32

CHAPTER 3: RESULTS AND DISCUSSION

3.1 Isolation, Screening and Identification of Fungi

The isolated fungi were subjected for the synthesis of silver nanoparticles. A total

of 15 fungi were screened for biosynthesis of AgNPs (Table- 3. 1). Amongst them

VRSRJ-09 showed UV-Vis spectroscopy peak at 416 nm within 1 h, showing maximum

biomass of 5.0 g/100 ml, which was identified as Aspergillus sp. This sps showed good

result and was identified based on morpholological and microscopic characters, conidia

are black, hyphae is white to dull yellow and reverse is light yellow, yellowish

conidiospores with smooth surface texture (Fig. 3. 1 a and b) (Dubey and Maheshwari,

2002). Based on morpholological and microscopic identification it was further sent to

Agharkar Research Institute, Pune for species identification, and was identified as

Aspergillus niger.

3.2 Silver Nanoparticles Biosynthesis

The fungal mycelia were separated after 72 h incubation by filtration, which is

pale yellow in colour and turns to brown colour when it is challenged against AgNO3.

The appearance of brown colour indicates the production of silver nanoparticles.

Afreen et al., (2011, b), Dattu et al., (2014 a) and Ranganath et al., (2012) also

reported that appearance of brown colour is an indication of AgNPs production. The

colour change was caused by the surface plasmon resonance (SPR) of AgNPs in the

visible region. AgNPs are known to exhibit size and shape dependent SPR bands which

are characterized by UV-Vis spectrum.

33

Organisms UV-Vis

Spect.(nm)

Biomass

(g/100 ml)

Reduction time

(h)

VRSRJ-01 399 4.0 5

VRSRJ-02 357 3.5 -

VRSRJ-03 319.5 3.9 -

VRSRJ-04 385 4.0 9

VRSRJ-05 369 2.9 -

VRSRJ-06 340.3 4.5 -

VRSRJ-07 438 3.0 -

VRSRJ-08 390 3.2 24

VRSRJ-09 416 5.0 1

VRSRJ-10 410 4.0 5

VRSRJ-11 412 4.2 6

VRSRJ-12 380 3.0 8

VRSRJ-13 392 2.9 14

VRSRJ-14 438 5.0 7

VRSRJ-15 429 4.0 18

Table -3. 1: Screening of fungi for AgNPs biosynthesis

(a) (b)

Fig. 3.1: Aspergillus niger on PDA, (a) front view and (b) reverse view

34

This clearly indicates the production of silver nanoparticles extracellularly. The

use of fungi in the synthesis of AgNPs is a relatively recent addition and holds promise

for large scale nanoparticles production. In fact, fungi secrete large amount of enzymes

involved in AgNPs synthesis and are simpler to grow both in laboratory and at industrial

scale (Murali et al., 2003, Anil et al., 2007 and Venkataraman et al., 2011). The use of

specific enzymes such as reductase secreted by fungi opens up exciting possibilities of

designing a rational biosynthesis strategy for metal nanoparticles of different chemical

composition (Anil et al., 2007 and Navin et al., 2011). Biosynthesis of AgNPs using

bacteria, fungi and actinomycetes were reported (Murali et al., 2003, Prashant et al.,

2008, Ranganath et. al., 2012, Prema et al., 2014 and Dattu et al., 2014, b).

A number of different genera of fungi have been investigated for biotechnological

process research and it has been shown that fungi are extremely good candidates for the

synthesis of AgNPs (Murali et al., 2003, Nelson et al., 2005, Xiangqian et al., 2011, Dattu

et al., 2013 and Jyothi et al., 2014). Application of biological systems for synthesis of

AgNPs has already been reported. However, the exact mechanism leading to the

synthesis of AgNPs is yet to be elucidated.

3.3 Characterization of Silver Nanoparticles

Extracellularly synthesized silver nanoparticles by A. niger under better suitable

growth conditions as said earlier, were characterized by various techniques such as-

Visual observation

UV-Vis spectrophotometer

TEM

ZETA-sizer

FTIR

SEM

35

EDS

AFM

XRD

Determination of tryptophan/tyrosine residues

3.3.1 Visual Observation

Visual observation of color change from light yellow to brown after addition of

AgNO3 to enzyme filtrate is the primary indication of AgNPs biosynthesis. In A. niger

there is clear colour change from light yellow to brown indicating the reduction of

aqueous Ag+ with culture supernatants their by showing production of AgNPs (Fig. 3.2).

Rati et al., (2011) reported change in colour to deep brown using the fungi P.

purpurogenum NPMF culture filtrate with silver nitrate due to the excitation of surface

plasmon which is typical of AgNPs, and also reported the increase in colour intensity of

culture filtrate was due to increased number of nanoparticles formed as a result of

reduction of silver ions. The colour change was caused by the SPR of AgNPs in the

visible region was also reported by Afreen et al., (2011 a) using the fungus R. stolonifer,

and also by Dattu et al., (2013) using endophytic fungi Penicillium sps.

Khabat et al., (2011) also reported yellowish-brown color in solution containing

the biomass is a clear indication of the formation of AgNPs in the reaction mixture using

the fungus Trichoderma reesei, the color of the solution is due to the excitation of surface

plasmon vibrations (essentially the vibration of the group conduction electrons) in the

AgNPs. Thus visual observation is the primary indication of reduction of aqueous Ag+

and formation of AgNPs.

36

3.3.2 UV-Vis Spectroscopy

The UV-Vis spectrum for AgNPs is obtained by exposing the sample to UV-light

from a light source. The specific surface plasmon resonance is responsible for their

unique remarkable optical phenomenon. A single peak maximum at 416 nm corresponds

to the surface plasmon resonance of AgNPs was observed in the UV-Vis spectrum (Fig.

3.3).

Priyabrata et al., (2001) reported the AgNPs absorb radiation in the visible region

of the electromagnetic spectrum (ca. 380-450 nm) due to excitation of surface plasmon

vibrations and it is responsible for the striking yellow brownish colour of silver

nanoparticles. Deepika et al., (2013) reported that in certain metals, such as silver and

gold, the plasmon resonance is responsible for their unique and remarkable optical

phenomena. Metallic (silver or gold) nanoparticles, typically 40–100 nm in diameter,

scatter optical light elastically with remarkable efficiency because of a collective

resonance of the conduction electrons in the metal known as surface plasmon resonance.

The surface plasmon resonance peak in UV absorption spectra is shown by these plasmon

resonant nanoparticles.

Venkataraman et al., (2011) also reported the magnitude, peak wavelength, and

spectral bandwidth of the plasmon resonance associated with a nanoparticle are

dependent on the particle’s size, shape, and material composition, as well as the local

environment. Dattu et al., (2014, a) reported the extracellular biosynthesis of AgNPs

using endophytic fungus Penicillium sp. with a maximum absorbance peak at 425nm and

also reported that increase in concentration of silver nitrate increases the particle size due

to aggregation of larger AgNPs.

37

Fig. 3.2: Visual observation of color change from light yellow to reddish brown from

A.niger

Fig. 3.3: UV-Vis spectrum of AgNPs from A. niger

38

In another study by Ivan and Salopek, (2004) the surface plasmon band at 405nm, the

appearance of brown color clearly indicates the formation of AgNPs. Similar types of

result were also observed by Jaidev and Narasimha (2010) using fungus A. niger isolated

from the areas where silver weaving threads were disposed in and around Venkatagiri

town in Nellore district of Andhra Pradesh.

3.3.3 Transmission Electron Microscopy (TEM)

A drop of AgNPs solution was placed on the carbon coated copper grids and kept

under vaccum before loading them onto a specimen holder. Then TEM micrographs were

taken by prepared grids to determine the size and shape of the produced AgNPs. Results

revealed that the particles are spherical in shape and size of the particles is between 20-

55nm (Fig. 3.4).

Guangquan et al., (2012) reported the fungus mediated green synthesis of AgNPs

using Aspergillus terreus and the particles were spherical, polydispersed with size

between 1-20 nm. Vandana et al., (2011) synthesized AgNPs from R. stolonifer and

reported that the size ranged between 5 and 50 nm suggesting that biological molecules

could possibly perform the function of stabilization of AgNPs and also reported that the

AgNPs synthesized by this route are fairly stable even after prolonged storage.

Navin et al., (2011) reported the extracellular biosynthesis and characterization of

AgNPs using Aspergillus flavus NJP08: A mechanistic perspective, revealed spherical

and monodispersed nanoparticles without agglomeration under low magnification with

particle size ranging from 10-35 nm. Sharanabasava et al., (2012) reported the well

distributed AgNPs with occasional aggregation, mainly spherical and the size range

between 5-30 nm using fungus Penicillium diversum.

39

Fig. 3.4: TEM micrograph of AgNPs

40

Faiez et al., (2011) also reported electron microscopic images of well dispersed

nanoparticles with diameter ranging from 15 to 45 nm from A. fumigates. Average size of

AgNPs produced using probiotic Rhodobacter sphaeroides as revealed by Hong et al.,

(2011) were 9.56 nm having spherical shape.

3.3.4 ZETA Sizer

The particle size distribution of the AgNPs was shown under different categories

like size distribution by volume and by intensity. The average diameter of the particles

was found to be 73nm (100% intensity) (Fig. 3.5 a) with a zeta potential −24mV (Fig. 3.5

b). The synthesized AgNPs were well distributed with respect to volume and intensity

indicating well dispersed AgNPs.

Synthesis of AgNPs using the supernatant of Bacillus cereus by Pratik et al.,

(2012) reported particles appear individually as point scatterers moving under Brownian

motion with mean size to be 39 nm under nanoparticle track analyzer . Hengyi et al.,

(2012) reported the slight negative zeta potential -28.94 mV showed the role of reactive

oxygen species on the antibacterial mechanism of hydrophobic AgNPs on E. coli

0157:H7. Stability study of the nanoparticles was reported by Panchaxari et al., (2011)

using zeta meter, zeta potential of the formulated nanoparticles was in the range of -25.16

to -32.71 mV which indicates good dispersion of the particles.

Shital (2011) reported the average diameter of the particles produced from the

Foeniculum vulgare to be 127, 100% intensity and width was found to be 37.25nm,

revealing monodispersity of the particles.

41

Fig. 3.5 (a): Particle Size Distribution of AgNPs by Intensity with Zeta Analyzer

Fig. 3.5: (b): Zeta Potential of AgNPs produced by A.niger

42

Jun et al., (2007) also reported surface zeta potential of AgNPs was measured to

be negative potential of -0.33mV and revealed existence of nitrate and borate ions in

colloidal solution were adsorbed on the surface of AgNPs which results in slight negative

zeta potential.

3.3.5 Fourier Transform Infrared Spectroscopy (FT-IR)

The interaction between protein and AgNPs was analyzed by Fourier Transform-

Infrared Spectroscopy (FT-IR) analysis. The AgNPs produced by A. niger suspension

was centrifuged at 10, 000 rpm/10 min and dried sample analysis was recorded on Perkin

Elmer one FT-IR spectrophotometer in the range 450 to 3000 cm-1

(Fig. 3.6). The

representative spectra in the region of 3000 to 450 cm-1

revealed the presence of different

functional groups like 3290.73-secondary amide (N-H stretch, H-bonded), 2928.01-

alkane (C-H stretching), 2161.23-alkyne (C≡C stretching), 1771.02- anhydride (C=O

stretching),1613.81- alkene (C=C stretching), 1538.60-aromatic (C-C stretching),

1386.45, 1313.48 and 1080.10- primary alcohol (C-O stretching) and 528.55-alkene

(=C-H bending) respectively .

Vandana et al., (2011) reported that proteins present in the extract of fungus R.

stolonifer can bind to the AgNPs through either free amino or carboxyl groups in the

proteins and also reported different functional groups absorbing characteristic frequencies

of FTIR radiation. Jaidev and Narasimha, (2010) reported the stable and well dispersion

of nanoparticles using FT-IR spectrum, which showed three distinct peaks, 3347.85,

1636.17 and 548.38 cm-1

. The peak at 3347.85 cm-1

refers to the stretching vibrations of

primary amines, while the peak at 1636.17 cm-1

refers to carbonyl stretch vibrations in

the amide linkage of proteins and 548.38 cm-1

is the fingerprint.

43

Hemath et al., (2010) reported the possible biomolecules responsible for the

reduction of the Ag+ ions by the cell free filtrate of Penicillium sp, using FT-IR spectrum.

The representative spectra of the particles obtained absorption peaks located at 3843.68

cm-1

(-NH group of amines), 3597.73 cm-1

(-OH group of phenols), 2080.65 cm-1

(aromatic –CH stretching), 1631.66 cm-1

(-NHCO of amide) and 767.16 (C-Cl).

Our findings also support the results of Monali et al., (2009), who reported that

proteins can bind to nanoparticles either through free amine groups or cysteine residues

or through the electrostatic attraction of negatively charged carboxylate groups in

enzymes present in the cell wall of mycelia, and therefore stabilization of the AgNPs by

protein occurs. Thus, FT-IR is an important and popular tool for structural elucidation

and compound identification.

3.3.6 Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy (SEM) was used to record the photomicrograph

images of synthesized AgNPs. A small volume of AgNPs suspension was taken for SEM

analysis on electromicroscope stub. The stubs were placed briefly in a drier and then

coated with gold in an ion sputter. Pictures were taken by random scanning of the stub.

Figure 3.7 (a) shows the distribution of AgNPs produced from A. niger. SEM micrograph

of single nanoparticle reveals the spherical and smooth morphology of the nanoparticle

(Fig. 3.7 b).

Monali et al., (2009) reported the fungus mediated synthesis of AgNPs of having

polydisperse with spherical nature.

44

Fig. 3.6: FT-IR spectra of silver nanoparticles from A. niger

Fig. 3.7 (a): SEM image of AgNPs, (b): Single nanoparticle

45

Jeevan et al., (2012) reported the spherical nanoparticles with average particle

size of 50 nm using extracellular synthesis of AgNPs by supernatant of P. aeruginosa. In

a study by Thangapandiyan and Prema (2012) spherical nanoparticles were observed

using SEM, but it was chemical synthesis.

3.3.7 Electron Dispersive Spectroscopy (EDS)

Energy Dispersive Spectroscopy (EDS) samples were prepared on a copper

substrate by drop coating of AgNPs. The elemental analysis was examined by EDS. The

presence of an optical absorption band at ~3eV reveals the presence of pure metallic

AgNPs along with the C and O signatures that might be from the stabilizing protein (Fig.

3.8).

As our result correlates with Wen-Ru et al., (2010) who reported the optical

absorption peak at 3 keV from commercially available AgNPs, EDS analysis gives the

additional evidence for the reduction of AgNPs; the optical absorption peak at 3 keV

reported by Upendra et al., (2011) is typical for the absorption of metallic AgNPs due to

surface Plasmon resonance, which confirms the presence of nanocrystalline elemental

silver using Psidium guajava. Pratik et al., (2012) also reported band at 3 keV which

reveals that the silver is present in the solution using Bacillus cereus.

Hong et al., (2011) reported the presence of signal of elemental silver optical

absorption band peak at 3 keV, which is typical of the absorption of metallic AgNPs

using probiotic bacteria R. sphaeroides.

46

Fig. 3.8: EDS spectrum of extracellular synthesized silver nanoparticles

47

3.3.8 Atomic Force Microscopy (AFM)

A small volume of biosynthesized AgNPs was subjected to atomic force

microscopic study. AFM (Fig. 3. 9 a) reveals the particles are smooth, spherical in shape

and the topography of the picture shows the particles from three different places seen in

(Fig. 3. 9 b).

Saha et al., (2010) synthesized AgNPs using fungus Bispolaris nodulosa reported

clear spherical shaped but some were hexahedral AgNPs with well distribution without

any aggregation. Sharanabasava et al., (2012) synthesized silver nanoparticles using P.

diversum and reported spherical nanoparticles with 10-50 nm size using AFM analysis.

Our results also correlates with Vandana et al., (2011) who showed the topography of

silver nanoparticles to be spherical in shape, with smooth surface and monodispersed

particles using R. stolonifer.

3.3.9 X-ray Diffraction

XRD (X- ray diffractometer) analysis reveals the crystalline nature of AgNPs.

The diffracted intensities were recorded from 0 to 80˚ (2Ɵ), the intense XRD peaks were

observed corresponding to (111), (200) planes at 38° and 46° of 2Ɵ. Results showed that

the particles have cubic structure. The peak at 32° might be related to Agcl which was

owing to the chloride ions involved during cell free filtrate preparation (Fig. 3.10). The

Bragg’s peak position and their intensities were compared with Standard JCPDS files.

Prema and Rincy, (2009) reported the X-ray diffraction pattern which showed the

presence of sharp reflections at (111), (200), (220) and (311).

48

Fig. 3.9 (a): AFM Micrograph of AgNPs, (b). 3D image of AgNPs

Fig. 3.10: X-ray diffraction of AgNPs synthesized from A. niger

49

Our result correlates with them at facets (111) and (200). While Guangquan et al.,

(2012) also revealed the intense XRD peaks corresponding to the (111). (200), (220),

(311) planes at 2θ angles of 38.28°, 44.38°, 64.54°, and 77.64° respectively. Our result

also corroborates with Faiez et al., (2011) who reported the pure silver facets with main

single peak at 38°, which is in good agreement with the published XRD standard for

silver metal.

Hong et al., (2011) reported crystalline nature of the nanoparticles in the fcc

structural confirmation by peaks at 2θ values of 38.45°, 44.48°, 64.69° and 77.62° in the

XRD pattern corresponding to (111), (200), (220) and (311) planes using R. sphaeroides.

Jyothi et al., (2014) also reported the intense XRD peaks corresponding to (101), (111)

and (200) lanes at 2θ angles of 32°, 38° and 43° which are indexed as crystalline silver

fcc phase.

3.3.10 Determination of Tryptophan/Tyrosine residues

UV-Vis spectra of fungal filtrate showed an emission band centered at 289 nm.

The nature of the emission band indicates that the proteins bound to the nanoparticles

surface and those present in the solution exist in the native form. In our studies the

excitation wavelength band close to maximal optical transitions of the

tyrosine/tryptophan was detected (Fig. 3.11).

Nelson et al., (2005) reported an absorption band at 265 nm and it was attributed

to aromatic amino acids of proteins. The band arises due to electronic excitation in

tryptophan and tyrosine residues in proteins. Release of proteins into solution by F.

oxysporum suggests a possible mechanism of nanoparticles synthesis.

50

Fig. 3.11: Emission spectra of reaction mixture of AgNO3- fungal filtrate of A.niger

51

Jaidev and Narasimha, (2010) also reported the absorption peak at 280 nm attributed to

tyrosine and tryptophan residues of the proteins using A. niger, and revealed the AgNPs

were found to be stable in their native form with no observable changes. Our results

correlate with Jaidev and Narasimha, (2010).