NPTEL – Nanotechnology - Nanobiotechnology

Biogenic nanoparticles
Dr. K. Uma Maheswari

Professor, School of Chemical & Biotechnology

SASTRA University

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Table of Contents
1 WHAT ARE BIOGENIC NANOPARTICLES? ............................................................................ 3
2 SYNTHESIS OF BIOGENIC METAL NANOPARTICLES ......................................................... 4
3 MECHANISM INVOLVED IN THE SYNTHESIS OF BIOGENIC METAL
NANOPARTICLES ......................................................................................................................... 7
4 REFERENCE ..............................................................................................................................10
5 ADDITIONAL READING ............................................................................................................10

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Module objective

This module aims to familiarize the learner with the concepts involved in the synthesis of
nanoparticles by microorganism, plants and higher organisms. An insight in to the
probable mechanisms involved in the synthesis is also discussed.

Preface
Synthesis of nanoparticles using physical and chemical methods has been found to
possess several difficulties in maintaining the size, shape and monodispersivity.
However, several microorganisms have been found to synthesize nearly monodisperse
nanoparticles with great control over size and shape. This module discusses the
significance of such biogenic synthesis and the mechanisms involved.

This lecture aims to introduce the learners to the concept of biogenic nanoparticles and
some of the probable mechanisms involving peptides in the synthesis of these biogenic
nanoparticles.

1 What are biogenic nanoparticles?

Nanoparticles synthesized using biological systems (eg. Microbes, fishes, plants etc.) are
referred to as biogenic nanoparticles. The process of synthesis of the nanostructures by
organisms is also known as biomineralization. As the process involves no harsh
chemicals and solvent systems, this method of nanoparticle synthesis is classed as a
‘Green synthesis’. These nanoparticles possess the advantage of having uniform size and
shape. Moreover, they have also been found to possess better stability owing to
stabilization by proteins and other biomolecules from the organism. The biogenic
nanoparticles may be sequestered in separate intracellular compartments inside the cells
and are referred to as intracellular biogenic particles. Separation of these nanoparticles
requires disruption of the cells. On the other hand, some organisms synthesize these
nanoparticles outside the cell or send them outside post-synthesis. Such nanoparticles are
referred to as extracellular biogenic nanoparticles.

Why do organisms synthesize nanoparticles? One of the major reasons involved in such
synthesis is detoxification. The major route to shield the cell(s) from the toxicity of
certain soluble ionic species such as metal ions is to convert them into insoluble forms
through reduction or precipitation.

In general, four major detoxification strategies have been identified in organisms.

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(a) Modifications in the cellular transport mechanisms to restrict entry of the toxic
ions into the cell
(b) Sequestration of the toxic species within the cell (intracellular sequestration) or
outside the cell (extracellular sequestration)
(c) Activation of energy-dependent efflux pathways to eliminate the toxic species
(d) Enzyme catalyzed oxidation or reduction of the toxic species to a less toxic form

In certain cases, the synthesis of nanoparticles is initiated to meet the cell’s requirements
for a functional component such as iron oxide or silica. Such synthesis occurs via
oxidation and condensation-association routes. Some of these nanostructures have
exquisite and complex morphology that is too difficult to be recreated using chemical
routes of synthesis. Moreover, large-scale production of these biogenic nanoparticles can
be easily accomplished and hence this area has been among the most widely explored
areas in nanoscience and technology.

2 Synthesis of biogenic metal nanoparticles

The synthesis of metal nanoparticles by biological systems is mainly a strategy employed
to protect the system against the toxic effects of the soluble metal ions. Several species of
microbes and plants exhibit tolerance to metal ions through this strategy. Both bacterial
and fungal species have been employed for synthesizing metal nanoparticles. The most
widely synthesized metal nanoparticles are silver and gold due to their widespread
applications in many fields of science and technology. The metal nanoparticles
synthesized using fungal species is referred to as ‘mycogenic nanoparticles’ while those
synthesized using bacterial species is known as ‘bacterioform nanoparticles’. Even
higher plants have been shown to be effective in synthesizing silver and gold
nanoparticles and the amount of reports available in this topic is too large to be
summarized.

Did you know?

The huge number of fungal species that have been used to synthesize different nanoparticles
with excellent stability and size control has led to the genesis of a new field known as
“Myconanotechnology”. The cost-effective large-scale production of metallic,
semiconductor and metal oxide nanoparticles is being developed for various applications in
this rapidly expanding field.

Some of the major organisms that have been employed to synthesize nanoparticles are
listed in the following Table.(Note:This Table is not meant to intimidate the learners with
its length but is given here mainly to highlight the enormous quantity of work that has
been carried out in this area!)

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Table 1: Biological systems used for synthesizing nanoparticles

Name of the biological species Nanoparticle Size (nm)

Acalypha indica Silver 20-30
Actinobacter spp. Magnetite
Aspergillus clavatus Silver 10-25
Aspergillus flavus Silver 8.92±1.61
Aspergillus fumigatus Silver 5-25
Aspergillus niger Silver 20
Apiin extracted from henna leaves Silver & Gold 39; 7.5-65
(respectively)
Avena sativa (oat) Gold 5-20 (pH 3-4) &
25-85 (pH 2)
Azadirachta indica Silver 50
Brassica juncea (mustard) Silver 2-35
Bacillus licheniformis Silver 50
Bacillus licheniformis (culture supernatant) Silver 50
Bacillus megaterium Silver 46.9
Bacillus sp. Silver 5-15
Bacillus subtilis Silver 5-60
Bacillus subtilis (culture supernatant) Silver 5-60
Brevibacterium casei Silver 50
Candida glabrata CdS Not available
Carica Papaya Silver 60-80
Cinnamomum camphora leaf Silver 55-80
Citrus limon (lemon) Silver <50
Cladosporium cladosporioides Silver 10-100
Clostridium thermoaceticum CdS Not available
Cochlospermum gossypium Silver 3
Coriandrum satiyum leaf extract Silver 26
Coriandrum sativum Gold 6.75-57.91
Corynebacterium sp. Silver 10-15
Cymbopogon flexuosus (lemongrass) Gold 200-500
Cycas sp. (cycas) Silver 2-6
Datura metel Silver 16-40
Desmodium triflorum Silver 5-20
Eclipta sp. Silver 2-6
Enhydra fluctuans Silver 100-400
Escherichia coli Silver 1-100
Escherichia coli CdS Not available
Eucalyptus camaldulensis (river red gum) Gold 1.25-17.5
Eucalyptus citriodora (neelagiri) Silver ˜20
Eucalyptus hybrida (safeda) Silver 50-150
Euphorbia hirta Silver 40-50
Ficus bengalensis (marri) Silver ˜20

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Fusarium acuminatum Silver 5-40

Fusarium oxysporum Silver 5-50
Fusarium oxysporum and Verticillium sp. Magnetite Not available
Fusarium semitectum Silver 10-60
Fusarium solani Silver 5-35
Garcinia mangostana (mangosteen) Silver 35
Geobacter sulfurreducens Silver 200
Gliricidia sepium Silver 10-50
Glycine max (soybean) leaf extract Silver 25-100
Honey Silver 4
Ipomoea aquatic Silver 100-400
Jatropha curcas (seed extract) Silver 15-50
Klebsiella pneumonia (culture supernatant) Silver 50
Lactic acid bacteria Silver 11.2
Lactobacillus Strains Silver 500
Ludwigia adscendeous (ludwigia) Silver 100-400
Magnetospirillium magneticum Fe3O4, Fe3S4 Not available
Mentha piperita (peppermint) Silver & Gold 5-30, 90; 150
Moringa oleifera Silver 57
Morganella sp. Silver 20±5
Murraya koenigii Silver & Gold 10; 20
Nelumbo nucifera (lotus) Silver 25-80
Ocimum sanctum (tulsi; root extract) Silver 10
Ocimum sanctum (tulsi; leaf extract) Silver 10-20
Penicillium brevicompactum WA 2315 Silver 23-105
Penicillium fellutanum Silver 1-100
Phanerochaete chrysosporium Silver 100
Phyllanthus amarus Silver 18-38
Proteus mirabilis Silver 10-20
Psidium guajava (guava) Gold 25-30
Pseudomonas stutzeri AG259 Silver 200
Scutellaria barbata D. Don (Barbated Gold 5-30
skullcup)
Schizosaccharomyces pombe CdS Not available
Sesbania drummondii (leguminous) Gold 6-20
Stapphylococcus aurens Silver 1-100
Syzygium aromaticum (clove) Gold 5-100
Syzygium cumini (jambul) Silver 29-92
Terminalia catappa (almond) Gold 10-35
Trichoderma asperellum Silver 13-18
Trichoderma viride Silver 5-40
Verticillium sp. Silver 25±12

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The differences in the reducing agents, stabilizing moieties as well as templating

molecules between different species cause variations in the size, size distribution as well
as crystal type of the nanoparticles.

3 Mechanism involved in the synthesis of biogenic metal

nanoparticles
Though different biogenic sources have been identified for synthesis of nanoparticles, the
mechanism of synthesis is yet to be deciphered clearly. Many theories have been
suggested to explain the mechanism of synthesis, especially for metal nanoparticles. As
biogenic metal nanoparticles have been found to be stable for three months and more, it is
evident that there should be a reducing agent as well as a stabilizing agent in the system
that would facilitate formation and stabilization of the metal nanoparticles. The
identification of these components from the complex cellular environment is extremely
challenging. The nature of the reducing agent and stabilizing agent also varies with the
species employed for the synthesis. In general, peptides, proteins, polyols and
heterocyclic compounds have been suggested to have an important role in the synthesis of
metal nanoparticles through an electron shuttle or charge capping mechanism.

Peptides have been investigated for their role as possible reducing agents as well as
stabilizing agents in many microbial species. Interestingly, several reports have indicated
that a single amino acid might not be as effective as a polypeptide sequence containing
the same amino acid residue for the synthesis. The metal ion is first reduced to form
metal nanoparticle, which acts as a nucleus for further growth of the metal crystal. The
peptide is initially thought to adsorb to the surface of the metal nanoparticle clusters
causing a localized reducing environment that results in the reduction of more metal ions
at the interface between the peptide and the metal nuclei. As a result, nanoparticles with
large size distributions are formed. Figure 1 depicts a cartoon on the formation of a silver
nanocrystal in the presence of peptide.

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Fig. 1: Formation of silver nanocrystal

nanocrystals in the presence of peptide

Formation
ormation of twinned crystals could also occur in this process.. A twinned crystal forms
when two crystals have some similar lattice points in a symmetrical manner. A similar
kind of mechanism has been proposed with proteins in plants and is referred to as the
limited nucleation
‘recognition-reduction-limited nucleation’.
’. The positively charged metal ions interact
electrostatically with negatively
gatively charged residues in the proteins followed by reduction
resulting in nucleation at specific sites. The protein template further directs the growth of
the crystals in a specific orientation
orientation.

Several amino acid residues have been implicated in the reduction and stabilization of
silver and gold nanoparticles. These include arginine, cysteine, lysine, methionine,
tyrosine and tryptophan. Tyrosi
Tyrosine
ne residue has been shown to reduce silver and gold ions
under alkaline conditions. Figure 2 gives the possiblele reaction involved in the formation
of metal nanoparticles in the presence of tyrosine residue.

Fig. 2: Role of tyrosine residue in the reduction of silver ions to form silver nanoparticles

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The phenolic group of tyrosine gets converted into a semi semi-quinone

quinone during this process.
The N-terminus
terminus of the peptide will interact with the metal nanoparticle conferring
stability. Is there any evidence for this theory? Yes! It was found that an
n engineered viral
protein template developed from the capsid of cowpea chlorotic mottle virus (CCMV)
containing tyrosine residue was found to effectively synthesize gold nanoparticles.
Removal of the tyrosine residues resulted in no reduction reaction and hence henc no gold
nanoparticle formation occurred
occurred.This
This observation strongly suggests that the presence of
tyrosine residue is a key factor in the formation of metal nanoparticles. Figure 3 shows a
schematic representation of a viral pro
protein containing tyrosine involved inn the synthesis
of metal nanoparticles.

Fig. 3: Viral capsid involved in the synthesis of gold nanoparticles

Tryptophan residue in peptides also has been shown to play a key role in the formation of
metal nanoparticles. The tryptophan residue forms a transient tryptophyl radical after
donating an electron to the metal ion during the reduction reaction. The highly unstable
radical forms ditryptophan, a fluorescent dimer and kynurenine, another fluorescent
molecule. This scheme of reactions is represented in Figure
Figure4.

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Fig. 4: Role of tryptophan residue in the formation of metal nanoparticles

4 Reference
Encyclopedia of Nanoscience& Nanotechnology, Volume 1. Edited by: H.S. Nalwa,
American Scientific Publishers, 2005

5 Additional Reading
Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by
peptide, bacteria, fungi and plants, Nelson Duran, Priscyla D. Marcata, Marcela Duran,
Alka Yadav, Aniket Gade, Mahendra Rai, Appl. Microbiol. Biotechnol. 90, (2011), 1609-
1609
1624

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