- Email: [email protected]
Biosynthesis and structural characterization of Ag nanoparticles from white rot fungi
Materials Science and Engineering C 33 (2013) 282–288
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biosynthesis and structural characterization of Ag nanoparticles from white rot fungi Yen San Chan, Mashitah Mat Don ⁎ School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang, Malaysia
a r t i c l e
i n f o
Article history: Received 25 March 2012 Received in revised form 12 July 2012 Accepted 29 August 2012 Available online 5 September 2012 Keywords: White rot fungi Silver nanoparticles Characterization Biosynthesis
a b s t r a c t Five species of white rot fungi were screened for their capability to synthesize Ag nanoparticles (AgNPs). Three modes of AgNP bioreduction were developed. Pycnoporus sanguineus is found as a potential candidate for the synthesis of AgNPs with a yield at 98.9%. The synthesized AgNPs were characterized using UV–vis spectroscopy, DLS, FTIR, TEM, and SEM. Results showed that AgNP absorption band was located at a peak of 420 nm. Both the SEM and TEM confirmed that the formation of AgNPs were mainly spherical with average diameters of 52.8–103.3 nm. The signals of silver atoms' presence in the mycelium were observed by SEM-EDS spectrum. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Since the last decade, white-rot fungi have been used intensively for bioremediation as they have the capability to transform or mineralize a wide range of environmentally hazardous compounds [1,2] through oxidative enzymatic mechanisms. The discovery of ligninolytic enzymes such as lignin peroxidase (LiP), manganese-peroxidase (MnP), horseradish-peroxidase (HrP) and laccase from white-rot fungi has triggered a biochemical research on lignin biodegradation [3–7]. However, the mechanistic action of these fungal secreted enzymes is nonspecific, non-stereospecific and extracellular in nature [8]. Although the application of these fungi involved mainly lignin degradation, it is known to degrade a wide range of hydrocarbons such as polyaromatic hydrocarbons (PAHs) [9], chlorinated aromatic hydrocarbons (CAHs) [10], polycyclic aromatics, polychlorinated biphenols [11], polychlorinated dibenzo-p-dioxins, pesticides DDT, lindane and azo dyes [5,6]. As stated by a few researchers, the most common white-rot fungi used in bioremediation are Phanerochaete chrysosporium [10], Schizophyllum commune [12] and Pycnoporus sanguineus [13,14]. Although the white rot fungi are commonly used in bioremediation, it was reported that these fungi can also serve as a platform for the bioreduction of Ag nanoparticles (AgNPs) [15]. Bioreduction is reported to be a biomimetic synthesis which utilized natural or biological principle, and implementing it into engineering [15]. The process involved absorption of metal ions onto the microbial surface by functional groups on the cell wall, and indirectly reduced by reducing sugars from hydrolysate of polysaccharides of the biomass into metal atoms [16]. Similar concept is applied on the adsorption of metal ions by fungal strain where
⁎ Corresponding author. Tel.: +60 4 5946468; fax: +60 4 5941013. E-mail addresses: [email protected], [email protected] (M. Mat Don). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.041
adsorption of metals can be classified as: extracellular accumulation, cell surface sorption and intracellular accumulation [17,18]. Duran et al. in 2005 in their research mentioned that the action of enzyme reduction or electron shuttle quinones in various microbes was responsible for the biomimetic process to occur. The role of the microbes in the bioreduction of metal is to provide a multitude of nucleation for establishing a highly dispersed nanoparticle system, slow down agglomeration, and provide a viscous medium [19]. In fact, the white-rot fungus, P. chrysosporium which is commonly used in bioremediation is reported to be an effective bioreduction agent for the synthesis of AgNPs. Thus showing that the fungi have the ability to reduce silver into AgNPs. Recently, AgNPs serve as noble metals and have drawn a considerable attention. Decreasing particle dimensions into nanosizes has manifested on the physical properties over bulk materials [20]. It is well known that silver has been historically recognized as a powerful biocide against bacteria, viruses and fungi. The particles are highly dispersed and posed a higher surface area which indirectly intensified antimicrobial properties and served as an effective antimicrobial agent [21]. In order to prepare these nanoparticles which serve as an effective antimicrobial agent over antibiotics, controlling the sizes and shapes as well as the stability is important [22]. Among various microorganisms screened, the white rot fungi namely Pleurotus sajor caju [23], Clostridium versicolor [24] and P. chrysosporium [15] produced stable AgNPs when challenged with silver nitrate in an aqueous medium. Though the capability of P. sanguineus for its AgNP production is less reported, it could be a potential route to produce an effective bioreduction agent using this fungus. This paper examines the capabilities of the Malaysian white rot fungi (P. sanguineus, S. commune, Lentinus sajor caju, Trametes feei, and Trametes pocas) that are isolated from the Malaysian rainforest to exhibit AgNPs in order to produce particles of smaller sizes. The differences in the modes of AgNP bioreduction and its characterization will also be investigated.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
2. Experimental 2.1. White rot fungi Five Malaysian white-rot fungi, namely P. sanguineus, S. commune, L. sajor caju, T. feei, and T. pocas are used in this study. They are obtained from the Forest Research Institute of Malaysia (FRIM), Kepong, Selangor, Malaysia. Cultures were maintained in the malt extract agar medium.
283
spectrophotometer (AA-6650). The light source used was a Hamamatsu Ag-hollow cathode lamp working at 10 mA current. Concentrations of AgNPs were identified using the same method as the experiment conducted on calibration curves. Concentrations of AgNPs were used to calculate the yield of the AgNPs produced based on Eq. (1) as shown.
Yield ¼
½AgNPs produced 100% ½AgNPs predicted
ð1Þ
2.2. Biosynthesis of silver nanoparticles The mycelial mat of the tested white rot fungi were mixed with 0.1% (v/v) Tween 80 before they were transferred aseptically into the cultivation flasks containing 50 mL of nutrient media. Cultivation media comprised (w/v): 0.7% KH2PO4, 0.2% K2HPO4, 0.01% MgSO4.7H2O, 0.01% (NH4)2SO4, 0.06% yeast extract and 1% glucose. The pH of the media was adjusted to 5.6 ±0.2 using 1 M HCl and 1 M NaOH. The mixture was then incubated at 30 °C, 200 rpm for 3 days. The harvested mycelia and culture broth were separated by centrifugation at 4500 rpm for 15 min; the supernatant and the pellet were then used for the synthesis of AgNPs. The mycelia pellets were washed thrice with deionized water. The washed pellets (1% w/v) and the culture supernatant (1% v/v) were then treated with a 0.001 M silver nitrate solution separately. Both mixtures were thereafter incubated at 30 °C in darkness at 200 rpm for 5 days. Three modes of AgNP bioreduction were conducted namely, (i) bioreduction of silver ion by the tested fungi-secreted proteins in culture supernatant (CS), (ii) bioreduction of silver ion by absorption of silver atom on the mycelia pellet (MP), and (iii) bioreduction of silver ion from the mycelia pellet which was released into the silver nitrate solution (SN), respectively. A control experiment containing only 0.001 M of silver nitrate solution was also performed. All experiments were carried out in triplicates and samples were drawn everyday throughout the 5 days of incubation. Samples obtained from the MP mode were re-suspended in deionized water and homogenized using a probe-top sonicator at 8.5 Hz for 5 min. The mixture of cells debris and AgNPs containing mixtures was centrifuged at 4500 rpm for 20 min. Supernatants containing AgNPs (MPS) were then used for further analysis.
where [AgNPs produced] is obtained from atomic absorption spectroscopy, and [AgNPs predicted] is obtained based on stoichiometry calculation.
Alkaline copper reagent was freshly prepared by mixing 1 mL of 1% (w/v) CuSO4 and 1 mL of 2% (w/v) Na tartrate with 98 mL of 2% (w/v) Na2CO3 in 0.1 M NaOH. 1.2 mL of protein sample was added into 6 mL of alkaline copper reagent. The mixture was mixed vigorously and the reaction was allowed to take place for 10 min. Later, 0.3 mL of Folin–Ciocalteu reagent was added by swirling, and then let it stand for another 30 min. The absorbance of the sample was recorded at A500.
The morphological properties of AgNPs produced by the tested fungal species were examined using a field emission scanning electron microscopy (FESEM) equipped with an energy dispersive X-ray (EDX) spectrometer (Zeiss Supra® 35VP, US), and a transmission electron microscopy (EFTEM, Zeiss Libra® 120 Plus, US). The micrographs of a spotted area were recorded and their corresponding EDX spectra were recorded by focusing on a cluster of particles. To examine the formation of AgNPs on the mycelial mats, freeze-dried mycelial mats were mounted on specimen stubs and coated with gold/palladium using a Bio-Rad Polaran Division SEM sputter coater, and examined under SEM operated at 10 kV at a magnification of 10,000×. For TEM, sample solutions CS and SN were dropped onto 300 mesh of carbon coated copper grid, and then allowed to dry prior to measurements. While fresh mycelial mats (MP) after culture were fixed using a McDowell–Trump fixative prepared in a 0.1 M phosphate buffer (pH 7.2) at 4 °C for 24 h. Later, it was post-fixed in a 1% (v/v) osmium tetraoxide for 2 h. The mycelia were dehydrated through a graded series of ethanol (50%, 75%, 95% and 100%; 95% and 100% levels were applied twice for 15 and 30 min, respectively) and 100% acetone twice for 10 min. After dehydration, the mycelium was infiltrated in a Spurr's mix resin in a rotator overnight. Infiltration in a new change of the Spurr's mix resin was performed for an interval of 6 h for 3 days. After the third day, samples were embedded at 60 °C. Subsequently, an ultramicrotomy process was performed on the embedded blocks using a Sorvall Ultramicrotome MT 5000 before they are being observed under the EFTEM. The infrared (IR) spectra of P. sanguineus and S. commune after the bioreduction of silver were obtained using the Fourier Transform Infrared Spectrometer (Shimadzu, IRPrestige-21). Reflectance technique was used in identifying the IR spectra. Potassium bromide (KBr) discs were prepared by grinding mycelium of P. sanguineus and S. commune with KBr in a ratio of 1:100 and compressing the mixture into a transparent disc of 13 mm in diameter. The transparent discs were directly placed into the infrared spectrometer in the wave number range of 400–4000 cm−1.
2.4. Characterization of silver nanoparticles
2.5. Antimicrobial activity
The bioreduction of Ag+ ion in sample solutions CS, SN and MPS was monitored by absorbance measurement using a double beam UV–vis spectrophotometer (Shimadzu UV-2550, US). The spectra of the surface plasmon resonance of AgNPs in the samples were measured at a resolution of 1 nm between 200 and 800 nm wavelengths. Subsequently, the average sizes of AgNPs in the sample solutions were measured using dynamic light scattering (DLS) a non-invasive back scatter (NIBS®) technology (Zetasizer Nano ZS, Malvern Instruments, Southborough, UK). All sample solutions were ultrasonicated (Transsonic Digital T 490 DH, Elma, Singe, Germany) and filtered using a 0.2 μm PTFE membrane syringe filter before analyses. 0.001 M of AgNO3 solution was used as the blank for both UV–vis and nanosize analyses. AgNP concentrations were analyzed on a Shimadzu atomic absorption
Minimum inhibitory concentration (MIC) assay was carried out using the microdilution method as described by Qi et al. [25], with a slight modification. The Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) and Gram positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus) were used as the tested microorganisms. 100 μL of AgNPs of known concentrations were transferred into 96 well microtiter plates containing 100 μL of Müeller Hinton broth. Dilutions were performed by the two-fold serial dilution method. Later, 100 μL of the tested bacteria was inoculated to all wells and the microtiter plates were incubated at 37 °C for 24 h. After the incubation period, the optical densities of cultures are measured at 595 nm using a microplate reader (Biorad Model 680). The minimum inhibitory concentration was determined as the lowest concentration of AgNPs that
2.3. Determination of protein concentration
284
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
inhibits the growth of microorganism which is represented by the absorbance.
a
3. Results and discussion 3.1. Biosynthesis and selection of AgNP producing fungus The bioreduction of silver occurred due to the presence of a reducing agent which reduced the silver ions in the AgNO3 solution into silver atoms of a nanosize dimension. It was reported that the enzyme reductase, nitrate reductase, hydrogenase or electron shuttle quinones were the potential enzymatic pathways for the bioreduction of silver [26,27]. Generally, the formation of silver can be primarily identified through visible observation in the change of color solution during the reaction from colorless to pale yellow or dark brown [28]. The change in color elucidates the presence of AgNPs in a solution due to the excitation of surface plasmon vibrations [29]. In this study, 5 species of the white-rot fungi namely P. sanguineus, S. commune, L. sajor caju, T. feei and T. pocas were screened for the synthesis of AgNPs. Results showed that sample solutions CS, SN and MPS of P. sanguineus and S. commune have shown a positive reaction for the biosynthesis of AgNPs through the increase in color intensity after 5 days of the incubation period. Fig. 1 showed a visual observation on the increase in color intensity of sample SN synthesized by the white rot fungus, S. commune after 5 days of incubation in shake flask cultures. The AgNP synthesis was further confirmed using the UV–vis spectral analysis. As reported by Sastry et al. [30], the UV–vis adsorption spectroscopy has proven to be one of the most useful and sensitive techniques for the analysis of nanoparticles. The electrochemical changes on the surface of nanosized metal particles are described as the optical properties of silver nanoparticles, and are being discussed within the Drude model [31]. In the Drude model, the ultraviolet region is the region of absorption for bulk plasmon frequency. Hence, the bioreduction of AgNPs can be identified indirectly using the UV– vis spectrum. In fact, the UV–vis spectroscopy method can also be used to identify the size evolution of AgNPs based on localized surface plasmon resonance band exhibiting at different wavelengths [32–34]. Fig. 2(a) and 2(b) showed the UV–vis spectra of the AgNPs synthesized by P. sanguineus extracellularly (SN) and through culture-free supernatant (CS), respectively. A strong, broad peak located between 430 nm and 450 nm was observed in the sample solution SN of P. sanguineus. However, a lower percentage of absorbance was observed in the solution CS compared to the SN of P. sanguineus. This could be due to a higher concentration of Ag synthesized extracellular than the culture-free supernatant synthesis of P. sanguineus. Similar UV-spectra were observed in the samples produced by S. commune but with a lower percentage of absorbance. According to the Beer–Lambert law, there is a correlation between the light transmission and absorption
D1
D2
D3
b
Fig. 2. UV–vis spectrum for AgNPs synthesized using P. sanguineus in samples (a) SN and (b) CS.
coefficient of the product. Hence, it explains the formation of absorption band by AgNPs at 420 nm. According to Sastry, [30,32], the presence of this peak indicated the sizes of metal nanoparticles which could be in the range of 2–100 nm. In addition, minor bands were also observed above 300 nm, which attributed to the electron excitation of protein residues [35]. Likewise, there is no clear change in the peak position of other tested species (L. sajor caju, T. feei, and T. pocas) as compared to P. sanguineus and S. commune. Although AgNPs are known to be effective broad spectrum biocides against a variety of drug-resistant bacteria, the mechanism of the inhibitory effects is still unclear. However, it was reported that the interaction of AgNPs with microorganisms results in inhibition
D4
D5
Fig. 1. Visual observation on increase in color intensity of sample SN synthesized by S. commune over 5 days of incubation.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
100 90 80 70
CS SN MPS
60 50 40 30 20 10 0 PS
SC
LSC
Fig. 3. Yield of AgNPs produced by P. sanguineus, S. commune and L. sajar caju after 5 days of incubation.
of growth, loss of infectivity to cell death and is much dependent on size of AgNPs synthesized, besides its shape and concentration [36–38]. Hence, identifying the nanosize of synthesized AgNPs is crucial in determining its applications. Fig. 3 summarized the selection of AgNP producing strain based on yield of AgNP production. The yield of AgNP production by P. sanguineus is shown to be the highest among the 3 modes of production tested compared to S. commune and L. sajar caju. In terms of particle size analysis, the average particle size of AgNPs produced by P. sanguineus and S. commune syntheses analyzed by dynamic light scattering are shown in Table 1. It was observed that the culture supernatant (CS) of the screened white-rot fungi has the ability to synthesize smaller diameter particle size in the range of 50 ± 10 nm to 65 ± 10 nm. In solution SN produced by P. sanguineus and S. commune showed that both white-rot fungi are able to produce a particle size of less than 80 nm. From definition, nanoparticles are nanosize particles ranging from 1 to 100 nm [39]. Hence showing that P. sanguineus gave the most promising AgNP production in terms of yield and particle size synthesized through extracellular (SN), culture-free supernatant (CS) and intracellular (MPS).
285
electron micrograph of AgNPs synthesized using mycelia pellet (MP) of S. commune and P. sanguineus after reacting with 0.001 M AgNO3. It was shown that the majority of the synthesized AgNPs present on the cell surface were spherical in shape. This could be due to the absorption of silver ions onto the surface of the mycelia pellet in the rotary shaker through an interaction with the mycelial surface functional groups such as carboxylate anion, carboxyl and peptide bond of proteins and hydroxyl of saccharides [40]. This finding is in agreement with the results of the Fourier transform infrared spectroscopy (FTIR) in this study which is shown in Fig. 5(a) and (b). The trough observed in regions 3410–3440 cm−1 results from the hydroxyl \OH. However, it was also reported that the signal of 3390 cm−1 may be due to the N\H asymmetric stretch mode of amines [41]. This indicated that the broad trough may be due to the presence of amino groups which are probably proteins. Similar observation was also reported by Mashitah et al., [42] for the binding mechanism of heavy metal biosorption by P. sanguineus where this broad trough is most likely due to the presence of protein. However, no carboxyl group was observed in the range of 1710–1740 cm−1. In the case of either biosorption or bioremediation, the functional groups of the fungal mycelial were responsible for binding the heavy metal [42]. However for the biosynthesis of AgNPs, an enzyme which was secreted by the fungi and acts as a bioreducing agent is needed. This showed that the functional groups of the tested fungi might be responsible for the bioreduction of silver, which are most likely to be the peptide bonds of proteins and hydroxyls of saccharides. Besides the functional groups it was also reported that when the mycelia matted together, they are more immobile and more capable of binding Ag+, hence in situ reduced to Ag0 [40,43]. Table 2 showed the IR spectra characteristics of P. sanguineus and S. commune mycelia after the
a
3.2. Structural and morphology characterizations of silver nanoparticles The results obtained by employing both analytical tools: UV–vis analysis and DLS analysis were useful as a prelude test to identify the presence of Ag during the biosynthesis process with fungal biomass or its culture supernatant. To corroborate the presence of AgNPs in fungal mycelia, SEM determination on the surface of treated fungal mycelia with AgNO3 were performed. Fig. 4(a) and (b) shows the scanning
b Table 1 Average particle size, polydispersity index of AgNPs, and protein concentrations produced by selected white-rot fungi. White-rot fungi Sample P. sanguineus
S. commune
L. sajor caju T. feei T. pocas
Ave. particle size ± SD, nm PDI
SN 70.2 ± 0.05 CS 63.8 ± 0.01 MPS 52.8 ± 0.06 SN 103.3 ± 0.06 CS 56.5 ± 0.02 MPS 53.9 ± 0.01 SN, CS, MPS ND SN, CS, MPS ND SN, CS, MPS ND
0.221 0.091 0.054 0.283 0.086 0.050 ND ND ND
[Protein] gL−1 20.41 27.37 30.51 15.26 28.12 29.85 ND ND ND
SN, extracellular synthesis; CS, culture-free supernatant synthesis; MPS, intracellular synthesis. ND, not determined due to negative or insignificant effect when treated with AgNO3. PDI, polydispersity index calculated based on ISO standard document 13321:1996E where, monodisperse b0.05, nearly monodisperse 0.05–0.08, mid range polydisperse 0.08–0.7, and very polydisperse >0.7.
Fig. 4. Scanning electron micrograph for the freeze-dried mycelium (a) S. commune (b) P. sanguineus.
286
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
Fig. 5. Fourier Transform spectroscopy spectra of mycelium (a) S. commune (b) P. sanguineus.
bioreduction of silver. It was observed that the bioreduction mechanism of both fungi is similar based on the presence of respective functional groups. The concentration of proteins produced by SN, CS and MPS of P. sanguineus, and S. commune was determined in Table 1. It showed that the higher the concentration of protein is secreted, the smaller the AgNPs produced. Although through SEM evaluation AgNPs were spherical in shape with smooth edges on the mycelia surface, Zaki et al. in 2010 stated that deposition of nanoparticles into the mycelia and mycelial membrane could be possible. Hence, a novel analysis by cutting mycelia fungi into a thin film (~10 nm) using an ultramicrotome was carried out and Table 2 Fourier transform infrared spectroscopy spectra characteristics and functional groups. Major peak
1 2 3 4 5
Wavelength (cm−1) P. sanguineus
S. commune
3434.08 2927.94 1621.07 1458.18 1072.42
3412.85 2924.52 1605.31 1428.08 1028.21
Assignment
Hydroxyl Aliphathic C\H group C_O stretching of COOH Symmetric bending of CH3 Carbohydrate group
observed under TEM. Fig. 6(a) and (b) shows the cross section of P. sanguineus and S. commune mycelia. In both cases, it was observed that majority of the AgNPs were located at the circumference of the fungal membrane. This showed that the enzyme that was secreted on the mycelial membrane served as the reducing agent for the biosynthesis of the silver nanoparticles [44]. However, from the micrographs, the AgNPs formed by the mycelial fungi tend to agglomerate. This phenomenon is possibly because of the unsuccessful binding of Ag+ to the surface of AgNPs and its being trapped in the fungi mycelia. To gain a further insight into the feature of the extracellular formation of AgNPs, an analysis of samples SN containing AgNPs were dropped onto carbon coated copper grid prior their observation under TEM. Fig. 6(c) and (d) showed that the AgNPs were freely dispersed in a silver nitrate solution. The AgNPs from the SN of P. sanguineus were observed to exhibit a higher degree of agglomeration compared to AgNPs from the SN of S. commune that are nearly spherical or ellipsoidal in shape for both cases. Polydispersity of AgNPs produced was further determined from polydispersity index calculated by Zetasizer Nano ZS as shown in Table 1. It showed that AgNPs produced by SN and CS were at the mid range of polydispersity while AgNPs by MPS were nearly monodispersed for both cases. Formation of freely dispersed AgNPs probably occurred during the initial incubation in the rotary shaker.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
a
b
c
d
287
Fig. 6. Transmission electron micrographs of a thin film of mycelium (a) S. commune (b) P. sanguineus coated on copper grid and AgNPs produced in sample SN by (c) S. commune and (d) P. sanguineus.
Generally, filamentous fungi which exhibited a higher surface area were more capable of binding Ag+. However, the tendency of surface proteins or chemical functional groups released into the solution during rotation is also higher in this study. The released reducing agents hence reduced Ag+ in the AgNO3 solution into Ag0 nanoparticles. It was observed in both UV–vis spectral analysis and SEM/TEM analysis that formations of extracellular AgNPs (SN) were more promising. This offers enormous advantages over intracellular synthesis of AgNPs in various applications.
3.3. Energy dispersive X-ray spectroscopy (EDS) analysis Table 3 illustrates the EDS patterns of the synthesized AgNPs by P. sanguineus and S. commune. Results showed that the EDS patterns consisted of Ag (66.05 wt.%), C (18.22 wt.%), Na (7.29 wt.%) and Cl (8.44 wt.%) which were present at the spotted area of the analysis. The signals of C, O, Na and Cl obtained were similar to the research by Zaki et al. that the signals are most probably due to the X-ray emission from the fungi mycelia and the remaining media [45]. In this Table 3 EDS analysis of AgNPs produced by mycelia P. sanguineus and S. commune after reaction with AgNO3. Fungus
Element C
P. sanguineus S. commune
study, P. sanguineus has shown to have the highest ability to synthesize AgNPs (28.21 Ar%) as compared to S. commune (22.81 Ar%). 3.4. Antimicrobial studies Silver has been well known for its antimicrobial activity since the last decades. Although it was reported that the antimicrobial effects could be due to inhibiting the enzymatic respiratory system of microbes and altering DNA synthesis [46,47], the mechanisms are still unclear. It is believed that the antimicrobial effects depend strongly on the superficial contact between silver and microorganisms. Due to that, a high surface area to volume ratio of the AgNPs could serve as a powerful antimicrobial agent. The antimicrobial effects of AgNPs produced by P. sanguineus and S. commune in this study were tested with Gram positive bacteria, S. aureus, S. epidermidis and the Gram negative bacteria, E. coli and K. pneumoniae. The MIC values for AgNPs synthesized by both P. sanguineus and S. commune were shown in Table 4. It was observed that the antimicrobial activity of AgNPs produced in the SN was more effective Table 4 MICs (μg/mL ± SD) of AgNPs. Fungi
PS Na
Cl
Ag
at.%
wt.%
at.%
wt.%
at.%
wt.%
at.%
wt.%
61.3 56.5
21.05 18.22
9.05 11.81
4.82 7.29
1.44 8.87
3.67 8.44
28.21 22.81
70.46 66.05
at.% and wt.% refer to atomic percentage and weight percentage of particular element.
SC
Sample
SN MPS CS SN MPS CS
Gram positive bacteria
Gram negative bacteria
S. epidermidis
S. aureus
E. coli
K. pneumoniae
0.03 ± 0.0 1.42 ± 0.2 1.43 ± 0.0 1.17 ± 0.1 7.03 ± 1.2 0.25 ± 0.0
4.27 ± 0.2 1.47 ± 0.2 0.37 ± 0.1 1.6 ± 0.2 9.5 ± 0.5 47 ± 2.1
0.09 ± 0.2 1.41 ± 0.3 0.71 ± 0.0 2.6 ± 0.1 30 ± 1.2 0.4 ± 0.0
1.02 ± 0.2 1.38 ± 0.3 0.41 ± 0.1 1.91 ± 0.1 12.8 ± 0.9 2.8 ± 0.6
PS refer to fungus P. sanguineus while SC refers to fungus S. commune.
288
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
than those of CS and MPS. The results also showed that AgNPs synthesized by both fungi were more effective antimicrobial agents as the MIC values lie between 0.03 and 47 μg/mL. This is in agreement with the results obtained by Kim [48] who indicated that the Gram positive bacteria, S. aureus was more resistant to AgNPs as compared to the Gram negative E. coli. Thus, it indicated that the tested white rot fungi of the present study produced AgNPs that have great bactericidal activities. In view of cytotoxicity, it was reported that AgNPs of 20 nm were more cytotoxic than silver ions [49], also smallest particle sizes of AgNPs were reported to have a greater ability to induce apoptosis in the Mc3T3-E1 cell line than larger AgNPs [50]. Hence, the particle size needs to be designed carefully for biomedical and biopharmaceutical uses. 4. Conclusions Among the five Malaysian white rot fungi tested, P. sanguineus and S. commune were capable in synthesizing AgNPs with an average particle size of range from 52.8 to 103.3 nm. Results showed that P. sanguineus is more favorable compared to S. commune as it has a higher atomic percentage of silver at 28.21% and a higher yield of AgNPs at 98.9%. The IR spectra of FTIR showed that the bioreduction of silver occurred when the silver ions interacted with the functional groups of the mycelial surface of the tested fungus. Thus, it indicated that the white rot fungi can be utilized for the production of AgNPs which have wider applications in pharmaceutical and medical fields. Acknowledgment This work is supported and funded by the Ministry of Higher Education Malaysia through Fundamental Research Grant Scheme (FRGS/1/10/ TK/USM/02/5) and by the USM fellowship which we gratefully acknowledge. References [1] M. Cameron, S. Timofeevski, S. Aust, Appl. Microbiol. Biotechnol. 54 (2000) 751–788. [2] A. Paszczynski, R. Crawford, Biotechnol. Prog. 11 (1995) 368–379. [3] J. Glenn, M. Morgan, M. Mayfield, M. Kuwahara, M. Gold, Biochem. Biophys. Res. Commun. 114 (1983) 1077–1083. [4] M. Kuwahara, J. Glenn, M. Morgan, M. Gold, FEBS Lett. 169 (1984) 247–250. [5] D. Wesenberg, I. Kyriakides, S. Agathos, Biotechnol. Adv. 22 (2003) 161–187. [6] A. Unyayar, M. Mazmanci, E. Erkurt, H. Atacag, A. Gizir, React. Kinet. Catal. Lett. 86 (2005) 99–107. [7] P. Kertsen, B. Kalyanaraman, K. Hamel, B. Reinhammar, T. Kirk, Biochem. J. 268 (1990) 475–480. [8] P. David, D. Steven, Environ. Sci. Technol. 28 (1994) 79A–87A. [9] T. Eggen, A. Majcherczyk, Int. Biodeterior. Biodegrad. 41 (1998) 111–117. [10] C. Wang, J. Xi, H. Hu, X. Wen, Biomed. Environ. Sci. 21 (2008) 474–478.
[11] P. Siripong, B. Oraphin, T. Sanro, P. Duanporn, Amer.–Eurasian J. Agric. & Environ. Sci. 5 (2009) 466–472. [12] W.M. Abd El-Rahim, O.A.M. El-Ardy, F.H.A. Mohammad, Desalination 249 (2009) 1206–1211. [13] E. Esposito, L.H. Innocentini-Mei, A. Ferraz, V.P. Canhos, N. Durán, J. Biotechnol. 29 (1993) 219–228. [14] M. Trovaslet, E. Enaud, Enzym. Microb. Technol. 41 (2007) 368–376. [15] N. Vigneshwaran, A. Kathe, P. Varadarajan, R. Nachane, R. Balasubramanya, Colloids Surf. B: Biointerfaces 53 (2006) 55–59. [16] Z. Lin, J. Wu, R. Xue, Y. Yang, Spectrochim. Acta A Mol. Biomol. Spectrosc. 61 (2005) 761–765. [17] N. Ahalya, T.V. Ramachandra, R.D. Kanamadi, Res. J. Chem. Environ. 7 (2003) 71–79. [18] C.C.V. Cruz, A.C.A. Da Costa, C.A. Henriques, A.S. Luna, Bioresour. Technol. 91 (2004) 249–257. [19] Y. Sun, Y. Xia, Science 298 (2002) 2176–2179. [20] S. Chulovskaya, V. Parfenyuk, Prot. Met. Phys. Chem. Surf. 45 (2009) 300–304. [21] V. Illic, Z. Saponjic, V. Vodnik, B. Pofkonjak, P. Jovancic, J. Nedeljkovic, M. Radetic, Carbohydr. Polym. 78 (2009) 564–569. [22] S. Kaviya, J. Santhananlakshmi, B. Viswanathan, J. Muthumary, K. Srinivasan, Spectrochim. Acta A Mol. Biomol. Spectrosc. 79 (2010) 594–598. [23] R. Nithya, R. Ragunathan, Dig. J. Nanomater. Biostruct. 4 (2009) 623–629. [24] R. Sanghi, P. Verma, Bioresour. Technol. 100 (2009) 501–504. [25] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Carbohydr. Res. 339 (2004) 2693–2700. [26] N. Duran, P.D. Marcato, O.L. Alves, G.I. Souza, E. Esposito, J. Nanobiotechnol. 3 (2005) 8. [27] J.C.G. Ottow, A. Von Klopotek, Appl. Microbiol. 18 (1969) 41–43. [28] M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Colloids Surf., B 73 (2009) 332–338. [29] A. Rai, A.K. Singh, A. Ahmad, M. Sastry, Langmuir 22 (2006) 736–741. [30] M. Sastry, K.S. Mayyaa, B. K., Colloids Surf., A 127 (1997) 221–228. [31] M. Sastry, V. Patil, S.R. Sainkar, J. Phys. Chem. B 102 (1998) 1404–1410. [32] A. Henglein, J. Phys. Chem. B 7 (1993) 5457–5471. [33] U. Kreibig, J. Phys. Chem. B 2000 (104) (1978) 9683–9688. [34] U. Kreibig, L. Grenzel, Surf. Sci. 156 (1985) 678–700. [35] A.M. Fayz, K. Balaji, P.T. Kalaichelvan, R. Venkatesan, Colloids Surf., B 74 (2009) 123–126. [36] S. Pal, Y. Tak, J. Song, Appl. Environ. Microbiol. 73 (2007) 1712–1720. [37] P. Asharani, M. Hanede, S. Valiyaveettil, BMC Cell Biol. 10 (2009) 65. [38] H. Yen, S. Hsu, C. Tsai, Small 5 (2009) 1553–1561. [39] S.E. McNeil, J. Leukoc. Biol. 78 (2005) 585–594. [40] Z. Lin, J. Wu, R. Xue, Y. Yong, Spectrochim. Acta A Mol. Biomol. Spectrosc. 61 (2005) 761–765. [41] T.R. Muraleedharan, I. Leela, C. Venkobachar, Environ. Technol. 15 (1994) 1015–1027. [42] M.D. Mashitah, Z. Zulfadhly, S. Bhatia, Artif. Cells Blood Substit. Immobil. Biotechnol. 27 (1999) 441–445. [43] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V. Varadarajan, Carbohydr. Res. 341 (2006) 2012–2018. [44] S.K. Das, E. Marsili, Rev. Environ. Sci. Biotechnol. 9 (2010) 199–204. [45] S. Zaki, M. El Kady, D. Abd-El-Haleem, Mater. Res. Bull. 46 (2010) 1571–1576. [46] D.W. Brett, Ostomy Wound Manage. 52 (2006) 34–41. [47] E. Hidalgo, C. Dominguez, Toxicol. Lett. 15 (1998) 169–179. [48] J.S. Kim, E. Kuk, K.N. Yu, J.H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.Y. Hwang, Y.K. Kim, Y.S. Lee, D.H. Jeong, M.H. Cho, Nanomedicine 3 (2007) 95–101. [49] M.V.D.Z. Park, A.M. Neigh, J.P. Vermeulen, L.J.J. de la Fonteyne, H.W. Verharen, J.J. Briedé, H. van Loveren, W.H. de Jong, Biomaterials 32 (2011) 9810–9817. [50] T.-H. Kim, M. Kim, H.-S. Park, U.S. Shin, M.-S. Gong, H.-W. Kim, J. Biomed. Mater. Res. Part A 100A (2012) 1033–1043.
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biosynthesis and structural characterization of Ag nanoparticles from white rot fungi Yen San Chan, Mashitah Mat Don ⁎ School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Penang, Malaysia
a r t i c l e
i n f o
Article history: Received 25 March 2012 Received in revised form 12 July 2012 Accepted 29 August 2012 Available online 5 September 2012 Keywords: White rot fungi Silver nanoparticles Characterization Biosynthesis
a b s t r a c t Five species of white rot fungi were screened for their capability to synthesize Ag nanoparticles (AgNPs). Three modes of AgNP bioreduction were developed. Pycnoporus sanguineus is found as a potential candidate for the synthesis of AgNPs with a yield at 98.9%. The synthesized AgNPs were characterized using UV–vis spectroscopy, DLS, FTIR, TEM, and SEM. Results showed that AgNP absorption band was located at a peak of 420 nm. Both the SEM and TEM confirmed that the formation of AgNPs were mainly spherical with average diameters of 52.8–103.3 nm. The signals of silver atoms' presence in the mycelium were observed by SEM-EDS spectrum. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Since the last decade, white-rot fungi have been used intensively for bioremediation as they have the capability to transform or mineralize a wide range of environmentally hazardous compounds [1,2] through oxidative enzymatic mechanisms. The discovery of ligninolytic enzymes such as lignin peroxidase (LiP), manganese-peroxidase (MnP), horseradish-peroxidase (HrP) and laccase from white-rot fungi has triggered a biochemical research on lignin biodegradation [3–7]. However, the mechanistic action of these fungal secreted enzymes is nonspecific, non-stereospecific and extracellular in nature [8]. Although the application of these fungi involved mainly lignin degradation, it is known to degrade a wide range of hydrocarbons such as polyaromatic hydrocarbons (PAHs) [9], chlorinated aromatic hydrocarbons (CAHs) [10], polycyclic aromatics, polychlorinated biphenols [11], polychlorinated dibenzo-p-dioxins, pesticides DDT, lindane and azo dyes [5,6]. As stated by a few researchers, the most common white-rot fungi used in bioremediation are Phanerochaete chrysosporium [10], Schizophyllum commune [12] and Pycnoporus sanguineus [13,14]. Although the white rot fungi are commonly used in bioremediation, it was reported that these fungi can also serve as a platform for the bioreduction of Ag nanoparticles (AgNPs) [15]. Bioreduction is reported to be a biomimetic synthesis which utilized natural or biological principle, and implementing it into engineering [15]. The process involved absorption of metal ions onto the microbial surface by functional groups on the cell wall, and indirectly reduced by reducing sugars from hydrolysate of polysaccharides of the biomass into metal atoms [16]. Similar concept is applied on the adsorption of metal ions by fungal strain where
⁎ Corresponding author. Tel.: +60 4 5946468; fax: +60 4 5941013. E-mail addresses: [email protected], [email protected] (M. Mat Don). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.08.041
adsorption of metals can be classified as: extracellular accumulation, cell surface sorption and intracellular accumulation [17,18]. Duran et al. in 2005 in their research mentioned that the action of enzyme reduction or electron shuttle quinones in various microbes was responsible for the biomimetic process to occur. The role of the microbes in the bioreduction of metal is to provide a multitude of nucleation for establishing a highly dispersed nanoparticle system, slow down agglomeration, and provide a viscous medium [19]. In fact, the white-rot fungus, P. chrysosporium which is commonly used in bioremediation is reported to be an effective bioreduction agent for the synthesis of AgNPs. Thus showing that the fungi have the ability to reduce silver into AgNPs. Recently, AgNPs serve as noble metals and have drawn a considerable attention. Decreasing particle dimensions into nanosizes has manifested on the physical properties over bulk materials [20]. It is well known that silver has been historically recognized as a powerful biocide against bacteria, viruses and fungi. The particles are highly dispersed and posed a higher surface area which indirectly intensified antimicrobial properties and served as an effective antimicrobial agent [21]. In order to prepare these nanoparticles which serve as an effective antimicrobial agent over antibiotics, controlling the sizes and shapes as well as the stability is important [22]. Among various microorganisms screened, the white rot fungi namely Pleurotus sajor caju [23], Clostridium versicolor [24] and P. chrysosporium [15] produced stable AgNPs when challenged with silver nitrate in an aqueous medium. Though the capability of P. sanguineus for its AgNP production is less reported, it could be a potential route to produce an effective bioreduction agent using this fungus. This paper examines the capabilities of the Malaysian white rot fungi (P. sanguineus, S. commune, Lentinus sajor caju, Trametes feei, and Trametes pocas) that are isolated from the Malaysian rainforest to exhibit AgNPs in order to produce particles of smaller sizes. The differences in the modes of AgNP bioreduction and its characterization will also be investigated.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
2. Experimental 2.1. White rot fungi Five Malaysian white-rot fungi, namely P. sanguineus, S. commune, L. sajor caju, T. feei, and T. pocas are used in this study. They are obtained from the Forest Research Institute of Malaysia (FRIM), Kepong, Selangor, Malaysia. Cultures were maintained in the malt extract agar medium.
283
spectrophotometer (AA-6650). The light source used was a Hamamatsu Ag-hollow cathode lamp working at 10 mA current. Concentrations of AgNPs were identified using the same method as the experiment conducted on calibration curves. Concentrations of AgNPs were used to calculate the yield of the AgNPs produced based on Eq. (1) as shown.
Yield ¼
½AgNPs produced 100% ½AgNPs predicted
ð1Þ
2.2. Biosynthesis of silver nanoparticles The mycelial mat of the tested white rot fungi were mixed with 0.1% (v/v) Tween 80 before they were transferred aseptically into the cultivation flasks containing 50 mL of nutrient media. Cultivation media comprised (w/v): 0.7% KH2PO4, 0.2% K2HPO4, 0.01% MgSO4.7H2O, 0.01% (NH4)2SO4, 0.06% yeast extract and 1% glucose. The pH of the media was adjusted to 5.6 ±0.2 using 1 M HCl and 1 M NaOH. The mixture was then incubated at 30 °C, 200 rpm for 3 days. The harvested mycelia and culture broth were separated by centrifugation at 4500 rpm for 15 min; the supernatant and the pellet were then used for the synthesis of AgNPs. The mycelia pellets were washed thrice with deionized water. The washed pellets (1% w/v) and the culture supernatant (1% v/v) were then treated with a 0.001 M silver nitrate solution separately. Both mixtures were thereafter incubated at 30 °C in darkness at 200 rpm for 5 days. Three modes of AgNP bioreduction were conducted namely, (i) bioreduction of silver ion by the tested fungi-secreted proteins in culture supernatant (CS), (ii) bioreduction of silver ion by absorption of silver atom on the mycelia pellet (MP), and (iii) bioreduction of silver ion from the mycelia pellet which was released into the silver nitrate solution (SN), respectively. A control experiment containing only 0.001 M of silver nitrate solution was also performed. All experiments were carried out in triplicates and samples were drawn everyday throughout the 5 days of incubation. Samples obtained from the MP mode were re-suspended in deionized water and homogenized using a probe-top sonicator at 8.5 Hz for 5 min. The mixture of cells debris and AgNPs containing mixtures was centrifuged at 4500 rpm for 20 min. Supernatants containing AgNPs (MPS) were then used for further analysis.
where [AgNPs produced] is obtained from atomic absorption spectroscopy, and [AgNPs predicted] is obtained based on stoichiometry calculation.
Alkaline copper reagent was freshly prepared by mixing 1 mL of 1% (w/v) CuSO4 and 1 mL of 2% (w/v) Na tartrate with 98 mL of 2% (w/v) Na2CO3 in 0.1 M NaOH. 1.2 mL of protein sample was added into 6 mL of alkaline copper reagent. The mixture was mixed vigorously and the reaction was allowed to take place for 10 min. Later, 0.3 mL of Folin–Ciocalteu reagent was added by swirling, and then let it stand for another 30 min. The absorbance of the sample was recorded at A500.
The morphological properties of AgNPs produced by the tested fungal species were examined using a field emission scanning electron microscopy (FESEM) equipped with an energy dispersive X-ray (EDX) spectrometer (Zeiss Supra® 35VP, US), and a transmission electron microscopy (EFTEM, Zeiss Libra® 120 Plus, US). The micrographs of a spotted area were recorded and their corresponding EDX spectra were recorded by focusing on a cluster of particles. To examine the formation of AgNPs on the mycelial mats, freeze-dried mycelial mats were mounted on specimen stubs and coated with gold/palladium using a Bio-Rad Polaran Division SEM sputter coater, and examined under SEM operated at 10 kV at a magnification of 10,000×. For TEM, sample solutions CS and SN were dropped onto 300 mesh of carbon coated copper grid, and then allowed to dry prior to measurements. While fresh mycelial mats (MP) after culture were fixed using a McDowell–Trump fixative prepared in a 0.1 M phosphate buffer (pH 7.2) at 4 °C for 24 h. Later, it was post-fixed in a 1% (v/v) osmium tetraoxide for 2 h. The mycelia were dehydrated through a graded series of ethanol (50%, 75%, 95% and 100%; 95% and 100% levels were applied twice for 15 and 30 min, respectively) and 100% acetone twice for 10 min. After dehydration, the mycelium was infiltrated in a Spurr's mix resin in a rotator overnight. Infiltration in a new change of the Spurr's mix resin was performed for an interval of 6 h for 3 days. After the third day, samples were embedded at 60 °C. Subsequently, an ultramicrotomy process was performed on the embedded blocks using a Sorvall Ultramicrotome MT 5000 before they are being observed under the EFTEM. The infrared (IR) spectra of P. sanguineus and S. commune after the bioreduction of silver were obtained using the Fourier Transform Infrared Spectrometer (Shimadzu, IRPrestige-21). Reflectance technique was used in identifying the IR spectra. Potassium bromide (KBr) discs were prepared by grinding mycelium of P. sanguineus and S. commune with KBr in a ratio of 1:100 and compressing the mixture into a transparent disc of 13 mm in diameter. The transparent discs were directly placed into the infrared spectrometer in the wave number range of 400–4000 cm−1.
2.4. Characterization of silver nanoparticles
2.5. Antimicrobial activity
The bioreduction of Ag+ ion in sample solutions CS, SN and MPS was monitored by absorbance measurement using a double beam UV–vis spectrophotometer (Shimadzu UV-2550, US). The spectra of the surface plasmon resonance of AgNPs in the samples were measured at a resolution of 1 nm between 200 and 800 nm wavelengths. Subsequently, the average sizes of AgNPs in the sample solutions were measured using dynamic light scattering (DLS) a non-invasive back scatter (NIBS®) technology (Zetasizer Nano ZS, Malvern Instruments, Southborough, UK). All sample solutions were ultrasonicated (Transsonic Digital T 490 DH, Elma, Singe, Germany) and filtered using a 0.2 μm PTFE membrane syringe filter before analyses. 0.001 M of AgNO3 solution was used as the blank for both UV–vis and nanosize analyses. AgNP concentrations were analyzed on a Shimadzu atomic absorption
Minimum inhibitory concentration (MIC) assay was carried out using the microdilution method as described by Qi et al. [25], with a slight modification. The Gram negative bacteria (Escherichia coli and Klebsiella pneumoniae) and Gram positive bacteria (Staphylococcus epidermidis and Staphylococcus aureus) were used as the tested microorganisms. 100 μL of AgNPs of known concentrations were transferred into 96 well microtiter plates containing 100 μL of Müeller Hinton broth. Dilutions were performed by the two-fold serial dilution method. Later, 100 μL of the tested bacteria was inoculated to all wells and the microtiter plates were incubated at 37 °C for 24 h. After the incubation period, the optical densities of cultures are measured at 595 nm using a microplate reader (Biorad Model 680). The minimum inhibitory concentration was determined as the lowest concentration of AgNPs that
2.3. Determination of protein concentration
284
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
inhibits the growth of microorganism which is represented by the absorbance.
a
3. Results and discussion 3.1. Biosynthesis and selection of AgNP producing fungus The bioreduction of silver occurred due to the presence of a reducing agent which reduced the silver ions in the AgNO3 solution into silver atoms of a nanosize dimension. It was reported that the enzyme reductase, nitrate reductase, hydrogenase or electron shuttle quinones were the potential enzymatic pathways for the bioreduction of silver [26,27]. Generally, the formation of silver can be primarily identified through visible observation in the change of color solution during the reaction from colorless to pale yellow or dark brown [28]. The change in color elucidates the presence of AgNPs in a solution due to the excitation of surface plasmon vibrations [29]. In this study, 5 species of the white-rot fungi namely P. sanguineus, S. commune, L. sajor caju, T. feei and T. pocas were screened for the synthesis of AgNPs. Results showed that sample solutions CS, SN and MPS of P. sanguineus and S. commune have shown a positive reaction for the biosynthesis of AgNPs through the increase in color intensity after 5 days of the incubation period. Fig. 1 showed a visual observation on the increase in color intensity of sample SN synthesized by the white rot fungus, S. commune after 5 days of incubation in shake flask cultures. The AgNP synthesis was further confirmed using the UV–vis spectral analysis. As reported by Sastry et al. [30], the UV–vis adsorption spectroscopy has proven to be one of the most useful and sensitive techniques for the analysis of nanoparticles. The electrochemical changes on the surface of nanosized metal particles are described as the optical properties of silver nanoparticles, and are being discussed within the Drude model [31]. In the Drude model, the ultraviolet region is the region of absorption for bulk plasmon frequency. Hence, the bioreduction of AgNPs can be identified indirectly using the UV– vis spectrum. In fact, the UV–vis spectroscopy method can also be used to identify the size evolution of AgNPs based on localized surface plasmon resonance band exhibiting at different wavelengths [32–34]. Fig. 2(a) and 2(b) showed the UV–vis spectra of the AgNPs synthesized by P. sanguineus extracellularly (SN) and through culture-free supernatant (CS), respectively. A strong, broad peak located between 430 nm and 450 nm was observed in the sample solution SN of P. sanguineus. However, a lower percentage of absorbance was observed in the solution CS compared to the SN of P. sanguineus. This could be due to a higher concentration of Ag synthesized extracellular than the culture-free supernatant synthesis of P. sanguineus. Similar UV-spectra were observed in the samples produced by S. commune but with a lower percentage of absorbance. According to the Beer–Lambert law, there is a correlation between the light transmission and absorption
D1
D2
D3
b
Fig. 2. UV–vis spectrum for AgNPs synthesized using P. sanguineus in samples (a) SN and (b) CS.
coefficient of the product. Hence, it explains the formation of absorption band by AgNPs at 420 nm. According to Sastry, [30,32], the presence of this peak indicated the sizes of metal nanoparticles which could be in the range of 2–100 nm. In addition, minor bands were also observed above 300 nm, which attributed to the electron excitation of protein residues [35]. Likewise, there is no clear change in the peak position of other tested species (L. sajor caju, T. feei, and T. pocas) as compared to P. sanguineus and S. commune. Although AgNPs are known to be effective broad spectrum biocides against a variety of drug-resistant bacteria, the mechanism of the inhibitory effects is still unclear. However, it was reported that the interaction of AgNPs with microorganisms results in inhibition
D4
D5
Fig. 1. Visual observation on increase in color intensity of sample SN synthesized by S. commune over 5 days of incubation.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
100 90 80 70
CS SN MPS
60 50 40 30 20 10 0 PS
SC
LSC
Fig. 3. Yield of AgNPs produced by P. sanguineus, S. commune and L. sajar caju after 5 days of incubation.
of growth, loss of infectivity to cell death and is much dependent on size of AgNPs synthesized, besides its shape and concentration [36–38]. Hence, identifying the nanosize of synthesized AgNPs is crucial in determining its applications. Fig. 3 summarized the selection of AgNP producing strain based on yield of AgNP production. The yield of AgNP production by P. sanguineus is shown to be the highest among the 3 modes of production tested compared to S. commune and L. sajar caju. In terms of particle size analysis, the average particle size of AgNPs produced by P. sanguineus and S. commune syntheses analyzed by dynamic light scattering are shown in Table 1. It was observed that the culture supernatant (CS) of the screened white-rot fungi has the ability to synthesize smaller diameter particle size in the range of 50 ± 10 nm to 65 ± 10 nm. In solution SN produced by P. sanguineus and S. commune showed that both white-rot fungi are able to produce a particle size of less than 80 nm. From definition, nanoparticles are nanosize particles ranging from 1 to 100 nm [39]. Hence showing that P. sanguineus gave the most promising AgNP production in terms of yield and particle size synthesized through extracellular (SN), culture-free supernatant (CS) and intracellular (MPS).
285
electron micrograph of AgNPs synthesized using mycelia pellet (MP) of S. commune and P. sanguineus after reacting with 0.001 M AgNO3. It was shown that the majority of the synthesized AgNPs present on the cell surface were spherical in shape. This could be due to the absorption of silver ions onto the surface of the mycelia pellet in the rotary shaker through an interaction with the mycelial surface functional groups such as carboxylate anion, carboxyl and peptide bond of proteins and hydroxyl of saccharides [40]. This finding is in agreement with the results of the Fourier transform infrared spectroscopy (FTIR) in this study which is shown in Fig. 5(a) and (b). The trough observed in regions 3410–3440 cm−1 results from the hydroxyl \OH. However, it was also reported that the signal of 3390 cm−1 may be due to the N\H asymmetric stretch mode of amines [41]. This indicated that the broad trough may be due to the presence of amino groups which are probably proteins. Similar observation was also reported by Mashitah et al., [42] for the binding mechanism of heavy metal biosorption by P. sanguineus where this broad trough is most likely due to the presence of protein. However, no carboxyl group was observed in the range of 1710–1740 cm−1. In the case of either biosorption or bioremediation, the functional groups of the fungal mycelial were responsible for binding the heavy metal [42]. However for the biosynthesis of AgNPs, an enzyme which was secreted by the fungi and acts as a bioreducing agent is needed. This showed that the functional groups of the tested fungi might be responsible for the bioreduction of silver, which are most likely to be the peptide bonds of proteins and hydroxyls of saccharides. Besides the functional groups it was also reported that when the mycelia matted together, they are more immobile and more capable of binding Ag+, hence in situ reduced to Ag0 [40,43]. Table 2 showed the IR spectra characteristics of P. sanguineus and S. commune mycelia after the
a
3.2. Structural and morphology characterizations of silver nanoparticles The results obtained by employing both analytical tools: UV–vis analysis and DLS analysis were useful as a prelude test to identify the presence of Ag during the biosynthesis process with fungal biomass or its culture supernatant. To corroborate the presence of AgNPs in fungal mycelia, SEM determination on the surface of treated fungal mycelia with AgNO3 were performed. Fig. 4(a) and (b) shows the scanning
b Table 1 Average particle size, polydispersity index of AgNPs, and protein concentrations produced by selected white-rot fungi. White-rot fungi Sample P. sanguineus
S. commune
L. sajor caju T. feei T. pocas
Ave. particle size ± SD, nm PDI
SN 70.2 ± 0.05 CS 63.8 ± 0.01 MPS 52.8 ± 0.06 SN 103.3 ± 0.06 CS 56.5 ± 0.02 MPS 53.9 ± 0.01 SN, CS, MPS ND SN, CS, MPS ND SN, CS, MPS ND
0.221 0.091 0.054 0.283 0.086 0.050 ND ND ND
[Protein] gL−1 20.41 27.37 30.51 15.26 28.12 29.85 ND ND ND
SN, extracellular synthesis; CS, culture-free supernatant synthesis; MPS, intracellular synthesis. ND, not determined due to negative or insignificant effect when treated with AgNO3. PDI, polydispersity index calculated based on ISO standard document 13321:1996E where, monodisperse b0.05, nearly monodisperse 0.05–0.08, mid range polydisperse 0.08–0.7, and very polydisperse >0.7.
Fig. 4. Scanning electron micrograph for the freeze-dried mycelium (a) S. commune (b) P. sanguineus.
286
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
Fig. 5. Fourier Transform spectroscopy spectra of mycelium (a) S. commune (b) P. sanguineus.
bioreduction of silver. It was observed that the bioreduction mechanism of both fungi is similar based on the presence of respective functional groups. The concentration of proteins produced by SN, CS and MPS of P. sanguineus, and S. commune was determined in Table 1. It showed that the higher the concentration of protein is secreted, the smaller the AgNPs produced. Although through SEM evaluation AgNPs were spherical in shape with smooth edges on the mycelia surface, Zaki et al. in 2010 stated that deposition of nanoparticles into the mycelia and mycelial membrane could be possible. Hence, a novel analysis by cutting mycelia fungi into a thin film (~10 nm) using an ultramicrotome was carried out and Table 2 Fourier transform infrared spectroscopy spectra characteristics and functional groups. Major peak
1 2 3 4 5
Wavelength (cm−1) P. sanguineus
S. commune
3434.08 2927.94 1621.07 1458.18 1072.42
3412.85 2924.52 1605.31 1428.08 1028.21
Assignment
Hydroxyl Aliphathic C\H group C_O stretching of COOH Symmetric bending of CH3 Carbohydrate group
observed under TEM. Fig. 6(a) and (b) shows the cross section of P. sanguineus and S. commune mycelia. In both cases, it was observed that majority of the AgNPs were located at the circumference of the fungal membrane. This showed that the enzyme that was secreted on the mycelial membrane served as the reducing agent for the biosynthesis of the silver nanoparticles [44]. However, from the micrographs, the AgNPs formed by the mycelial fungi tend to agglomerate. This phenomenon is possibly because of the unsuccessful binding of Ag+ to the surface of AgNPs and its being trapped in the fungi mycelia. To gain a further insight into the feature of the extracellular formation of AgNPs, an analysis of samples SN containing AgNPs were dropped onto carbon coated copper grid prior their observation under TEM. Fig. 6(c) and (d) showed that the AgNPs were freely dispersed in a silver nitrate solution. The AgNPs from the SN of P. sanguineus were observed to exhibit a higher degree of agglomeration compared to AgNPs from the SN of S. commune that are nearly spherical or ellipsoidal in shape for both cases. Polydispersity of AgNPs produced was further determined from polydispersity index calculated by Zetasizer Nano ZS as shown in Table 1. It showed that AgNPs produced by SN and CS were at the mid range of polydispersity while AgNPs by MPS were nearly monodispersed for both cases. Formation of freely dispersed AgNPs probably occurred during the initial incubation in the rotary shaker.
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
a
b
c
d
287
Fig. 6. Transmission electron micrographs of a thin film of mycelium (a) S. commune (b) P. sanguineus coated on copper grid and AgNPs produced in sample SN by (c) S. commune and (d) P. sanguineus.
Generally, filamentous fungi which exhibited a higher surface area were more capable of binding Ag+. However, the tendency of surface proteins or chemical functional groups released into the solution during rotation is also higher in this study. The released reducing agents hence reduced Ag+ in the AgNO3 solution into Ag0 nanoparticles. It was observed in both UV–vis spectral analysis and SEM/TEM analysis that formations of extracellular AgNPs (SN) were more promising. This offers enormous advantages over intracellular synthesis of AgNPs in various applications.
3.3. Energy dispersive X-ray spectroscopy (EDS) analysis Table 3 illustrates the EDS patterns of the synthesized AgNPs by P. sanguineus and S. commune. Results showed that the EDS patterns consisted of Ag (66.05 wt.%), C (18.22 wt.%), Na (7.29 wt.%) and Cl (8.44 wt.%) which were present at the spotted area of the analysis. The signals of C, O, Na and Cl obtained were similar to the research by Zaki et al. that the signals are most probably due to the X-ray emission from the fungi mycelia and the remaining media [45]. In this Table 3 EDS analysis of AgNPs produced by mycelia P. sanguineus and S. commune after reaction with AgNO3. Fungus
Element C
P. sanguineus S. commune
study, P. sanguineus has shown to have the highest ability to synthesize AgNPs (28.21 Ar%) as compared to S. commune (22.81 Ar%). 3.4. Antimicrobial studies Silver has been well known for its antimicrobial activity since the last decades. Although it was reported that the antimicrobial effects could be due to inhibiting the enzymatic respiratory system of microbes and altering DNA synthesis [46,47], the mechanisms are still unclear. It is believed that the antimicrobial effects depend strongly on the superficial contact between silver and microorganisms. Due to that, a high surface area to volume ratio of the AgNPs could serve as a powerful antimicrobial agent. The antimicrobial effects of AgNPs produced by P. sanguineus and S. commune in this study were tested with Gram positive bacteria, S. aureus, S. epidermidis and the Gram negative bacteria, E. coli and K. pneumoniae. The MIC values for AgNPs synthesized by both P. sanguineus and S. commune were shown in Table 4. It was observed that the antimicrobial activity of AgNPs produced in the SN was more effective Table 4 MICs (μg/mL ± SD) of AgNPs. Fungi
PS Na
Cl
Ag
at.%
wt.%
at.%
wt.%
at.%
wt.%
at.%
wt.%
61.3 56.5
21.05 18.22
9.05 11.81
4.82 7.29
1.44 8.87
3.67 8.44
28.21 22.81
70.46 66.05
at.% and wt.% refer to atomic percentage and weight percentage of particular element.
SC
Sample
SN MPS CS SN MPS CS
Gram positive bacteria
Gram negative bacteria
S. epidermidis
S. aureus
E. coli
K. pneumoniae
0.03 ± 0.0 1.42 ± 0.2 1.43 ± 0.0 1.17 ± 0.1 7.03 ± 1.2 0.25 ± 0.0
4.27 ± 0.2 1.47 ± 0.2 0.37 ± 0.1 1.6 ± 0.2 9.5 ± 0.5 47 ± 2.1
0.09 ± 0.2 1.41 ± 0.3 0.71 ± 0.0 2.6 ± 0.1 30 ± 1.2 0.4 ± 0.0
1.02 ± 0.2 1.38 ± 0.3 0.41 ± 0.1 1.91 ± 0.1 12.8 ± 0.9 2.8 ± 0.6
PS refer to fungus P. sanguineus while SC refers to fungus S. commune.
288
Y.S. Chan, M. Mat Don / Materials Science and Engineering C 33 (2013) 282–288
than those of CS and MPS. The results also showed that AgNPs synthesized by both fungi were more effective antimicrobial agents as the MIC values lie between 0.03 and 47 μg/mL. This is in agreement with the results obtained by Kim [48] who indicated that the Gram positive bacteria, S. aureus was more resistant to AgNPs as compared to the Gram negative E. coli. Thus, it indicated that the tested white rot fungi of the present study produced AgNPs that have great bactericidal activities. In view of cytotoxicity, it was reported that AgNPs of 20 nm were more cytotoxic than silver ions [49], also smallest particle sizes of AgNPs were reported to have a greater ability to induce apoptosis in the Mc3T3-E1 cell line than larger AgNPs [50]. Hence, the particle size needs to be designed carefully for biomedical and biopharmaceutical uses. 4. Conclusions Among the five Malaysian white rot fungi tested, P. sanguineus and S. commune were capable in synthesizing AgNPs with an average particle size of range from 52.8 to 103.3 nm. Results showed that P. sanguineus is more favorable compared to S. commune as it has a higher atomic percentage of silver at 28.21% and a higher yield of AgNPs at 98.9%. The IR spectra of FTIR showed that the bioreduction of silver occurred when the silver ions interacted with the functional groups of the mycelial surface of the tested fungus. Thus, it indicated that the white rot fungi can be utilized for the production of AgNPs which have wider applications in pharmaceutical and medical fields. Acknowledgment This work is supported and funded by the Ministry of Higher Education Malaysia through Fundamental Research Grant Scheme (FRGS/1/10/ TK/USM/02/5) and by the USM fellowship which we gratefully acknowledge. References [1] M. Cameron, S. Timofeevski, S. Aust, Appl. Microbiol. Biotechnol. 54 (2000) 751–788. [2] A. Paszczynski, R. Crawford, Biotechnol. Prog. 11 (1995) 368–379. [3] J. Glenn, M. Morgan, M. Mayfield, M. Kuwahara, M. Gold, Biochem. Biophys. Res. Commun. 114 (1983) 1077–1083. [4] M. Kuwahara, J. Glenn, M. Morgan, M. Gold, FEBS Lett. 169 (1984) 247–250. [5] D. Wesenberg, I. Kyriakides, S. Agathos, Biotechnol. Adv. 22 (2003) 161–187. [6] A. Unyayar, M. Mazmanci, E. Erkurt, H. Atacag, A. Gizir, React. Kinet. Catal. Lett. 86 (2005) 99–107. [7] P. Kertsen, B. Kalyanaraman, K. Hamel, B. Reinhammar, T. Kirk, Biochem. J. 268 (1990) 475–480. [8] P. David, D. Steven, Environ. Sci. Technol. 28 (1994) 79A–87A. [9] T. Eggen, A. Majcherczyk, Int. Biodeterior. Biodegrad. 41 (1998) 111–117. [10] C. Wang, J. Xi, H. Hu, X. Wen, Biomed. Environ. Sci. 21 (2008) 474–478.
[11] P. Siripong, B. Oraphin, T. Sanro, P. Duanporn, Amer.–Eurasian J. Agric. & Environ. Sci. 5 (2009) 466–472. [12] W.M. Abd El-Rahim, O.A.M. El-Ardy, F.H.A. Mohammad, Desalination 249 (2009) 1206–1211. [13] E. Esposito, L.H. Innocentini-Mei, A. Ferraz, V.P. Canhos, N. Durán, J. Biotechnol. 29 (1993) 219–228. [14] M. Trovaslet, E. Enaud, Enzym. Microb. Technol. 41 (2007) 368–376. [15] N. Vigneshwaran, A. Kathe, P. Varadarajan, R. Nachane, R. Balasubramanya, Colloids Surf. B: Biointerfaces 53 (2006) 55–59. [16] Z. Lin, J. Wu, R. Xue, Y. Yang, Spectrochim. Acta A Mol. Biomol. Spectrosc. 61 (2005) 761–765. [17] N. Ahalya, T.V. Ramachandra, R.D. Kanamadi, Res. J. Chem. Environ. 7 (2003) 71–79. [18] C.C.V. Cruz, A.C.A. Da Costa, C.A. Henriques, A.S. Luna, Bioresour. Technol. 91 (2004) 249–257. [19] Y. Sun, Y. Xia, Science 298 (2002) 2176–2179. [20] S. Chulovskaya, V. Parfenyuk, Prot. Met. Phys. Chem. Surf. 45 (2009) 300–304. [21] V. Illic, Z. Saponjic, V. Vodnik, B. Pofkonjak, P. Jovancic, J. Nedeljkovic, M. Radetic, Carbohydr. Polym. 78 (2009) 564–569. [22] S. Kaviya, J. Santhananlakshmi, B. Viswanathan, J. Muthumary, K. Srinivasan, Spectrochim. Acta A Mol. Biomol. Spectrosc. 79 (2010) 594–598. [23] R. Nithya, R. Ragunathan, Dig. J. Nanomater. Biostruct. 4 (2009) 623–629. [24] R. Sanghi, P. Verma, Bioresour. Technol. 100 (2009) 501–504. [25] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Carbohydr. Res. 339 (2004) 2693–2700. [26] N. Duran, P.D. Marcato, O.L. Alves, G.I. Souza, E. Esposito, J. Nanobiotechnol. 3 (2005) 8. [27] J.C.G. Ottow, A. Von Klopotek, Appl. Microbiol. 18 (1969) 41–43. [28] M. Sathishkumar, K. Sneha, S.W. Won, C.W. Cho, S. Kim, Y.S. Yun, Colloids Surf., B 73 (2009) 332–338. [29] A. Rai, A.K. Singh, A. Ahmad, M. Sastry, Langmuir 22 (2006) 736–741. [30] M. Sastry, K.S. Mayyaa, B. K., Colloids Surf., A 127 (1997) 221–228. [31] M. Sastry, V. Patil, S.R. Sainkar, J. Phys. Chem. B 102 (1998) 1404–1410. [32] A. Henglein, J. Phys. Chem. B 7 (1993) 5457–5471. [33] U. Kreibig, J. Phys. Chem. B 2000 (104) (1978) 9683–9688. [34] U. Kreibig, L. Grenzel, Surf. Sci. 156 (1985) 678–700. [35] A.M. Fayz, K. Balaji, P.T. Kalaichelvan, R. Venkatesan, Colloids Surf., B 74 (2009) 123–126. [36] S. Pal, Y. Tak, J. Song, Appl. Environ. Microbiol. 73 (2007) 1712–1720. [37] P. Asharani, M. Hanede, S. Valiyaveettil, BMC Cell Biol. 10 (2009) 65. [38] H. Yen, S. Hsu, C. Tsai, Small 5 (2009) 1553–1561. [39] S.E. McNeil, J. Leukoc. Biol. 78 (2005) 585–594. [40] Z. Lin, J. Wu, R. Xue, Y. Yong, Spectrochim. Acta A Mol. Biomol. Spectrosc. 61 (2005) 761–765. [41] T.R. Muraleedharan, I. Leela, C. Venkobachar, Environ. Technol. 15 (1994) 1015–1027. [42] M.D. Mashitah, Z. Zulfadhly, S. Bhatia, Artif. Cells Blood Substit. Immobil. Biotechnol. 27 (1999) 441–445. [43] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V. Varadarajan, Carbohydr. Res. 341 (2006) 2012–2018. [44] S.K. Das, E. Marsili, Rev. Environ. Sci. Biotechnol. 9 (2010) 199–204. [45] S. Zaki, M. El Kady, D. Abd-El-Haleem, Mater. Res. Bull. 46 (2010) 1571–1576. [46] D.W. Brett, Ostomy Wound Manage. 52 (2006) 34–41. [47] E. Hidalgo, C. Dominguez, Toxicol. Lett. 15 (1998) 169–179. [48] J.S. Kim, E. Kuk, K.N. Yu, J.H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.Y. Hwang, Y.K. Kim, Y.S. Lee, D.H. Jeong, M.H. Cho, Nanomedicine 3 (2007) 95–101. [49] M.V.D.Z. Park, A.M. Neigh, J.P. Vermeulen, L.J.J. de la Fonteyne, H.W. Verharen, J.J. Briedé, H. van Loveren, W.H. de Jong, Biomaterials 32 (2011) 9810–9817. [50] T.-H. Kim, M. Kim, H.-S. Park, U.S. Shin, M.-S. Gong, H.-W. Kim, J. Biomed. Mater. Res. Part A 100A (2012) 1033–1043.