Scholarly article on topic 'Extracellular biosynthesis of silver nanoparticles using Rhizopus stonililifer'

Extracellular biosynthesis of silver nanoparticles using Rhizopus stonililifer Academic research paper on "Chemical sciences"

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Saudi Journal of Biological Sciences
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{Nanoparticles / Silver / " Rhizopus stolonifer "}

Abstract of research paper on Chemical sciences, author of scientific article — Khalid AbdelRahim, Sabry Younis Mahmoud, Ahmed Mohamed Ali

Abstract Synthesis of silver nanoparticles (AgNPs) has become a necessary field of applied science. Biological method for synthesis of AgNPs by Rhizopus stolonifer aqueous mycelial extract was used. The AgNPs were identified by UV–visible spectrometry, X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectrometry (FT-IR). The presence of surface plasmon band around 420nm indicates AgNPs formation. The characteristic of the AgNPs within the face-centered cubic (fcc) structure are indicated by the peaks of the X-ray diffraction (XRD) pattern corresponding to (111), (200) and (220) planes. Spherical, mono-dispersed and stable AgNPs with diameter around 9.47nm were prepared and affirmed by high-resolution transmission electron microscopy (HR-TEM). Fourier Transform Infrared (FTIR) shows peaks at 1426 and 1684cm−1 that affirm the presence of coat covering protein the AgNPs which is known as capping proteins. Parameter optimization showed the smallest size of AgNPs (2.86±0.3nm) was obtained with 10−2 M AgNO3 at 40°C. The present study provides the proof that the molecules within aqueous mycelial extract of R. stolonifer facilitate synthesis of AgNPs and highlight on value-added from R. stolonifer for cost effectiveness. Also, eco-friendly medical and nanotechnology-based industries could also be provided. Size of prepared AgNPs could be controlled by temperature and AgNO3 concentration. Further studies are required to study effect of more parameters on size and morphology of AgNPs as this will help in the control of large scale production of biogenic AgNPs.

Academic research paper on topic "Extracellular biosynthesis of silver nanoparticles using Rhizopus stonililifer"

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Extracellular biosynthesis of silver nanoparticles using Rhizopus stonililifer

Original article

Saudi Journal of Biological Sciences



Khalid AbdelRahim, Sabry Younis Mahmoud, Ahmed Mohamed Ali

S1319-562X(16)00083-8 SJBS 687

To appear in:

Saudi Journal of Biological Sciences

Received Date: 5 December 2015

Revised Date: 23 February 2016

Accepted Date: 29 February 2016

Please cite this article as: K. AbdelRahim, S.Y. Mahmoud, A.M. Ali, Extracellular biosynthesis of silver nanoparticles using Rhizopus stonililifer, Saudi Journal of Biological Sciences (2016), doi: 10.1016/j.sjbs.2016.02.025

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Extracellular biosynthesis of silver nanoparticles using

Rhizopus stonililifer

Khalid AbdelRahim1'2*, Sabry Younis Mahmoud3 and Ahmed Mohamed Ali3,

(1) Botany and Microbiology department, College of Science, King Saud University

Box 2455, Riyadh 11451, Saudi Arabia E. Mail: (corresponding author)*

(2) Botany department, Faculty of science, Sohag University, Sohag 82524, Egypt.

(3) Department of Medical laboratory technology, College of Applied Medical Science,

University of Dammam, 1704,Hafr Al Batin-319 91, Saudi Arabia.

Corresponding author: Khalid AbdelRahim, Botany and Microbiology department, College of Science, King Saud University P.O. Box 2455, Riyadh 11451, Saudi Arabia. :, Tel.: 0114675818, Fax: 0114675833.


Synthesis of silver nanoparticles (AgNPs) has become a necessary field of applied science. Biological method for synthesis of AgNPs by Rizobus stonilifer aqueous mycellical extract was used. The AgNPs were identified by UV-visible spectrometry, X-ray diffraction (XRD), Transmission electron microscopy (TEM) and Fourier transform Infrared spectrometry (FT-IR). Presence of surface plasmon band around 420 nm indicates AgNPs formation . The characteristic of the of AgNPs within the face-centered cubic (fcc) structure are indicated by the peaks of the X-ray diffraction (XRD) pattern corresponding to (111), (200) and (220) planes. Spherical, mono-dispersed and stable AgNPs with diameter around 9.47 nm were prepared and affirmed by high- resolution transmission electron microscopy (HR-TEM). Fourier Transform Infrared (FTIR) shows peaks at 1426 and 1684 cm-1 that affirm presence of coat covering protein the AgNPs which is known as capping proteins. Parameters optimization showed smallest size of AgNPs ( 2.86 ± 0.3 nm ) was obtained with 10-2 M AgNO3 at 40 ° C. Present study provides the proof that the molecules within aqueous mycellial extract of R. stonilifer facilitate synthesis of AgNPs and highlight on value-added from R. stonilifer for cost effective. Also, eco-frindely medical and nanotechnology-based industries could also be provided. Size of prepared AgNPs could be controlled by temperature and AgNO3 concentration. Further studies are required to study effect of more

parameters on size and morphology of AgNPs as this will help in control of large scale production of biogenic AgNPs.

Keywords: Nanoparticles, Silver, Rhizopus stonlifer


of varied nanomaterials. Now, various kinds of metal nanomaterials are being prepared by copper, zinc, titanium, magnesium, gold, alginate and silver (Basavaraj et al., 2012). AgNPs became the main focus of intensive research because of their wide selection of applications in areas like catalyst , optics, antimicrobials, and biomaterial production (Qin et al., 2011; Rai et al., 2009; Rao et al., 2000; Zhang et al., 2011; Zhong-jie et al., 2005). Silver nanoparticles showed new or improved properties due to their unique size, morphology, and distribution. At the moment, there's a growing demand develop eco-friendly nanoparticles using safe chemicals in the synthesis protocol.

Researcher turned to biological system for synthesis of nanoparticles as alternatives to chemical and physical methods. This as a result of several unicellular and multicellular organisms are well-known producing inorganic materials either intra- or extra-cellular (Simkiss and Wilbur 1989, Mann 1996). Biosynthesis of nanoparticles such as nanosilver and control in their size composition and mono-disparity is an important area of research in nanoscience, Silver nanoparticles are widest used among all nanomaterials. so biological and biomimetic approaches for biological synthesis of AgNPs are under exploring. biomass or extracellular materials from microorganisms like Fusarium oxysporum , E. coli , Aspergillus flavus, licheniformis are used for biotransformation of silver ions to AgNPs (Kim et al., 1998, Cho et al., 2005, Ahmed et al., 2003, Shahverdi et al., 2007). The aim of this study is biosynthesis of AgNPs using fungi R. stolonifer which is cheap , safe, nonpolluting and 'table method. Filamentous fungi are more preferred than bacteria and unicellular organisms as they are easy to handle and able to synthesis AgNPs extracellular (Kalishwaralal et al., 2008). Synthesized nanoparticles by fungi are more stable with better mono-disparity (Balaji et al., 2009). in this study we prepared stabilized AgNPs by aqueous extract of R. stolonifer, characterized by UV-Visible absorption spectra, XRD,FTIR and confirmed by TEM. Also we investigate effect of temperature and AgNO3 concentration on size of prepared AgNO3.

to bios accepta

2-Material methods


All used chemicals in present study obtained from Sigma-Aldrich, USA. Fresh deionized water was used during the experimental work.

2.2- R. stolonifer isolation and identification

_____Fresh dei(

R. stolonifer was isolated from naturally infected tomato fruits according to Balali et al., (Mukherjee et al., 2008). Potato dextrose Agar (PDA) medium supplemented with (30 mg L-1) Chloromaphenicol to discourage bacterial contamination was prepared routinely and used for fungal isolation. Monosporic cultures were obtained by cultivation of serial dilutions from pure cultures after that individual spores were collected and grown on PDA. Isolates were identified according to their cultural and morphological characteristics based on identification standards (Govindaraju et al., 2010) .

2.3-Biosynthesis of silver nanoparticles

Biomass was produced by cultivation of R. stolonifer in Malt Glucose peptone (MGYP) broth composed of yeast extract and malt extract zero.3% each, glucose1%, peptone 0.5%. Culture was incubated at 40oC on an orbital shaker 180 rpm for 3 days (Balali et al., 1995). Culture was filtered and resulted biomass was washed extensively by deionized water to get rid of adhered media parts. Mycelia extract prepared by suspension of fungal biomass in 100 ml deionized water and incubated as described above for 72 hours, after that mycelia spension was filtered using (Whatman paper No.1), Resulted filtrate (mycelia extract) was mixed with AgNO3 solution (1mM AgNO3 final concentration) and incubated on orbital shaker 180 rpm at 40° C for two days.

2.4-UV-Visible spectrometry measurement

Biotransformation of silver ions was monitored by UV-visible spectroscopy measurement of the reaction medium. Three milliliters of supernatant were taken after 6,12, 24, 36 and48

hours and absorbance was scanned by Labomed, UV-vis double beam (Labomed , Inc, USA) within the wave length ranged from 200 to 600 nm. The absorption of the visible depend directly on color of the chemicals in solution.

2.5-X-Ray Diffraction (XRD) measurement

XRD technique was used for checking quality of prepared nanoparticles were. XRD pattern of drop-coated films of synthesized nanoparticles on glass material was recorded in wide selection of Bragg angles 20 at a scanning rate of 20 min-1, using Philips PW 1830 instrument (Philips, Inc, USA) adjusted at 40 kv and 30 mA with metal Cu ka radiation (X=1.5405 A).

2.6-TEM measurements

netal Cu

The morphology and size of AgNPs was determined using TEM by Transferring aliquot of aqueous suspension of AgNPs onto a carbon coated copper grid and allowed to be air dried (Domsch et al., 1993). The grid was then scanned employing a Phillips EM 208S transmission microscope (Philips, Inc, USA) adjusted at 100 kV.

2.7-Fourier rework Infrared (FT-IR) spectrometry analysis

The sample was scanned by FT-IR spectrometry using PerkinElmer spectrophotometer (Los Angeles, CA). Briefly 2 milligrams of sample mixed 200 mg Potassium bromide ( KBr) (FT-IR grade) and pressed into a pellet and placed into the sample holder and FT-IR spectra were scanned in rang 4000-400 cm-1 in FTIR spectrometry at a resolution of one cm-1.

Effect of parameters on controlling the size AgNPs

To study size control of AgNps we investigate effect of temperature and AgNO3 concentration on size of AgNps, to achieve this purpose we used different concentrations of AgNO3 (10-1, 10-2 and 10-3 M), temperatures (10, 20, ,40, , 60 ,80° C) . Biosynthesized

gNPs size measurements was determined by TEM. 2.8-Statistical analysis

Origin professional eight (Microsoft ,USA) software was used statistical Gaussian approximation to find full width at half maximum (FWHM) and calculation of One way Anova , P value < 0.05 is considered statistically significant.

3-Results and discussion

The pure colonies were obtained and known as R. stonilifer supported the microscopic results. Addition of 100 ml of R. stonilifer mycellial aqueous extract to aqueous solution of silver nitrate at final concentration1 mM, pale yellow color of R. stonilifer mycellial filtrate to reddish brown color within 48 h (Figure 1). This result indicates the formation and deposition of silver AgNPs while original color of silver nitrate (negative control) remain unchanged. Usually, AgNPs formation detected by color change observation of reaction medium from colorless to yellowness or dark brown (Karbasian et al., 2008). Color change of solution is due to excitation of surface Plasmon vibrations of AgNPs (Gericke and Pinches 2006).

Figure 1. Visible observation of AgNPs biosynthesis. (A) ErlenMeyer flask with R. stonilifer mycellial filtrate after exposure to AgNO3 solution (1 mM) for a few minutes (no or change), and (B) ErlenMeyer flask with R. stonilifer mycellial filtrate after exposure to AgNO3 solution (1 mM) for 48 h (reddish-brown color).

UV-Visible spectrometry showed optical absorption spectra of AgNPs ranged from 300 to 600 nm. The absorption spectra show one outstanding symmetric peak around 420 nm , That is as a result of surface plasmon resonance of AgNPs (Figure 2). This spectroscopic pattern results from interactions of free electrons limited to tiny metallic spherical objects with episode electromagnetic wave. To study effect of time on AgNPs production, we measured

UV-Vis spectra at different time, we found that absorbance at 420 nm increased with the incubation time of the silver nitrate with the mycelium extract. The statistical analysis showed a significant difference (P value = 0.001) in the production of AgNPs (Figure 3) highest production of AgNPS was after 48 hours of incubations . UV- spectroscopy showed increased absorbance with time and AgNPS were synthesized by 24 h and there was almost no increase in absorbance after 48 h for the four tested Aspergillus species (Neveen, 2013). Increase in intensity of the absorbance peak with time indicates the continued reduction of the silver ions and increase in concentration of AgNPs (Birla et al., 2013). Electronic style of AgNPs are notably sensitive to their form and size, resulting in clear effects in its visible spectrum pattern. One of interesting criteria is increasing of bandwidth of resonance with the decrease of the dimensions of the particles as a result of electron scattering induction at the surface. Resonance shifting and the variation of its bandwidth are important information for nanoparticles characterization. Presence of Plasmon band at 420 nm due to dipole plasmon resonance shows that the AgNPs has spherical shape (Sathishkumar et al., 2009; Kannan et al., 2011). The full-width at half-maximum (FWHM) provide useful tool for determination size of nanoparicles and their distribution in the medium based on the concept of Brown et al (Mock et al., 2002), In our study the FWHM of the AgNPs is 79.42 nm (Figure 2). It's concluded that a FWHM of 79.42 nm is mostly indication of a small size distribution. Thus, the synthesis of AgNPs using R. stonilifer is a promising and appropriate technique for preparation of uniform AgNPs with a small size distribution .

1.4 ■

£ 1.0.

<n 0.8 ■


300 350

Gauss of B

Equation y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x -xc)/w)A2)

Adj. R-Squ 0.98731

Value Standard Er

B y0 0.31237 0.01596

B xc 425.067 0.75549

B w 67.4538 1.95995

B A 110.372 3.5187

B sigma 33.7269

B FWHM 79.4208

B Height 1.30555


Figure 2 . UV-vis spectra of prepared Ag NPs, B- Optical absorption spectra and its Gauss of B. Inserted table show Statistical Gaussian approximation was performed to find FWHM).

1.4 1.2

0) 1.0 o c ra

-9 0.8

0.4 0.2 0.0

48 hours 24 hours 12 hours 6 hours


Figure 3. UV-visible absorption spectra of produced AgNPs using Rhizopus stonililifer mycelium extract at different incubation times, synthesis of SNPs is

the function of time.

XRD analysis of the synthesized AgNPs showed three diffraction peaks at 20 = 37.65°, 44.85° and 64.89° that are corresponding to Bragg's reflections of the face-centered cubic (fcc) structure of metallic silver, (111), (200) and (220) respectively (Figure 3). All diffraction peaks are in proper agreement with the quality value (JCPDS card No. 04-0783). This XRD line width are often used to estimate the dimensions of the particle by the Debye-Scherrer equation d = 0.9A/ 3 cos 0 , wherever d is that the particle size, A is that the wavelength of x-ray radiation (1.5406A), 3 is that the FWHM of the height (in radians) and 20 is that the Bragg angle. The calculated average particle size was around 9.46 ± 2.64 nm.

TEM is considered a high resolution tool that gives actual information concerning particle size and shape (Jin et al., 2001; Sathishkumar et al., 2009). Recent HRTEM has the ability to image atoms directly in specimens at resolutions about one A, smaller than inter-atomic space. This technique is very necessary for characterizing materials at a length scale from

atoms to hundreds nanometers. Figure 4 shows HRTEM imaging of AgNPs. All AgNPs have a spherical form and diameter about 6.04 nm. Spherical form of prepared AgNPs are highly agree with the fact given by (Brown et al., 2000) This confirm that surface plasmon peak around 420 nm indicate that the AgNPs have spherical form. The AgNPs were mono-dispersed and stable.

Figure 5. HRTEM pictures of the mono distributed spherical AgNPs (x=100 nm) (inserted picture: selected area electron diffraction pattern).

Fourier transform infrared spectrum indicted that mycelia aqueous extract of R. stonilifer contain active biomolecules which are responsible for biotransformation of silver ion to metallic AgNPs, that revealed distinct peak within the range of 3750-500 cm-1 (Fig. 4). The broad peak at 3,500 cm-1 is resulting from strong stretching vibration of phenolic hydroxyl OH (Link and El-Sayed, 2000). The band at 2624 is due to NH group from peptide linkage in the mycelia aqueous extract of R. stonilifer (Wang, 2000). Peak at 1684 is characteristic of amid group NHCO. Infrared (IR) analysis study has confirmed that carbonyl group resulted from amino acid residue and peptide protein can strongly bind to metal, So protein may acts as capping protein of AgNPs prevents agglomeration and stabilizes particles within the medium. This prove suggests that the biological molecules are responsible for biotransformation of silver ions to AgNPs and its stabilization in aqueous medium. It is well known character of protein that can bind to AgNPs through free amine group and stabilization of AgNPs may due to surface bound protein (Gopinath et al., 2012). The peaks at1426 and 1634 cm-1 are corresponding to carbonyl stretch vibrations in the amid linkages of proteins (Mubarakali et al., 2001).The carbonyl group from amino acid residues and peptides remains have strong capability to bind to silver (Gole et al., 2001). Also it is reported that proteins will bind to nanoparticles either through free amino or cystein group in proteins (Basavaraja et al., 2008). The peaks at 1952, 1426 and 1684 cm-1 are corresponding AgNPs binding between oxygen from hydroxyl group and amid carbonyl groups of R. stonilifer mycelia extract (Mandal et al., 2005) (Figure 5).

From FTIR results we conclude that presence of protein in reaction medium provide reducing agent and coat covering for AgNPs known as capping proteins. Capping protein prevents agglomeration of AgNPs in the medium and responsible for formation high stable AgNPs. Similar results obtained with bacteria (Kumar et al., 2011) and Algae (Sudha et al., 2013). However polymers and surfactants were widely used as capping agent in preparation of AgNPs , protein capping provide advantage over polymer and surfactant as it is coast effective, safe, ecofriendly and does not need special conditions. Several harmful chemical by-products, metallic aerosol, irradiation, etc. are commonly produced during use of chemical and physical in AgNPs synthesis processes . These, along with the facts that these

processes are expensive, time consuming, and typically done on small laboratory scale, render these methods less suitable for large-scale production (Mansoori, 2005; Sahu and Biswas, 2010). Another advantage of protein capping when compared to polymer and surfactant it acts as the anchoring layer for drug or genetic materials to be transported into human cells (Hu et al., 2011). The presence of a nontoxic protein cap also increases uptake and retention inside human cells (Rodriguez et al., 2013). The presence of natural capping proteins eliminates the postproduction steps of capping which is necessary for most of applications of nanoparticles in the field of medicine (Chowdhury et al, 2014)

Figure 6. Fourier-transform infrared spectra of AgNPs biosynthesized by R. stonlifer after 48 hours from biosynthesis reaction.

Previous studies reported that size of AgNPs and micrometer scale Ag could be controlled by temperature or by molar ratio (Wang et al., 2010; Sun and Luo, 2005). In order to know how mycelium extract control size of AgNPs, we examined the effect of metal concentrations and temperature. Our findings showed no AgNPs was produced at 10° C or at 80° C this due to denaturation or inactivation of enzymes and active molecules which are involved in biogenesis of AgNPs either by low or high temperature. Small mono-dispersed AgNPs with average size 2.86 ± 0.3 nm were produced at 40 ° C (Figure 7) , Large AgNPs with average particle size 25.89 ± 3.8 and 48.43 ± 5.2 nm and were produced at 20 ° C and 60 ° C respectively (Figure 8,9). This increase in AgNPs size is due to low activity of enzymes involved in AgNPs biogenesis as a result of unsuitable temperature. Quite similar results

reported at a temperature (50 °C), most of AgNPs were small. Further incubation at higher temperatures, the enzymes have denatured nature this lead to increase in particles size according to loss of enzyme activity (Sherif et al., 2015). Closed results obtained by (Birla et al., 2013). Increase of temperature, the kinetic energy of the AgNPs in the solution also increases and collision frequency between the particles also rises resulting in higher rate of agglomeration (Sarkar et al., 2007).

Figure 7. HRTEM image (x=100 nm) and size distribution histogram of AgNPs prepared

t 40 ° C and 10-2 M AgNO3 .

Figure 8. HRTEM image (x=100 nm) and size distribution histogram of AgNPs prepared

at 20 ° C .

Figure 9. HRTEM image (x=200 nm) and size distributio ram of AgNPs prepared

In order to investigate effect of AgNO3 concentration on size of AgNPs we applied different

—1 -2 —3

concentrations of AgNO3 (10 , 10 and 10 M) and temperature was fixed at 40 ° C.

Smallest AgNPs size (2.86 ± 0.3 nm) was obtained at concentration 10 M of AgNO3 (Figure 7). Large AgNPs with average particle size 54.67 ± 4.1 and 14.23 ± 1.3 nm and were prepared at concentration 10-1, 10-3 M of AgNO3 respectively (Figure 10,11). Usually, the optimum concentration of silver nitrate (1mM)is used for the synthesis of AgNO3 (Ahmad et al., 2003). Smallest AgNPs were obtained at10-2 M of metal ion and excess addition of metal ions with concentration 10-1M results in formation of very large particles exhibiting irregularly shaped that most of the cell enzymes were consumed by reduction of particles, h capacity of the cells for silver reduction (Sherief et al.,2015).

at 60 ° C

Figure 10. HRTEM image (x=200 nm) and size distribution histogram of AgNPs prepared at concentration 10-1 M of AgNO3.

Figure 11. HRTEM image (x=200 nm) and size distribution histogram of AgNPs prepared

at concentration 10 M of AgNO3. 4- Conclusion

In present study mono-dispersed AgNPs was prepared by mycellial aqueous extract of R . stolonifer. It have spherical form with average size of 9.46 ±2.64 nm. The AgNPs were characterized by UV-visible, XRD, TEM and FT-IR spectra. Biological synthesis of AgNPs using R. stolonifer is cheap, nonpolluting and safe technique is a good alternative to chemical

and physical methods. Our finding confirmed that mycellial aqueous extract of R . stolonifer could be powerful tool for biosynthesis and stabilization of silver ion to AgNPs However our results showed that temperature and metal ion concentration are playing an important role in AgNPs size control further studies are required to study kinetic of AgNPs reaction and effect of more parameters on size, geometry and morphology of AgNPs. These studi are important for controlling AgNPs large scale production and production high quality uniform AgNPs.

6- Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no (RG - 1435 - 060).


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