World J Microbiol Biotechnol DOI 10.1007/s11274-014-1634-z
ORIGINAL PAPER
Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria Tawfik M. Muhsin • Ahmad K. Hachim
Received: 28 November 2013 / Accepted: 7 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of this study was to biosynthesis silver nanoparticles from the fungus Nigrospora sphaerica isolated from soil samples and to examine their activity against five human pathogenic strains of bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus using disc diffusion method. The synergistic effect of silver nanoparticles in combination with commonly used antibiotic Gentamycin against the selected bacteria was also examined. The synthesized silver nanoparticles from freecell filtrate were characterized by using UV–Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). UV–Vis spectrophotometer analysis showed a peak at 420 nm indicating the synthesis of silver nanoparticles, FTIR analysis verified the detection of protein capping of silver nanoparticles while SEM micrographs revealed that the silver nanoparticles are dispersed and aggregated and mostly having spherical shape within the size range between 20 and 70 nm. The synthesized silver nanoparticles exhibited a varied growth inhibition activity (15–26 mm diam inhibition zones) against the tested pathogenic bacteria. A remarkable increase of bacterial growth inhibition (26–34 mm diam) was detected when a combination of silver nanoparticles and Gentamycin was used. A significant increase in fold area of antibacterial activity was observed when AgNPs in combination with Gentamycin was applied. The synthesized silver nanoparticles produced by the fungus N. sphaerica is a promising
T. M. Muhsin (&) A. K. Hachim Department of Biology, College of Education for Pure Sciences, University of Basra, Basra, Iraq e-mail: [email protected]
to be used as safe drug in medical therapy due to their broad spectrum against pathogenic bacteria. Keywords Antibacterial agents Fungal free-cell filtrate Mycosynthesis Pathogenic bacteria Silver nanoparticles Synergistic effect
Introduction Biosynthesis of metal nanoparticles is a recent approach has been emerged from nanobiotechnology and becoming widely applicable in different fields. At present there is an increase research interests to explore natural sources for biosynthesis of metal nanoparticles that can be used in industrial, agricultural, environmental and medical applications (Kearns et al. 2006; Thomas et al. 2007; Sharma et al. 2009; Kashyap et al. 2013). Among the metals, silver nanoparticles has been synthesized by various physical and chemical methods and their biosynthesis method has been lately developed using natural sources mainly microorganisms (Maliszewska et al. 2009; Sadhasivum et al. 2010). Nevertheless, fungi are good natural source and considered as bionanofactories for synthesis of silver nanoparticles and other metal nanoparticles (Sastry et al. 2003; Rai et al. 2009). However, biosynthesis of silver nanoparticles by fungi is of great interest due to the fungal properties such as they are easy to culture with large biomass productivity, secretion of extracellular enzymes, high amount of protein production and consequently a higher production of nanoparticles (Sastry et al. 2003; Rai et al. 2009; Kashyap et al. 2013). Moreover, silver nanoparticles (AgNPs) are one of the most important metals that have been used as antimicrobial agent and so far many fungal species have been examined for their capability to
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synthesize silver nanoparticles and their antimicrobial activities (Feng et al. 2000; Aymonier et al. 2002; Morones et al. 2005; Lee et al. 2003; Riddin et al. 2006; Kim et al.2007; Yokoyama and Welchons 2007; Ingle et al. 2008; Fayaz et al. 2010; Gade et al. 2010). The present work aimed to synthesize and characterize silver nanoparticles from the soil fungus Nigrospora sphaerica and examine their activity against five strains of human pathogenic bacteria and the synergistic activity combined with the commercial antibiotic Gentamycin.
agitated in water bath (120 rpm) for 72 h at 25 °C. After incubation the fungal cell filtrate was filtered by Whatman filter paper No. 1. The fungal free cell filtrate was treated with 1 mM AgNO3 solution and incubated at room temperature at dark condition. Flasks containing free cell filtrate without silver nitrate solution were made as control. Triplicate flasks for treated and untreated fungal filtrate were made. Detection and characterization of silver nanoparticles UV–visible spectrophotometric assay
Materials and methods Fungal isolation Soil samples were collected from subsurface of cultivated localities in Basra (southern Iraq) during the year 2013 using a sterile spatula then placed in sterile plastic bags and brought to the Laboratory. Fungal isolation was performed by serial dilution method (Warcup 1950). 1 mL of each dilution was transferred aseptically into Petri dish containing Potato Dextrose Agar (PDA), the plates were incubated at 25 °C for 7 da and axenic fungal cultures of the growing colonies were made for each fungal isolate. Identification of the recovered fungi was confirmed according to the available taxonomic literature (Ellis 1971, 1976; Domsch et al. 1980; Watanabe 2002). Fungal culture Among the isolated fungi the species Nigrospora sphaerica (Sacc.) Mason was selected in this study for synthesis of silver nanoparticles. The fungal culture was prepared according to Ahmad et al. (2003) as following; one disc of 8 mm diam was cut from five days old culture colony by a sterilized Cork borer and inoculated into 250 mL Erlenmeyer flasks containing 100 mL a liquid medium of MGYP (Malt extract 3 g, Glucose 10 g, Yeast 3 g and Peptone 5 g per 1 L of distilled water) after being autoclaved at 121 °C for 15 min. The culture flasks were incubated at 25 ± 1 °C for 10 day then after incubation the mycelia were separated from the culture broth by centrifugation (600 rpm) at 10 °C for 10 min and the harvested mycelia was washed thrice with sterile distilled water to remove any medium component from the biomass. Biosynthesis of silver nanoparticles For extracellular production of silver nanoparticles typically 10 g of fungal mycelia biomass (wet weight) under a sterilized condition was brought into contact with 100 mL sterile double distilled water in an Erlenmeyer flask and
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After 24 h of incubation of the fungal free cell filtrate treated with AgNO3 solution, the bioreduction of Ag? in aqueous solution was monitored using UV–Vis spectrophotometer (APEL PD-303) at regular intervals. During the reduction process, 0.1 mL of filtrate was taken and diluted three times with deionized water. After dilution, it was centrifuged at 800 rpm for 5 min and the supernatant was scanned using UV–Vis spectrophotometer at the wave length of 300–900 nm. UV–Vis spectra were recorded at 24, 48, and 72 h at a resolution of 1 nm. Untreated free cell filtrate was used as a control. Fourier transform infrared (FTIR) After 72 h of incubation, the free cell filtrate was subjected to Fourier Transform Infrared (FTIR) (Shimadzu UV-1700, Japan) analysis. After a complete reduction of aqueous silver ions within the fungal filtrate, the filtrate was mixed with acetone (1:5 vol/vol) with a continuous shaking then centrifuged at 4,000 rpm for 15 min forming a pellet according to the described method (Raheman et al. 2011). The supernatant was discarded and 2 mL of acetone was added into the pellet and shaken thoroughly then poured into a Petri plate. Acetone was evaporated in order to obtain the powder of silver nanoparticles. Characterization of AgNPs was carried out by using FTIR at the range of 400–4,000 cm-1at a resolution of 4 cm-1. SEM analysis Scanning electron microscopic (SEM) (Netherland INSPECT S50) analysis of the fungal cell filtrate treated with AgNO3 was performed. Thin films of the filtrate samples were prepared on a carbon coated copper grid by just dropping a very little amount of the filtrate on the grid and the extra solution was removed by a blotting paper then the films on the grids were allowed to dry overnight at room temperature under a sterilized condition. SEM micrographs of the silver nanoparticles were exposed at different magnifications.
World J Microbiol Biotechnol Fig. 1 Nigrospora sphaerica culture grown on PDA medium (a) and microscopic structures (b) showing fungal hyphae and conidia (940 magnification)
Fig. 2 Untreated fungal free cell filtrate (a) and treated with 1 mM silver nitrate solution (b) 1.4
Fig. 4 SEM micrographs showing the silver nanoparticles in fungal free-cell filtrate appeared as spherical shape (arrows) at magnification (913,000) with size range between 20 and 70 nm
24 hr 1.2
48 hr 72 hr
Absorbance
1
F.C.F 0.8 0.6 0.4 0.2 0 300
400
500
600
700
800
900
Wavelength in nm
Fig. 3 UV-Vis spectrum of fungal free cell filtrate (fcf) containing silver nanoparticles recorded at different exposure times
Antibacterial activity of silver nanoparticles Agar well diffusion assay The potentiality of silver nanoparticles was examined for their antibacterial
efficiency using agar well diffusion assay method (Perez et al. 1990). Five strains of pathogenic bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus were tested. Swabs from each bacterial culture grown overnight were streaked on sterilized Mueller–Hinton agar (MHA) plates. Wells (5 mm diam) were made in agar plates using sterilized stainless steel Cork borer. The wells were loaded with two concentrations (50 and 100 lL) of silver nanoparticles solutions. The plates were incubated at 37 °C for 24 h and examined for the appearance of inhibition zones around the wells and the diameters of inhibition zones were measured. Minimum inhibitory concentration (MIC) and MBC assay Minimum inhibitory concentration (MIC) assay was carried out using the micro dilution method according to Qi et al. (2004). 100 lL of AgNPs was transferred into 96-well microtitre plates containing 100 lL of Mueller–
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World J Microbiol Biotechnol Fig. 5 FTIR analysis of silver nanoparticles synthesis in fungal free cell filtrates. Before (a) and after (b) silver bioreduction
Hinton broth. 100 lL of the tested bacteria E. coli (ATCC 25922) and S. aureus (NCTC 6571) was inoculated into each well and incubated at 37 °C for 24 h. After the incubation period a small amount of bacterial suspension was streaked on MHA plates and incubated at the same condition. The minimum inhibitory concentration was determined as the lowest concentration of AgNPs that inhibits the growth of bacteria. While the minimum bactericidal concentration (MBC) was determined as the lowest concentration of AgNPs that kills the bacteria and no growth was observed on the agar medium. Assay of antibacterial activity of AgNPs in combination with Gentamycin Disc diffusion method was used to assay the antibacterial activity of synthesized AgNPs combined with commonly used antibiotic Gentamycin as described by Birla et al. (2009). A standard antibiotic disc of Gentamycin was impregnated with 20 lL of freshly prepared AgNPs and placed onto the MHA medium
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inoculated with each tested bacteria. Similarly, the antibacterial activity test for Gentamycin and AgNPs was carried out as control. Discs of Gentamycin and of AgNPs were separately placed on the MHA medium inoculated with each tested bacteria. The plates were incubated at 37 °C for 24–48 h. After incubation, the zones of bacterial growth inhibition for the combination of AgNPs with Gentamycin treatment and for control plates were measured. Four replicates for each treatment were made. Assessment of increase in fold area The increase in fold area was assessed by calculating the mean surface area of the inhibition zone exhibited by the antibiotic alone and in a combination with AgNPs. The fold increase area was calculated by the equation; (B2 - A2)/A2, where A refers to the inhibition zone diam exhibited by the antibiotic activity alone and B refers to the inhibition zone diam exhibited by activity of a combination of antibiotic and AgNPs (Birla et al. 2009).
World J Microbiol Biotechnol Fig. 6 Appearance of inhibition zones on agar plates using of silver nanoparticles synthesized by the fungus N. sphaerica against E. coli (a), S. aureus (b), P. aeruginosa (c), S. Typhi (d) and P. mirabilis (e) at concentration (1) control, (2) 50 lL and (3) 100 lL of silver nanoparticles
Results
pale yellow into dark brown color which indicates the synthesis of silver nanoparticles (Fig. 2).
Fungal culture and silver nanoparticles synthesis UV–Vis spectra of AgNPs The isolated fungus N. sphaecia grows rapidly on PDA medium and covered the Petri plate within three days (Fig. 1). The fungal free cell filtrate (fcf) obtained from the fungal cultures grown in MGYP liquid medium over ten days after being treated with AgNO3 solution turned from
The UV–Vis spectra recorded from the fungal free cell filtrates amended with 1 mM AgNO3 solution revealed significant variations in spectra of silver nanoparticles synthesis at different intervals of reaction (Fig. 3). The
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Inhbition zones (mm diam)
30 25
E. coli
P .aeruginosa
S . aureus
S. typhi
inhibition zones dim) than at 100 lL concentration (15–26 mm inhibition zones diam) (Fig. 7). The highest growth inhibitory activity of AgNPs was against P. aeruginosa and lowest against P. mirabilis. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were very low against the two strains of bacteria E. coli and S. aureus (Table 1).
P. mirabilis
20 15 10 5
Antibacterial activity of AgNPs combined with gentamycin
0
AgNPs (50 ul)
AgNPs (100 ul)
Fig. 7 Inhibition zones exhibited by two concentrations of silver nanoparticles from the fungus N. sphaerica against five strains of pathogenic bacteria
Table 1 The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of AgNPs against two strains of bacteria
Bacteria
MIC (lg/L)
MBC (lg/L)
E. coli
0.156
0.312
S. aureus
0.0024
0.0048
absorbance pattern of the Ag–fcf monitored at the range of 300–900 nm revealed an increase of absorbance with increasing time of incubation at 430 nm. Highest spectrum of AgNPs synthesis after 72 h of incubation was detected. SEM analysis SEM images with different magnifications showed that the silver nanoparticles are dispersed or aggregated and mostly showed spherical shape and their size ranging between 20 and 70 nm (Fig. 4). FTIR spectroscopy FTIR analysis of silver nanoparticles synthesized from the fungus N. sphaerica showed the presence of peak at 1,660.6, 1,384.79, 1,112.8 and 1,014.4 cm-1 (Fig. 5). The bands at 1,660.6 refer to the bonding vibrations of the amide I and II of proteins while the bands at 1,384.79 cm-1 and 1,112.8 cm-1 indicate the presence of C–N stretching vibrations of aromatic and aliphatic amines. Antibacterial activity of silver nanoparticles The mycosynthesized silver nanoparticles exhibited an antibacterial activity against the tested Gram positive and Gram negative bacteria (Fig. 6). The present results showed that the bacterial growth inhibition at 50 lL/mL concentration of AgNPs was slightly lower (13–24 mm
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The antibacterial activity of AgNPs combined with commercial antibiotic Gentamycin was significantly increased against the growth of the tested strains of bacteria compared with activity of either of them alone (Fig. 8). Over all the tested bacteria, the growth inhibition zones ranged between 26 and 34 mm diam (Table 2). Among the examined bacteria, the growth of S. aureus and P. mirabilis was remarkably inhibited by silver nanoparticles combined with gentamycin as indicated by the increase in fold area values (0.133 and 0.28, respectively) (Table 2).
Discussion The last decade witnessed an increased research interests focusing on the biosynthesis of silver nanoparticles as an agent widely used in biomedical applications (Sharma et al. 2009; Kearns et al. 2006). However, silver nanoparticles is of a particular interest as antimicrobial agent (Yokoyama and Welchons 2007; Fayaz et al. 2010). The present study showed that the soil fungus N. sphaerica exhibited high potentiality for synthesis of silver nanoparticles in cultures as indicated by the color change from yellow into dark brown after 72 h of incubation after being treated with 1 mM AgNO3 solution. These findings are in similarity with other previous studies using different fungal species such as Fusarium oxysporum, F. acuminatum and Phoma glomerata for the synthesis of silver nanoparticles (Riddin et al. 2006; Ingle et al. 2008; Birla et al. 2009). It has been reported that the color change after addition of AgNO3 into the fungal free-cell filtrate is due to the excitation of surface plasmon resonance vibration of silver that confirmed the reduction of silver ions (Chitra and Annadurai 2013). UV–Vis spectrophotometry is commonly used technique for analyzing AgNPs synthesis (Henglein 1993). The present data revealed that UV–Vis spectrophotometry analysis showed a sharp peak with high absorbance at 420 nm which confirmed the AgNPs synthesis by the examined fungus. The absorption peak in UV spectrum is equivalent to the surface plasmon resonance and the maximum biosynthesis of AgNPs was at 72 h of
World J Microbiol Biotechnol Fig. 8 Antibacterial activity indicated by the inhibition zones (mm diam) exhibited by biosynthesized silver nanoparticles against E. coli (a), S. aureus (b), P. aeruginosa (c), S. typhi (d) and P. mirabilis (e) using AgNPs alone (1), commercial antibiotic gentamycin (2) and a combination of AgNPs with gentamycin (3)
incubation. This is in conformity with some other studies (Ingle et al. 2008; Chitra and Annadurai 2013). Based on literatures, apparently, there are some variations among the fungal free cell filtrate incubation periods and their UV spectra recorded for the biosynthesis of silver nanoparticles by using different sources of fungi. For example, Chitra and Annadurai (2013) reported that the synthesis of silver nanoparticles by the fungus Trichoderma viride occurred at
incubation time ranged between 30 min and 96 h and a sharp spectra peak appeared at 400 nm. On the other hand, the size and shape of silver nanoparticles synthesized by fungi are also varied. Such variations in characterization of silver nanoparticles biosynthesis might be related to a selected fungal species and the culture conditions applied. The present study revealed that the size of the detected AgNPs range between 20 and 70 nm with spherical shape
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World J Microbiol Biotechnol Table 2 Inhibition zones exhibited by the silver nanoparticles combined with commercial antibiotic gentamycin against five bacteria strains and the calculated increase in fold area Bacteria
Inhibition zone (mm diam) AgNPs
Gentamycin
AgNPs ? gentamycin
Increase in fold area
E. coli
18*
25
26
0.08
S. aureus
17
31
33
0.133**
P. aeruginosa S. typhi
20 15
26 25
28 26
0.16** 0.08
P. mirabilis
20
30
34
0.28**
* Values represent means of four replicates ** Significant difference at P \ 0.001
and are dispersed or aggregated as confirmed by SEM images. Another study (Martinez-Castanon et al. 2008) has reported that the size of silver particles range between 7 and 89 nm. A study of Saha et al. (2011) reported that the size of silver nanoparticles synthesized from the fungus N. oryzae ranged between 30 and 90 nm. It has been stated that the absorption spectrum of spherical shape of silver nanoparticles present a maximum between 420 and 450 nm (Pal et al. 2007). Nevertheless, the size and shape of silver nanoparticles might be affected by the sample preparation method for SEM analysis such as drying process (Sadawiski et al. 2008) as we applied in the present study. Analysis of FTIR indicated the release of proteins into fungal filtrate which leads to a reduction of silver ions present in the free cell filtrate. The reduction of the Ag? ions is might be due to the enzyme reductase that released by the fungus as reported by Gole et al. (2001). It has been shown that FTIR analysis has confirmed that amino acid residues and peptides of proteins are binding with the silver nanoparticles forming a capping agent of nanoparticles and stabilizing them in the culture medium (Vigneshwaran et al. 2007). In this study two concentrations 50 lL/mL and 100 lL/mL of silver nanoparticles synthesized by the fungus N. sphaerica were tested against the selected human pathogenic bacterial strains and rendered a significant bacterial growth inhibition as indicated by the appearance of clear zones. However, variations among the antibacterial activity exhibited by the silver nanoparticles were observed. Higher inhibition zones diameters was recorded for P. mirabilis and P. aeruginosa than other tested bacteria. The present study also revealed that silver nanoparticles from N. sphaerica exhibited higher inhibition activity against the pathogenic bacteria S. aureus and S. typhi than that reported by Raheman et al. (2011) who examined the endophytic fungus Pestalotia sp. Although the mechanism of AgNPs effects against the bacteria is not yet well understood, however, it can be attributed to the action of Ag on
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bacterial cell causing cell membrane damage, DNA denaturation, enzymes retardation or due to some other effects. The minimum inhibitory concentration (MIC) of the silver nanoparticles was determined and revealed that the MIC values were 0.156 and 0.0024 lg/L mL against E. coli and S. aureus, respectively. The MIC values were very low demonstrating the high potential activity of biosynthesized silver nanoparticles against Gram positive and Gram negative bacteria. Similar observations were found by other workers using AgNPs synthesized from other fungi against Gram positive and Gram negative bacteria (Martinez-Castanon et al. 2008; Birla et al. 2009). The present study has also demonstrated that the antibacterial activity of the synthesized silver nanoparticles combined with the commercial antibiotic Gentamycin. The efficiency of silver nanoparticles with Gentamycin was found to be increased significantly reaching 33 and 34 mm diam inhibition zone against S. aureus and Proteus mirabilis, respectively. The antibacterial activity of a combination of AgNPs ? Gentamycin was expressed as increase in fold area according to Birla et al. (2009).The increase in fold area was 0.133 and 0.28 for S. aureus and Proteus mirabilis, respectively. Our results support some other studies examined the synergistic effects of AgNPs, synthesized from various fungal species, in a combination with different commercial antibiotics tested against Gram positive and Gram negative bacteria (Shahverdi et al. 2007; Fayaz et al. 2010; Gajbhiye et al. 2009). A conclusion can be derived from the present study that the soil fungus N. sphaerica has a potentiality for silver nanoparticles synthesis which exhibiting a high growth inhibition activity against Gram positive and negative pathogenic bacteria. Generally, fungi represent a natural resources and ecofriendly and can be implemented in medical therapy. Thus, further researches to explore more potent fungi for silver nanoparticles synthesis are needed. Also an optimization of fungal growth conditions used for biosynthesis of silver nanoparticles and their antimicrobial activities are
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recommended. Further mechanistic studies to understand the synthesis of silver nanoparticles from fungi are important. Acknowledgments The authors would like to thank the authorities of Biology Department, College of Education for Pure Sciences, Basra University (Iraq) for supporting this research work as a part of M Sc. research program scholarship awarded to the second author.
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ORIGINAL PAPER
Mycosynthesis and characterization of silver nanoparticles and their activity against some human pathogenic bacteria Tawfik M. Muhsin • Ahmad K. Hachim
Received: 28 November 2013 / Accepted: 7 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of this study was to biosynthesis silver nanoparticles from the fungus Nigrospora sphaerica isolated from soil samples and to examine their activity against five human pathogenic strains of bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus using disc diffusion method. The synergistic effect of silver nanoparticles in combination with commonly used antibiotic Gentamycin against the selected bacteria was also examined. The synthesized silver nanoparticles from freecell filtrate were characterized by using UV–Vis spectrophotometer analysis, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). UV–Vis spectrophotometer analysis showed a peak at 420 nm indicating the synthesis of silver nanoparticles, FTIR analysis verified the detection of protein capping of silver nanoparticles while SEM micrographs revealed that the silver nanoparticles are dispersed and aggregated and mostly having spherical shape within the size range between 20 and 70 nm. The synthesized silver nanoparticles exhibited a varied growth inhibition activity (15–26 mm diam inhibition zones) against the tested pathogenic bacteria. A remarkable increase of bacterial growth inhibition (26–34 mm diam) was detected when a combination of silver nanoparticles and Gentamycin was used. A significant increase in fold area of antibacterial activity was observed when AgNPs in combination with Gentamycin was applied. The synthesized silver nanoparticles produced by the fungus N. sphaerica is a promising
T. M. Muhsin (&) A. K. Hachim Department of Biology, College of Education for Pure Sciences, University of Basra, Basra, Iraq e-mail: [email protected]
to be used as safe drug in medical therapy due to their broad spectrum against pathogenic bacteria. Keywords Antibacterial agents Fungal free-cell filtrate Mycosynthesis Pathogenic bacteria Silver nanoparticles Synergistic effect
Introduction Biosynthesis of metal nanoparticles is a recent approach has been emerged from nanobiotechnology and becoming widely applicable in different fields. At present there is an increase research interests to explore natural sources for biosynthesis of metal nanoparticles that can be used in industrial, agricultural, environmental and medical applications (Kearns et al. 2006; Thomas et al. 2007; Sharma et al. 2009; Kashyap et al. 2013). Among the metals, silver nanoparticles has been synthesized by various physical and chemical methods and their biosynthesis method has been lately developed using natural sources mainly microorganisms (Maliszewska et al. 2009; Sadhasivum et al. 2010). Nevertheless, fungi are good natural source and considered as bionanofactories for synthesis of silver nanoparticles and other metal nanoparticles (Sastry et al. 2003; Rai et al. 2009). However, biosynthesis of silver nanoparticles by fungi is of great interest due to the fungal properties such as they are easy to culture with large biomass productivity, secretion of extracellular enzymes, high amount of protein production and consequently a higher production of nanoparticles (Sastry et al. 2003; Rai et al. 2009; Kashyap et al. 2013). Moreover, silver nanoparticles (AgNPs) are one of the most important metals that have been used as antimicrobial agent and so far many fungal species have been examined for their capability to
123
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synthesize silver nanoparticles and their antimicrobial activities (Feng et al. 2000; Aymonier et al. 2002; Morones et al. 2005; Lee et al. 2003; Riddin et al. 2006; Kim et al.2007; Yokoyama and Welchons 2007; Ingle et al. 2008; Fayaz et al. 2010; Gade et al. 2010). The present work aimed to synthesize and characterize silver nanoparticles from the soil fungus Nigrospora sphaerica and examine their activity against five strains of human pathogenic bacteria and the synergistic activity combined with the commercial antibiotic Gentamycin.
agitated in water bath (120 rpm) for 72 h at 25 °C. After incubation the fungal cell filtrate was filtered by Whatman filter paper No. 1. The fungal free cell filtrate was treated with 1 mM AgNO3 solution and incubated at room temperature at dark condition. Flasks containing free cell filtrate without silver nitrate solution were made as control. Triplicate flasks for treated and untreated fungal filtrate were made. Detection and characterization of silver nanoparticles UV–visible spectrophotometric assay
Materials and methods Fungal isolation Soil samples were collected from subsurface of cultivated localities in Basra (southern Iraq) during the year 2013 using a sterile spatula then placed in sterile plastic bags and brought to the Laboratory. Fungal isolation was performed by serial dilution method (Warcup 1950). 1 mL of each dilution was transferred aseptically into Petri dish containing Potato Dextrose Agar (PDA), the plates were incubated at 25 °C for 7 da and axenic fungal cultures of the growing colonies were made for each fungal isolate. Identification of the recovered fungi was confirmed according to the available taxonomic literature (Ellis 1971, 1976; Domsch et al. 1980; Watanabe 2002). Fungal culture Among the isolated fungi the species Nigrospora sphaerica (Sacc.) Mason was selected in this study for synthesis of silver nanoparticles. The fungal culture was prepared according to Ahmad et al. (2003) as following; one disc of 8 mm diam was cut from five days old culture colony by a sterilized Cork borer and inoculated into 250 mL Erlenmeyer flasks containing 100 mL a liquid medium of MGYP (Malt extract 3 g, Glucose 10 g, Yeast 3 g and Peptone 5 g per 1 L of distilled water) after being autoclaved at 121 °C for 15 min. The culture flasks were incubated at 25 ± 1 °C for 10 day then after incubation the mycelia were separated from the culture broth by centrifugation (600 rpm) at 10 °C for 10 min and the harvested mycelia was washed thrice with sterile distilled water to remove any medium component from the biomass. Biosynthesis of silver nanoparticles For extracellular production of silver nanoparticles typically 10 g of fungal mycelia biomass (wet weight) under a sterilized condition was brought into contact with 100 mL sterile double distilled water in an Erlenmeyer flask and
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After 24 h of incubation of the fungal free cell filtrate treated with AgNO3 solution, the bioreduction of Ag? in aqueous solution was monitored using UV–Vis spectrophotometer (APEL PD-303) at regular intervals. During the reduction process, 0.1 mL of filtrate was taken and diluted three times with deionized water. After dilution, it was centrifuged at 800 rpm for 5 min and the supernatant was scanned using UV–Vis spectrophotometer at the wave length of 300–900 nm. UV–Vis spectra were recorded at 24, 48, and 72 h at a resolution of 1 nm. Untreated free cell filtrate was used as a control. Fourier transform infrared (FTIR) After 72 h of incubation, the free cell filtrate was subjected to Fourier Transform Infrared (FTIR) (Shimadzu UV-1700, Japan) analysis. After a complete reduction of aqueous silver ions within the fungal filtrate, the filtrate was mixed with acetone (1:5 vol/vol) with a continuous shaking then centrifuged at 4,000 rpm for 15 min forming a pellet according to the described method (Raheman et al. 2011). The supernatant was discarded and 2 mL of acetone was added into the pellet and shaken thoroughly then poured into a Petri plate. Acetone was evaporated in order to obtain the powder of silver nanoparticles. Characterization of AgNPs was carried out by using FTIR at the range of 400–4,000 cm-1at a resolution of 4 cm-1. SEM analysis Scanning electron microscopic (SEM) (Netherland INSPECT S50) analysis of the fungal cell filtrate treated with AgNO3 was performed. Thin films of the filtrate samples were prepared on a carbon coated copper grid by just dropping a very little amount of the filtrate on the grid and the extra solution was removed by a blotting paper then the films on the grids were allowed to dry overnight at room temperature under a sterilized condition. SEM micrographs of the silver nanoparticles were exposed at different magnifications.
World J Microbiol Biotechnol Fig. 1 Nigrospora sphaerica culture grown on PDA medium (a) and microscopic structures (b) showing fungal hyphae and conidia (940 magnification)
Fig. 2 Untreated fungal free cell filtrate (a) and treated with 1 mM silver nitrate solution (b) 1.4
Fig. 4 SEM micrographs showing the silver nanoparticles in fungal free-cell filtrate appeared as spherical shape (arrows) at magnification (913,000) with size range between 20 and 70 nm
24 hr 1.2
48 hr 72 hr
Absorbance
1
F.C.F 0.8 0.6 0.4 0.2 0 300
400
500
600
700
800
900
Wavelength in nm
Fig. 3 UV-Vis spectrum of fungal free cell filtrate (fcf) containing silver nanoparticles recorded at different exposure times
Antibacterial activity of silver nanoparticles Agar well diffusion assay The potentiality of silver nanoparticles was examined for their antibacterial
efficiency using agar well diffusion assay method (Perez et al. 1990). Five strains of pathogenic bacteria viz. Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella typhi and Staphylococcus aureus were tested. Swabs from each bacterial culture grown overnight were streaked on sterilized Mueller–Hinton agar (MHA) plates. Wells (5 mm diam) were made in agar plates using sterilized stainless steel Cork borer. The wells were loaded with two concentrations (50 and 100 lL) of silver nanoparticles solutions. The plates were incubated at 37 °C for 24 h and examined for the appearance of inhibition zones around the wells and the diameters of inhibition zones were measured. Minimum inhibitory concentration (MIC) and MBC assay Minimum inhibitory concentration (MIC) assay was carried out using the micro dilution method according to Qi et al. (2004). 100 lL of AgNPs was transferred into 96-well microtitre plates containing 100 lL of Mueller–
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World J Microbiol Biotechnol Fig. 5 FTIR analysis of silver nanoparticles synthesis in fungal free cell filtrates. Before (a) and after (b) silver bioreduction
Hinton broth. 100 lL of the tested bacteria E. coli (ATCC 25922) and S. aureus (NCTC 6571) was inoculated into each well and incubated at 37 °C for 24 h. After the incubation period a small amount of bacterial suspension was streaked on MHA plates and incubated at the same condition. The minimum inhibitory concentration was determined as the lowest concentration of AgNPs that inhibits the growth of bacteria. While the minimum bactericidal concentration (MBC) was determined as the lowest concentration of AgNPs that kills the bacteria and no growth was observed on the agar medium. Assay of antibacterial activity of AgNPs in combination with Gentamycin Disc diffusion method was used to assay the antibacterial activity of synthesized AgNPs combined with commonly used antibiotic Gentamycin as described by Birla et al. (2009). A standard antibiotic disc of Gentamycin was impregnated with 20 lL of freshly prepared AgNPs and placed onto the MHA medium
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inoculated with each tested bacteria. Similarly, the antibacterial activity test for Gentamycin and AgNPs was carried out as control. Discs of Gentamycin and of AgNPs were separately placed on the MHA medium inoculated with each tested bacteria. The plates were incubated at 37 °C for 24–48 h. After incubation, the zones of bacterial growth inhibition for the combination of AgNPs with Gentamycin treatment and for control plates were measured. Four replicates for each treatment were made. Assessment of increase in fold area The increase in fold area was assessed by calculating the mean surface area of the inhibition zone exhibited by the antibiotic alone and in a combination with AgNPs. The fold increase area was calculated by the equation; (B2 - A2)/A2, where A refers to the inhibition zone diam exhibited by the antibiotic activity alone and B refers to the inhibition zone diam exhibited by activity of a combination of antibiotic and AgNPs (Birla et al. 2009).
World J Microbiol Biotechnol Fig. 6 Appearance of inhibition zones on agar plates using of silver nanoparticles synthesized by the fungus N. sphaerica against E. coli (a), S. aureus (b), P. aeruginosa (c), S. Typhi (d) and P. mirabilis (e) at concentration (1) control, (2) 50 lL and (3) 100 lL of silver nanoparticles
Results
pale yellow into dark brown color which indicates the synthesis of silver nanoparticles (Fig. 2).
Fungal culture and silver nanoparticles synthesis UV–Vis spectra of AgNPs The isolated fungus N. sphaecia grows rapidly on PDA medium and covered the Petri plate within three days (Fig. 1). The fungal free cell filtrate (fcf) obtained from the fungal cultures grown in MGYP liquid medium over ten days after being treated with AgNO3 solution turned from
The UV–Vis spectra recorded from the fungal free cell filtrates amended with 1 mM AgNO3 solution revealed significant variations in spectra of silver nanoparticles synthesis at different intervals of reaction (Fig. 3). The
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World J Microbiol Biotechnol
Inhbition zones (mm diam)
30 25
E. coli
P .aeruginosa
S . aureus
S. typhi
inhibition zones dim) than at 100 lL concentration (15–26 mm inhibition zones diam) (Fig. 7). The highest growth inhibitory activity of AgNPs was against P. aeruginosa and lowest against P. mirabilis. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were very low against the two strains of bacteria E. coli and S. aureus (Table 1).
P. mirabilis
20 15 10 5
Antibacterial activity of AgNPs combined with gentamycin
0
AgNPs (50 ul)
AgNPs (100 ul)
Fig. 7 Inhibition zones exhibited by two concentrations of silver nanoparticles from the fungus N. sphaerica against five strains of pathogenic bacteria
Table 1 The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of AgNPs against two strains of bacteria
Bacteria
MIC (lg/L)
MBC (lg/L)
E. coli
0.156
0.312
S. aureus
0.0024
0.0048
absorbance pattern of the Ag–fcf monitored at the range of 300–900 nm revealed an increase of absorbance with increasing time of incubation at 430 nm. Highest spectrum of AgNPs synthesis after 72 h of incubation was detected. SEM analysis SEM images with different magnifications showed that the silver nanoparticles are dispersed or aggregated and mostly showed spherical shape and their size ranging between 20 and 70 nm (Fig. 4). FTIR spectroscopy FTIR analysis of silver nanoparticles synthesized from the fungus N. sphaerica showed the presence of peak at 1,660.6, 1,384.79, 1,112.8 and 1,014.4 cm-1 (Fig. 5). The bands at 1,660.6 refer to the bonding vibrations of the amide I and II of proteins while the bands at 1,384.79 cm-1 and 1,112.8 cm-1 indicate the presence of C–N stretching vibrations of aromatic and aliphatic amines. Antibacterial activity of silver nanoparticles The mycosynthesized silver nanoparticles exhibited an antibacterial activity against the tested Gram positive and Gram negative bacteria (Fig. 6). The present results showed that the bacterial growth inhibition at 50 lL/mL concentration of AgNPs was slightly lower (13–24 mm
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The antibacterial activity of AgNPs combined with commercial antibiotic Gentamycin was significantly increased against the growth of the tested strains of bacteria compared with activity of either of them alone (Fig. 8). Over all the tested bacteria, the growth inhibition zones ranged between 26 and 34 mm diam (Table 2). Among the examined bacteria, the growth of S. aureus and P. mirabilis was remarkably inhibited by silver nanoparticles combined with gentamycin as indicated by the increase in fold area values (0.133 and 0.28, respectively) (Table 2).
Discussion The last decade witnessed an increased research interests focusing on the biosynthesis of silver nanoparticles as an agent widely used in biomedical applications (Sharma et al. 2009; Kearns et al. 2006). However, silver nanoparticles is of a particular interest as antimicrobial agent (Yokoyama and Welchons 2007; Fayaz et al. 2010). The present study showed that the soil fungus N. sphaerica exhibited high potentiality for synthesis of silver nanoparticles in cultures as indicated by the color change from yellow into dark brown after 72 h of incubation after being treated with 1 mM AgNO3 solution. These findings are in similarity with other previous studies using different fungal species such as Fusarium oxysporum, F. acuminatum and Phoma glomerata for the synthesis of silver nanoparticles (Riddin et al. 2006; Ingle et al. 2008; Birla et al. 2009). It has been reported that the color change after addition of AgNO3 into the fungal free-cell filtrate is due to the excitation of surface plasmon resonance vibration of silver that confirmed the reduction of silver ions (Chitra and Annadurai 2013). UV–Vis spectrophotometry is commonly used technique for analyzing AgNPs synthesis (Henglein 1993). The present data revealed that UV–Vis spectrophotometry analysis showed a sharp peak with high absorbance at 420 nm which confirmed the AgNPs synthesis by the examined fungus. The absorption peak in UV spectrum is equivalent to the surface plasmon resonance and the maximum biosynthesis of AgNPs was at 72 h of
World J Microbiol Biotechnol Fig. 8 Antibacterial activity indicated by the inhibition zones (mm diam) exhibited by biosynthesized silver nanoparticles against E. coli (a), S. aureus (b), P. aeruginosa (c), S. typhi (d) and P. mirabilis (e) using AgNPs alone (1), commercial antibiotic gentamycin (2) and a combination of AgNPs with gentamycin (3)
incubation. This is in conformity with some other studies (Ingle et al. 2008; Chitra and Annadurai 2013). Based on literatures, apparently, there are some variations among the fungal free cell filtrate incubation periods and their UV spectra recorded for the biosynthesis of silver nanoparticles by using different sources of fungi. For example, Chitra and Annadurai (2013) reported that the synthesis of silver nanoparticles by the fungus Trichoderma viride occurred at
incubation time ranged between 30 min and 96 h and a sharp spectra peak appeared at 400 nm. On the other hand, the size and shape of silver nanoparticles synthesized by fungi are also varied. Such variations in characterization of silver nanoparticles biosynthesis might be related to a selected fungal species and the culture conditions applied. The present study revealed that the size of the detected AgNPs range between 20 and 70 nm with spherical shape
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World J Microbiol Biotechnol Table 2 Inhibition zones exhibited by the silver nanoparticles combined with commercial antibiotic gentamycin against five bacteria strains and the calculated increase in fold area Bacteria
Inhibition zone (mm diam) AgNPs
Gentamycin
AgNPs ? gentamycin
Increase in fold area
E. coli
18*
25
26
0.08
S. aureus
17
31
33
0.133**
P. aeruginosa S. typhi
20 15
26 25
28 26
0.16** 0.08
P. mirabilis
20
30
34
0.28**
* Values represent means of four replicates ** Significant difference at P \ 0.001
and are dispersed or aggregated as confirmed by SEM images. Another study (Martinez-Castanon et al. 2008) has reported that the size of silver particles range between 7 and 89 nm. A study of Saha et al. (2011) reported that the size of silver nanoparticles synthesized from the fungus N. oryzae ranged between 30 and 90 nm. It has been stated that the absorption spectrum of spherical shape of silver nanoparticles present a maximum between 420 and 450 nm (Pal et al. 2007). Nevertheless, the size and shape of silver nanoparticles might be affected by the sample preparation method for SEM analysis such as drying process (Sadawiski et al. 2008) as we applied in the present study. Analysis of FTIR indicated the release of proteins into fungal filtrate which leads to a reduction of silver ions present in the free cell filtrate. The reduction of the Ag? ions is might be due to the enzyme reductase that released by the fungus as reported by Gole et al. (2001). It has been shown that FTIR analysis has confirmed that amino acid residues and peptides of proteins are binding with the silver nanoparticles forming a capping agent of nanoparticles and stabilizing them in the culture medium (Vigneshwaran et al. 2007). In this study two concentrations 50 lL/mL and 100 lL/mL of silver nanoparticles synthesized by the fungus N. sphaerica were tested against the selected human pathogenic bacterial strains and rendered a significant bacterial growth inhibition as indicated by the appearance of clear zones. However, variations among the antibacterial activity exhibited by the silver nanoparticles were observed. Higher inhibition zones diameters was recorded for P. mirabilis and P. aeruginosa than other tested bacteria. The present study also revealed that silver nanoparticles from N. sphaerica exhibited higher inhibition activity against the pathogenic bacteria S. aureus and S. typhi than that reported by Raheman et al. (2011) who examined the endophytic fungus Pestalotia sp. Although the mechanism of AgNPs effects against the bacteria is not yet well understood, however, it can be attributed to the action of Ag on
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bacterial cell causing cell membrane damage, DNA denaturation, enzymes retardation or due to some other effects. The minimum inhibitory concentration (MIC) of the silver nanoparticles was determined and revealed that the MIC values were 0.156 and 0.0024 lg/L mL against E. coli and S. aureus, respectively. The MIC values were very low demonstrating the high potential activity of biosynthesized silver nanoparticles against Gram positive and Gram negative bacteria. Similar observations were found by other workers using AgNPs synthesized from other fungi against Gram positive and Gram negative bacteria (Martinez-Castanon et al. 2008; Birla et al. 2009). The present study has also demonstrated that the antibacterial activity of the synthesized silver nanoparticles combined with the commercial antibiotic Gentamycin. The efficiency of silver nanoparticles with Gentamycin was found to be increased significantly reaching 33 and 34 mm diam inhibition zone against S. aureus and Proteus mirabilis, respectively. The antibacterial activity of a combination of AgNPs ? Gentamycin was expressed as increase in fold area according to Birla et al. (2009).The increase in fold area was 0.133 and 0.28 for S. aureus and Proteus mirabilis, respectively. Our results support some other studies examined the synergistic effects of AgNPs, synthesized from various fungal species, in a combination with different commercial antibiotics tested against Gram positive and Gram negative bacteria (Shahverdi et al. 2007; Fayaz et al. 2010; Gajbhiye et al. 2009). A conclusion can be derived from the present study that the soil fungus N. sphaerica has a potentiality for silver nanoparticles synthesis which exhibiting a high growth inhibition activity against Gram positive and negative pathogenic bacteria. Generally, fungi represent a natural resources and ecofriendly and can be implemented in medical therapy. Thus, further researches to explore more potent fungi for silver nanoparticles synthesis are needed. Also an optimization of fungal growth conditions used for biosynthesis of silver nanoparticles and their antimicrobial activities are
World J Microbiol Biotechnol
recommended. Further mechanistic studies to understand the synthesis of silver nanoparticles from fungi are important. Acknowledgments The authors would like to thank the authorities of Biology Department, College of Education for Pure Sciences, Basra University (Iraq) for supporting this research work as a part of M Sc. research program scholarship awarded to the second author.
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