF, BECF, and cines. However, the possible agents involved in the reduction BBF were determined after 24 h by DLS analysis. GNP and stabilization of GNP need further investigation. Studies synthesized using BECF was comparatively of a very concerning the mechanism and biochemistry of GNP synthe- small size range with mean hydrodynamic radius (Rh) sis can provide a complete knowledge on the molecular of 9.92±2.124 nm as compared to 38.54±10.347 and mechanism which can help control the size, shape, and 40.92±5.682 nm in the case of ECF and BBF respectively monodispersity of nanoparticles. (Supplementary Fig. 3). The obtained data are in good agreement with UV–vis spectra (Fig. 2) in which the SPR Acknowledgment The authors would like to thank the reviewers for band of BECF is blue-shifted in comparison to those of ECF their valuable suggestions in improving the manuscript. E. Priyadarshini and BBF. would like to express her sincere thanks to the Department of Science and The comparison of FTIR spectra (Supplementary Fig. 4)of Technology, New Delhi, India, for fellowship under the DST-Inspire Scheme. the fungal extract (ECF) before and after GNP synthesis was analyzed to identify the functional groups and biomolecules responsible for the reduction of Au ions and subsequent stabilization of the formed GNP. 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Biogenic synthesis of floral-shaped gold nanoparticles using a novel strain, Talaromyces flavus

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Springer Journals
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Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-013-0744-4
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Abstract

Ann Microbiol (2014) 64:1055–1063 DOI 10.1007/s13213-013-0744-4 ORIGINAL ARTICLE Biogenic synthesis of floral-shaped gold nanoparticles using anovel strain, Talaromyces flavus Eepsita Priyadarshini & Nilotpala Pradhan & Lala Behari Sukla & Prasanna Kumar Panda & Barada Kanta Mishra Received: 21 June 2013 /Accepted: 22 October 2013 /Published online: 19 November 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 . . Abstract A biogenic route was adopted towards the synthesis Keywords Biogenic synthesis Gold nanoparticle . . of gold nanoparticles using the extract of a novel strain, Talaromyces flavus Extracellular Nanobiotechnology Talaromyces flavus. Reduction of chloroauric acid by the fungal extract resulted in the production of gold nanoparticle, which was further confirmed by the concordant results obtain- Introduction ed from UV–visible spectroscopy, energy dispersive spectros- copy (EDS), and dynamic light scattering (DLS) analysis. The last decade has been witnessing a fascinating area of Morphology and the crystal nature of the synthesized nano- research termed nanotechnology: “the synthesis and fabrica- particles were characterized using transmission electron mi- tion of nanomaterials and their applicability” (Eustis and croscopy (TEM), X-ray diffraction (XRD) and selected area El-Sayed 2006). The European Union Commission defines electron diffraction (SAED). A direct correlation was observed nanoparticles as “particles having one or more dimensions in between nanoparticle formation and the concentration of re- the size range of 1−100 nm” and nanomaterials as “natural, ducing agent present in the fungal extract. The time-dependent incidental or manufactured material containing particles, in an kinetic study revealed that the bioreduction process follows an unbound state or as an aggregate or an agglomerate and autocatalytic reaction. Crystalline, irregular, and mostly flower- where, for 50 % or more of the particles in the number size shaped gold nanoparticles with a mean hydrodynamic radius of distribution, one or more external dimensions is in the size 38.54±10.34 nm were obtained. pH played a significant role on range 1–100 nm.” The increasing application of nanomaterials production of mono-dispersed nanoparticle. FTIR analysis par- concomitant with their growing demand has led to the devel- tially deciphered the involvement of –NH , −SH, and –CO 2 opment of a plethora of routes for their synthesis and size groups as the probable molecules in the bio-reduction and modification. Among the noble metals (silver, gold, and plat- stabilization process. Compared to the conventional methods, inum), gold has been of paramount importance since ancient a time-resolved, green, and economically viable method for times. However, gold nanoparticles (GNP) in comparison to floral-shaped nanoparticle synthesis was developed. bulk gold have attracted specific attention because of their interesting optical and catalytic properties (Sardar et al. 2009; Eustis and El-Sayed 2006). Electronic supplementary material The online version of this article GNP have found application in diverse fields like optics (doi:10.1007/s13213-013-0744-4) contains supplementary material, which is available to authorized users. (Hong and Hall 2012), electronics (Schmid and Simon 2005), electrochemistry (Huang et al. 2005), catalysis (Stratakis and E. Priyadarshini (*) N. Pradhan Academy of Scientific and Innovative Research, CSIR-Institute of Garcia 2012), sensing and detection agents (Scampicchio Minerals and Materials Technology, Bhubaneswar 751013, India et al. 2006), and in biomedical spheres (Dykman and e-mail: lisa.eepsita@gmail.com Khlebtsov 2012) because of their stability, resistance to oxi- : : dation, and biocompatibility. Recently, GNP are being widely N. Pradhan L. B. Sukla P. K. Panda Bioresources Engineering Department, CSIR-Institute of Minerals used for catalysis and SERS studies, which demand the use of and Materials Technology, Bhubaneswar 751013, India complex multi-shaped nanostructures like star-shape, urchin- like, and floral-shaped (Boca et al. 2011;Nehl etal. 2006). B. K. Mishra Such ashape enhances thetotal surfaceareaaswellas CSIR- Institute of Minerals and Materials Technology, Bhubaneswar 751013, India show an enhanced Raman scattering effect. Floral-shaped 1056 Ann Microbiol (2014) 64:1055–1063 GNP in comparison with spherical GNP is known to exhibit cotton blue stain using a phase contrast microscope (Nikon strong surface plasmon resonance along with a very high H550L). Molecular characterization was done by 28S rDNA electromagnetic field, localized at its protrusions (Nehl et al. gene sequencing. DNA was isolated and the fragment of 2006; Kim et al. 2009). However, the existing methods for gene was amplified using the universal primers DF: floral-shaped GNP synthesis involves the use of toxic chem- 5 ’ -ACCCCGCTGAACTTAAG C - 3 ’ an d D R: ical reagents and tedious multistep procedures (Wang et al. 5’-GGTCCGTGTTTCAAGACGG-3'. The obtained se- 2008) making the process hazardous and expensive. quence was then subjected to BLAST analysis and highly Several physicochemical methods such as laser ablation, homologous gene sequences were selected and represented sonoelectrochemistry, photochemical and chemical reduction in the form of phylogenetic tree. exist for GNP synthesis (Sylvestre et al. 2004;Serwas et al. 2008; Dong and Zhou 2007; Scampicchio et al. 2006). Biosynthesis of gold nanoparticles However, researchers are currently focusing on the biological route that utilizes an environmentally friendly solvent medium A loopful of fungal spore was aseptically inoculated in 100 ml and a nontoxic material for stabilization. Biological systems of sterile cornmeal medium (HIMEDIA) composed of corn- have proved to be natural nano-factories leading to the pro- meal (20 g/l), peptic digest of animal tissue (10 g/l), yeast duction of stably-capped GNPs (Sharma et al. 2009). Bacillus extract (4 g/l), and dextrose (10 g/l). The flasks were incubated subtilis was the first reported biological system for GNP at 35 °C for 72 h in an orbital shaker incubator at 150 rpm synthesis (Beveridge and Murray 1980). Later, eukaryotes (Kuhner, Switzerland). The biomass obtained was filtered like fungi, algae and plant extracts were also found to effec- using Whatman filter paper and washed thoroughly with tively reduce gold ions to GNP (Soni and Prakash 2012; sterile distilled water to remove all media components. The Luangpipat et al. 2011; Ghosh et al. 2012). Several fungi like washed biomass was further incubated in 100 ml of sterile Aspergillus niger, Aspergillus nidulans, Penicillum sp. and distilled water under similar conditions for 72 h. Incubation in Verticillium sp. have also been used for GNP synthesis (Soni sterile distilled water subjects it to a stress condition, which and Prakash 2012; Prusinkiewicz et al. 2012;Liangwei etal. allows the extracellular secretion of proteins and metabolites. 2011; Mukherjee et al. 2001). Fungi are greatly preferred for The filtrate obtained after incubation was termed the extracel- their capacity to secrete high amount of proteins, along with lular fluid (ECF). In another set of experiment, ECF was the advantage of easy handling, which make the downstream boiled for 30 min and was termed the boiled extracellular process simple (Sastry et al. 2003). fluid (BECF). In yet another set of experiment, the mycelia The current work describes the synthesis of extracellular along with the ECF obtained after 72 h were boiled for 30 min. floral-shaped GNP, by the reduction of gold ions using The resulting solution was filtered and termed the boiled the aqueous extract of the fungus Talaromyces flavus. biomass filtrate (BBF). All three filtrates were used for GNP Talaromyces flavus, a novel fungal strain, has been reported production. to inhibit various soil-borne plant pathogens such as Sclerotinia The ECF, BECF, and BBF were treated with chloroauric sclerotiorum, Rhizoctonia solani, and Verticillium dahlia. acid (HAuCl ) and incubated for GNP synthesis. The concen- (Boosalis 1956; Fravel et al. 1986; Mclaren et al. 1994). tration of HAuCl was maintained at 1 mM, barring a few However, to the best of our knowledge, its potential to reduce experiments. Three sets of control, comprising of cornmeal gold ions to GNP has not yet been reported in the literature. medium, ECF taken at 0 and 72 h of incubation were run This is the first report of a simple, green and rapid protocol for simultaneously. The first two sets of the control were treated floral-shape GNP production using a fungal extract. The effect with 1 mM HAuCl and the last set was run without HAuCl 4 4 of different parameters required for the optimization of yield, addition. controlled synthesis and minimization of the time for synthesis The effect of ionic strength on the stability of GNP synthe- were studied. sized by biological and chemical method was studied by treating the synthesized GNP colloidal suspension with NaCl. Chemical synthesis of GNP was carried out using Materials and methods sodium citrate as the reducing agent (Turkevitch et al. 1951). The critical coagulation concentration (CCC) represents the Fungal strain and its characterization NaCl concentration up to which the synthesized GNP remain stable without aggregation (Xie et al. 2003). In brief, CCC was The fungal strain used in the study was isolated from a mine- determined by treating the synthesized GNP (biological and contaminated site and subsequently maintained as pure chemical) with gradual increasing concentration of NaCl, culture on potato dextrose agar (HIMEDIA, India) slants. until a shift in the original SPR peak was observed. The The strain was designated as IF3. Morphological characteri- stability with respect to the SPR band was established by zation of the fungal strain was carried out by lactophenol recording the UV–visible absorbance of the mixture. Ann Microbiol (2014) 64:1055–1063 1057 Characterization of the synthesized GNP and analysis when resolved on agarose gel. The consensus sequence of of Extracellular fluid 611 bp of D2 region of the 28S rDNA gene generated was used to carry out BLAST. Based on the maximum identity Biosynthesis of GNP was visually observed by the change score, the first ten sequences were selected and the phyloge- in color of the reaction mixture (ECF treated with HAuCl ), netic tree was constructed using MEGA 4 software. On the andconcordantproductionofGNP wasconfirmedusing basis of nucleotide homology and phylogenetic analysis UV–vis Spectroscopic analysis (CECIL) in the range of (Fig. 1c), the isolate was identified to be Talaromyces flavus 190–1,100 nm. Transmission electron microscopy (TEM) strain NRRL 2098 (GenBank Accession No.: EU021596.1). analysis of synthesized GNP was carried out to determine their size and shape. TEM microscope (FEI, TECNAI-G2) Biosynthesis of gold nanoparticles using the fungal extracts 20-TWIN operating at 200 kVequipped with a GATAN CCD and its kinetic analysis camera was used for TEM-EDX analysis. The sample for TEM analysis was prepared by coating a drop of the colloidal On treating the ECF with HAuCl , a visible change in color gold solution onto a carbon-coated copper grid and allowing from colorless to burgundy was observed within 24 h of the solvent to evaporate under an IR lamp prior to analysis. To incubation. Change in color of the solution indicated the bio- investigate the possible functional groups involved in GNP reduction of chloroauric acid with the resultant formation of synthesis, Fourier transform infrared spectroscopy (FTIR, GNP.The formationofGNP wasconfirmedbyUV–vis Perkin-Elmer Model Spectrum 1) analysis was carried out. spectra with the appearance of a characteristic SPR peak in −1 The spectra were measured in the range of 4,000–400 cm at the range of 520–580 nm (Haiss et al. 2007). The control set −1 aresolutionof2cm . A few drops of the sample were layered comprising cornmeal medium showed GNP production indi- on top of a circular glass slide and allowed to dry, resulting in a cating that the media components were also capable of reduc- thin film that was then analyzed. The crystalline nature and ing gold ions. However, the second set of control (ECF taken phase analysis of the synthesized GNP were studied using at 0 h of incubation) when treated with HAuCl was not synchotron X-ray diffraction measurement. Diffraction capable of GNP production, indicating complete washout of pattern measurements were carried out on a Philips X’pert the medium components during biomass washing. The last set Pro, Panalytical X-ray powder diffractometer, having Cu-K of the control (ECF obtained after 72 h of incubation without (λ=1.54 Å) radiation, working at 40 kV/30 mA. The X-ray addition of HAuCl ) did not show any change in color; patterns were obtained in the 2θ range of 10–79°, using however, when treated with HAuCl , it showed GNP produc- 0.0050 step size. Dynamic light scattering (DLS) measure- tion. The results thus clearly indicate that the fungal biomol- ments were carried out to determine the hydrodynamic radius ecules secreted into the ECF during the 72 h of incubation of synthesized GNP using a Dawn Heleos II (Wyatt) system were solely responsible for the reduction of the gold ions to operating at a wavelength of 658 nm. GNP. Similarly, BECF and BBF were also treated with All the three filtrates obtained (ECF, BECF, and BBF) were HAuCl to study the role of denatured proteins on GNP 4, subjected to protein assay. Total protein content before and synthesis. Interestingly, characteristic SPR peaks for GNP after GNP production was determined by the Folin-Lowry were obtained in both cases (Fig. 2). In addition, GNP syn- method (Lowry et al. 1951). thesis in BBF was comparatively fast, i.e. within 5 h of HAuCl addition. This rapid production may be due to the additional release of intracellular reducing components into Result and discussion the extract when subjected to boiling. The kinetics of GNP biosynthesis was investigated to study Identification of the strain capable of GNP synthesis the effect of reaction time on gold ion reduction. For this, aliquots of the reaction mixture were withdrawn at regular The morphological and molecular identity of the fungus is time intervals and scanned to obtain the absorption spectra. depicted in Fig. 1. When grown on PDA plate, light yellow- The evolution of optical density (OD) or absorbance at colored mycelia with green spores and orange-yellow colora- 540 nm for GNP synthesized using ECF and BECF is pre- tion were observed behind the plate (Fig. 1a). Figure 1b shows sented in Fig. 3a. It is apparent that, initially, GNP synthesis the morphological feature of the fungal strain as observed using ECF and BECF was slow, up to 120 and 240 min, under a phase contrast microscope. respectively, after which the reaction was quite rapid for ECF, Molecular characterization of the fungal strain was done by which showed a continuous increase in absorbance up to 24 h 28S rDNA sequencing. The fragment of the D1/D2 region of beyond which there was a slow increase. Comparatively, a the LSU (large subunit 28S rDNA) gene obtained from the slower pace of reaction was observed for BECF which isolated plasmid DNA was amplified by PCR. The inset in showed a smaller but continuous formation of GNP even Fig. 1c shows the single discrete amplicon band of 650 bp beyond 48 h. Even at 48 h of incubation, reaction is not 1058 Ann Microbiol (2014) 64:1055–1063 a b c Penicillium marneffei AB363758.1 Penicillium marneffei AB219804.1 Penicillium marneffei AB363757.1 Penicillium marneffei (AB363756.1) Penicillium marneffei (AB363759.1) 700 bp Penicillium marneffei (AB363755.1) Acremonium cellulolyticus Y-94 (AB474751.1) Penicillium cf. verruculosum RS7PF (EU579531.1) Penicillium verruculosum (AF510496.1) IF3 Talaromyces flavus strain NRRL 2098 (EU021596.1) Fig. 1 a, b Morphological characteristics of Talaromyces flavus. c Phylogenetic tree depicting the evolutionary relationship of Talaromyces flavus.The inset in (c) shows the agarose gel image of PCR amplicon (Lane 1 D1/D2 region of LSU (large subunit 28S rDNA) amplicon band; lane 2 DNA marker) complete as Au ions were still available for reduction. The The observed rate constant (k ) is obtained from the slope of obs reaction process may be slower because of less proteins/native the plot (Fig. 3b). The observed rate constant (k )for ECF obs −1 proteins and/or reducing agent available in the BECF after and BECF was found to be 0.032 and 0.014 h , respectively. boiling. Secondly, the obtained curves were sigmoidal in shape suggesting an autocatalytic reaction. An autocatalytic reaction involves a continuous but slow nucleation process Optimization of conditions for gold nanoparticle biosynthesis followed by fast growth. For an autocatalytic reaction, the The effect of initial concentration of gold salt (HAuCl )on reaction rate is obtained by plotting ln[a/(1-a)] with respect to time (where a =OD(t)/OD(∞); OD(t) and OD(∞)are the GNP formation was studied by varying its concentration in the reaction mixture. Figure 4a shows the UV–vis spectra of GNP O.D. at times t and ∞, respectively) and ln[a/(1-a)] is expected to change linearly with progressing time (Huang et al. 1993). synthesized at different gold salt concentrations. At a low HAuCl concentration of 0.5 mM, two distinct peaks at 535 and 740 nm was observed. The appearance of a peak in the 2.5 longer wavelength region is because of longitudinal plasmon ECF only resonance implying the formation of anisotropic particles (Yu ECF + HAuCl4 et al. 1997). With the gradual increase in gold salt concentra- BECF + HAuCl4 BBF + HAuCl4 tion, a red shift in the transverse SPR peak was observed along 1.5 with the complete disappearance of the longitudinal plasmon resonance peak. At 1 mM concentration, the SPR peak was observed at around 540 nm with the highest absorbance intensity. At a higher concentration of 1.5 and 2 mM, a broad SPR peak at around 555 nm was observed. This indicates that 0.5 increasing the HAuCl concentration beyond a certain limit leads to the formation of large and aggregated GNP. The obtained kinetic data showed that the observed rate constant 240 440 640 840 −1 was maximum for 1 mM concentration (k =0.032 h ). The Wavelength (nm) obs rate constant was found to increase with concentration, up to Fig. 2 UV–vis absorption spectra of GNP synthesized using boiled fungal extracts (condition: 1 mM HAuCl ,24 h) 1 mM HAuCl , and thereafter decreased with further increase 4 4 Absorbance Ann Microbiol (2014) 64:1055–1063 1059 2.5 ECF BECF BECF ECF a b 1.6 -1 Kobs (ECF) = 0.032h 1.5 1.2 0.5 0.8 -1 Kobs (BECF) = 0.014h 0.4 -0.5 0 -1 0 500 1000 1500 2000 2500 3000 0 10203040506070 80 Time (mins) Time (hours) Fig. 3 a Evolution of absorbance intensity with time for GNP synthesized using ECF and BECF at 1 mM HAuCl concentration. b Autocatalytic kinetic plot of resulting optical data of GNP synthesized using ECF and BECF in concentration. The k was 0.030, 0.032, 0.022, and the formation of GNP (data not shown), which indicates obs −1 0.019 h for 0.5, 1, 1.5, and 2 mM HAuCl concentrations, the possible involvement of protein molecules as reduc- respectively. Figure 4b illustrates the effect of initial concen- ing or stabilizing agents in GNP synthesis. This study tration of HAuCl on the observed rate constant. The result further proves that the components present in the fungal thus indicates that the reduction rate depends on the concen- ECF carry out the bioreduction process and subsequent tration of initial gold salt. As 1 mM HAuCl concentration GNP production. showed the maximum intensity of GNP production, it was The influence of biomass amount on GNP production was consequently taken as the optimized concentration for further studied by varying the sucrose concentration in the growth experiments. (cornmeal) medium. High concentrations of carbon source are The concentration of ECF was varied to study the effect of known to enhance the growth of microorganisms leading to biomolecules on GNP biosynthesis. In brief, ECF was con- increased amounts of biomass, which in turn would enhance centrated by lyophilization and treated in increasing concen- the secretion of biomolecules. As hypothesized, a gradual trations (5, 10, 15, and 20 mg/ml) with 1 mM HAuCl The increase in sucrose concentration favored an increase in fungal UV–vis spectra presented in Fig. 5 reveals that, with the biomass, resulting in higher protein content in the ECF (and gradual increase in ECF concentration, the absorbance inten- may be other biomolecules responsible for GNP formation). sity of the synthesized GNP also increases, thereby Figure 6a presents the relationship between the sucrose con- confirming a direct relationship between the concentration of centration in the growth medium, the corresponding biomass fungal biomolecules and GNP synthesis. A decrease in protein weight, and protein concentration. At 2 % sucrose concentra- concentration in the reaction mixture was also observed after tion, a sharp SRP peak was observed at 545 nm that signified 0.04 ECF only ab 0.5mM 0.03 1mM 1.5 1.5mM 2mM 0.02 0.01 0.5 230 330 430 530 630 730 830 930 0.5 1 1.5 2 Wavelength ( nm) HAuCl4 Conc (mM) Fig. 4 a UV–vis absorption spectra of GNP synthesized at different concentrations of HAuCl b The effect of initial concentration of HAuCl on the 4 4 observed rate constant Absorbance (540 nm) Absorbance ln a/(1-a) -1 k h obs 1060 Ann Microbiol (2014) 64:1055–1063 5mg/ml 10mg/ml 1.2 15mg/ml 20mg/ml 0.8 0.4 300 500 700 900 56789 10 Wavelength (nm) pH Fig. 5 UV–vis absorption spectra of GNP synthesized at different con- Fig. 7 Effect of initial pH of ECF on absorption maxima (SPR) of centrations of ECF after 24 h of reaction synthesized GNP homogeneity and monodispersity of the synthesized nanopar- using dilute HCl and NaOH prior to HAuCl addition, keep- ticles. With a gradual increase in sucrose concentration, nar- ing other parameters constant. At different pH, the SPR peaks row peaks with enhanced intensity were observed. Even a were centered at different wavelength maxima. GNP synthe- decrease in reduction time was observed. With 5 % sucrose sized at inherent pH (pH 8) and pH 9 exhibited evident peaks concentration, visible GNP production was observed within at 540 nm. A peak with the maximum intensity was obtained 3 h of treating the ECF with HAuCl . Increasing the sucrose at the inherent pH. However, with a decrease in the pH of concentration in media thus helps in a rapid reduction the reaction mixture, the SPR peak shifted towards a and enhanced GNP production. Figure 6b shows the longer wavelength and a decrease in intensity was also absorbance intensity of synthesized GNP at different su- observed (Fig. 7). It has been reported that mostly crose concentrations. Higher sucrose concentration led to high amino, sulfydryl, and carboxylic acid groups are in- protein concentration which in turn resulted in enhanced volved in nanoparticle formation. They are positively GNP production. Thus, it can be inferred that proteins charged at low pH due to excessive protonation, altering the most likely play a role in the reduction and stabilization surface charge of the biomolecules and ultimately resulting in of GNP. mild reduction and heterogeneity of the synthesized nanopar- Literature survey emphasized that pH is yet another ticles (Ahmad et al. 2002). Therefore, acidic pH triggers the important factor in controlling the size of nanoparticles during formation of polydispersed nanoparticles. A gradual increase their synthesis (Nayak et al. 2010). With this in mind, the in pH leads to an increase in the reduction rate with controlled effect of the pH of the reaction mixture on GNP production nucleation, and results in the formation of monodispersed and was investigated. The initial pH of fungal ECF was varied homogenous nanoparticles. 60 14 2 Protein conccentration (mg/ml) a b Biomass weight (g/100 ml) 1.6 1.2 0.8 0.4 0 0 0 012345 012345 Sucrose (%) added to Medium Sucrose Conc. (%) Fig. 6 a Effect of increasing sucrose concentration in the growth medium on biomass weight and protein concentration of ECF. b Effect of different sucrose concentration on absorbance intensity of synthesized GNP Protein (mg/ml) Absorbance Biomass (g/100 ml) Absorbance maxima (SPR) Absorbance at 545 nm Ann Microbiol (2014) 64:1055–1063 1061 Fig. 8 UV–vis absorption 1.5 ab Original spectra depicting the effect of 10mM NaCl 1.2 ionic strength (NaCl 30mM NaCl concentration) on a biological 40mM NaCl 0.9 and b chemically synthesized GNP 0.6 0.9 0.3 300 400 500 600 700 Wavelength (nm) 0.6 Original 40mM NaCl 100mM NaCl 220mM NaCl 0.3 300 400 500 600 700 800 900 Wavelength (nm) Effect of ionic strength on stability of biological diffractogram (Supplementary Fig. 1). The biosynthesized and chemically synthesized GNP GNP were in an elemental form, Au , as established by the XRD analysis. The distinct Bragg reflection peaks at 2θ Nanoparticles undergo coagulation at high salt (ionic strength) values of 38.0, 44.08, and 64.4 closely matched the standard concentrations. Coagulation occurs due to the compression of XRD data for gold (JCPDS Card No: 00-001-1172) and the electrical double layer in the presence of high salt concen- corresponded to the crystal facets of (111), (200), and (220). trations (Chandran et al. 2012). In the present study, it was The formed GNPs were consistent with the face-centered observed that coagulation of chemically synthesized GNP cubic (fcc) structure of gold, and thus synthesized GNP were occurred at much lower concentrations of NaCl compared to undoubtedly crystalline in nature. An additional peak at 2θ biological GNP. The biological GNP were stable up to value of 27.69 corresponding to that of AuCl was also ob- 220 mM of NaCl concentration (Fig. 8a), whereas chemically served (JCPDS Card No: 00-018-0568), depicting its exis- synthesized GNP coagulated at 40 mM of NaCl (Fig. 8b). This tence as the dissolved precursor ion. suggests the high stability of biological GNP because of TEM images of GNP synthesized using ECF and BECF efficient capping that strongly shields the GNP, thereby after 24 h confirms the metal particles to be in nanoscale, but resisting coagulation. were irregularly shaped and of varying size (Fig. 9a, b). The GNP formed using ECF were of floral shape. They possess a gold core surrounded by asymmetrical protrusions. Synthetic Characterization of synthesized gold nanoparticle procedures undertaken for floral-shaped nanoparticle synthe- sis involves multiple steps and the use of hazardous The exact phase composition and crystalline structure of the chemicals, whereas, in the present case, floral-shaped GNP biosynthesized GNP were analyzed from the XRD a b c Fig. 9 Transmission electron micrographs of GNP synthesized using a ECF, b BECF, and c nucleation points of GNPs in ECF Absorbance Absorbance 1062 Ann Microbiol (2014) 64:1055–1063 synthesis involved a single step protocol and was rapid. Since Thus, FTIR spectra partially deciphered the involvement floral-shaped nanoparticles are known to exhibit a high sur- of –NH, –CO, and –SH groups as the possible capping face area and enhanced plasmon resonance, this single step agents. biogenic synthesis of floral GNPs using T. flavus can be a major breakthrough in catalysis as well as biomedical appli- cations. In the case of BECF, the synthesized GNP were Conclusion and future perspectives polydispersed with a mixture of hexagonal, spherical, and triangular-shaped nanoparticles. Figure 9c shows the presence In this paper, we report a rapid, ecofreindly, and feasible single of some minute particles (even smaller than 1 nm) along with step route for floral-shaped GNP biosynthesis using a novel large particles. The former indicates the points of nucleation fungal strain, Talaromyces flavus. The synthesized nanoparti- which fuse to form bigger particles. cles were of floral-shaped pattern with a mean hydrodynamic Energy dispersive spectroscopic (EDS) analysis radius of 38.54±10.34. nm The bioreduction of gold ions to (Supplementary Fig. 2) showed the presence of strong signals form GNP followed an autocatalytic reaction model, with an corresponding to elemental gold along with signals for C, O, −1 observed rate constant of 0.032 h . A direct relationship and Cu. Copper peaks were detected due to the use of copper between the concentration of fungal biomolecules and GNP grids in the TEM analysis, and the carbon and oxygen peaks biosynthesis was confirmed. FTIR analysis revealed the in- are due the fungal biomolecules. The SAED image exhibits volvement of –NH, –CO, and –SH groups in the stabilization several diffraction ring patterns and further confirms the crys- of synthesized GNPs. Thus, in addition to antimicrobial prop- talline nature of the synthesized GNP. The particles were erties of the Talaromyces flavus extract, its capacity to syn- arranged in different planes which is in agreement with the thesize floral-shaped GNPs is an additional breakthrough XRD spectra. benefit in the fields of catalysis, SERS study, and biomedi- The sizes of the GNP synthesized using ECF, BECF, and cines. However, the possible agents involved in the reduction BBF were determined after 24 h by DLS analysis. GNP and stabilization of GNP need further investigation. Studies synthesized using BECF was comparatively of a very concerning the mechanism and biochemistry of GNP synthe- small size range with mean hydrodynamic radius (Rh) sis can provide a complete knowledge on the molecular of 9.92±2.124 nm as compared to 38.54±10.347 and mechanism which can help control the size, shape, and 40.92±5.682 nm in the case of ECF and BBF respectively monodispersity of nanoparticles. (Supplementary Fig. 3). The obtained data are in good agreement with UV–vis spectra (Fig. 2) in which the SPR Acknowledgment The authors would like to thank the reviewers for band of BECF is blue-shifted in comparison to those of ECF their valuable suggestions in improving the manuscript. E. Priyadarshini and BBF. would like to express her sincere thanks to the Department of Science and The comparison of FTIR spectra (Supplementary Fig. 4)of Technology, New Delhi, India, for fellowship under the DST-Inspire Scheme. the fungal extract (ECF) before and after GNP synthesis was analyzed to identify the functional groups and biomolecules responsible for the reduction of Au ions and subsequent stabilization of the formed GNP. 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Journal

Annals of Microbiology – Springer Journals

Published: Nov 19, 2013

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