CHAPTER 1: INTRODUCTION

1.1 Introduction
In the recent years nanotechnology has attracted the great attention due to the application in various
field such as energy, medicine, electronic and space industries. Basically, there are two methods for
the synthesis of nanoparticles: top down and bottom up. In top down approach, material is broken
into small pieces slowly and in bottom up approach, molecules and atom are club together to
synthesis nanoparticles. For biological synthesis of nanoparticles, generally bottom up approach is
used [1]. The properties of nanoparticles change drastically due to the small size and high surface
area.
1.1.1 Why to go for green synthesis of metal nanoparticle?
There are many physical and chemical method such as chemical reduction, electrochemical
reduction, photochemical reduction, heat evaporation and so on for the synthesis of nanoparticles. In
many cases, surface passivator reagents are needed to prevent the nanoparticles from agglomeration
and these passivators such as thiourea, thiophenol, etc. are toxic in nature [2]. The use of toxic
chemical and non-polar solvent leads to the inability to use nanoparticles in medical field. Moreover,
in physical and chemical method, monodisperse nanoparticles were made which were not stable.
Also, these methods were found to be quite expensive when compared with the green synthesis of
nanoparticles.

Therefore, the development of nontoxic, clean, biocompatible and eco-friendly method to synthesis
of nanoparticles is needed. Not only environment friendly but biosynthesis of nanoparticles is
considered cost effective, safe and sustainable. Researchers are now focusing on the biosynthesis of
metal nanoparticle using both unicellular and multicellular organisms [3]. Both living and dead
microorganisms are gaining importance by the virtue of facile assembly of nanoparticles. The
inspiration for green chemistry and bio processes comes from nature through yeast, fungi, bacteria
and plant extracts in the synthesis of biocompatible metal and semiconductor nanoparticles.
Integration of green chemistry principles to nanotechnology is one of the key issues in nanoscience
research [4]. Synthesis of nanoparticles through the plant extracts could be beneficial over other
environmentally benign biological processes by eliminating the elaborate process of maintaining
cell cultures.
Jose-Yacaman and co-workers first described the formation of gold and silver nanoparticles by
living plants [5]. The above synthetic procedure by plant extract or biomass exemplifies the
promising application of the green synthesis of metal nanoparticles. Very recently green silver
nanoparticles have been synthesized using various natural products like green tea (Camellia
sinensis), neem (Azadirachta indica) leaf broth, natural rubber, starch, Aloe Vera plant extract,
lemongrass leaves extract, leguminous shrub (Sesbania drummondii), latex of Jatropha curcas etc.

Among microorganisms, prokaryotic bacteria have primarily concerned the most attention. An
important demonstration was reported by Klaus et al. [6] who describe the formation of silver-
based particles at the cell poles of propagating Pseudomonas stutzeri AG259. Sastry and co-
workers [7] have opened the field to the synthesis of metal nanoparticles by eukaryotic organisms
like Verticillium sp. They established that the shift from bacteria to fungi had the added benefit that
processing and handling of the biomass would be much simpler. Some well-known examples of
bio-organisms synthesizing inorganic materials include magneto tactic bacteria (which synthesize
magnetic nanoparticles), S-layer bacteria, etc.

CHAPTER 2: Synthesis of Nanoparticles Using Leaf Extract

2.1 Abstract

The chemical reaction usually involves organic compounds like Flavonoids, Alkaloids, Terepenoids,
Polyphenols etc, reacting with metal ions to create a metal nano-particle. The chemical constituents
of plant extracts in the process act as reducing agents as well as stabilizing agent for the nano-
particle. There are a number of ways of synthesizing nanoparticles, one such way is using
biomolecules from plant extracts to reduce metal ions to nanoparticles in a single step. The process
is quick, easy and can readily be scaled up. The process also has an added advantage of being
environmentally benign, as it majorly involves water soluble plant metabolites. Majorly silver and
gold nanoparticles are processed using these metabolites. There are a number of methods of making
nanoparticles using plant extracts.

2.2 Introduction

Nanotechnology is the term given to those areas of science and engineering where phenomena that take
place at dimensions in the nanometre scale are utilised in the design, characterisation,
production and application of materials, structures, devices and systems. Nano technology is one of the
most rapidly progressing fields of technology and it has opened up numerous new frontiers of research
for us. Its advent into the field targeted drug delivery, therapeutic actions and as bio sensors has captured
the imagination of the scientific community and various methods are being devised to from new
nanoparticles with more specifications, scientists are striving to come up with methods which let us
control the shape, size, specificity and other characteristics of the particles more closely. One of the most
useful and revolutionary technique coming up presently is synthesis of nanoparticles using plant extracts
and their subsequent action. The formation of nano particles using plant extracts has a major edge over
methods in terms of its interaction and effect on the environment; it is completely environmentally
friendly and does not pose any threats even from its waste. The time required for the formation of
particles is also within acceptable limits and with the ease of getting the requisite plants make it one of
the best options available in this field to develop the particles. In this report we will expound various
methods and the uses of manufacturing nanoparticles, which are fast becoming indispensable to us, using
plants.

2.3 Significance of Plant Metabolites and Uses

This report concerns synthesis of metal nanoparticles using plant metabolites. Even though nano
particles can be develop using physiochemical techniques, they are lack of being environmentally
benign causes a lot of problem. Especially when they are intended to use is for the development of
medicines. Environment factors are not only reason biological synthesis is preferred, also because it
can be used to produce large quantities of nanoparticles that are free of contamination and have a
well defined sized and morphology. The use of plant metabolites to reduce the metal ions has been
known for a long time, although the nature of the reducing agents had been unknown for a long
time. Processes for making nanoparticles using plant extracts are readily scalable and may be less
expensive compare with the relatively expensive methods based on microbial processes or whole
plant.

An important significance of plant extracts, in context of synthesizing nanoparticles, is that they act
as both reducing and stabilizing agents. The nature of nanoparticles synthesized depends on the
source of plant extract. This aspect can also be utilized in making nanoparticles of interest. This
happen because different plant source has different concentrations and combinations of organic
reducing agents.

Plants being used to reduce metal ion has been done for a long time, dating back as early 1900s. But
this practice was restricted to the use of whole plant extracts or plant tissues only. Compared to this
the use of plant extracts to synthesize nanoparticles in much simpler. They act as both reducing and
stabilizing agents.

2.4 Use of Plant Extracts in Nano Particle Synthesis

During the process of production of metal nanoparticles, the plant extract is usually mixed with a
solution of metal salt at room temperature. It is quick reaction and usually takes minutes to
complete. Nanoparticles of gold. silver and various other metals have been synthesized in the same
way. Various plants’ extract are used for synthesis of metal nanoparticles. There are:
 Fig 2-1 Synthesis of nanoparticles using leaf extract

Nanoparticle properties and production time depend on various characteristics of Plant extract,
namely:

 its concentration
 the concentration of the metal salt
 the pH
 temperature
 contact time

2.5 Advantages of using Plant extracts

 The production of nano particles using the chemical methods has been raising concern
among the environmentalists as they have an adverse effect on their ecology, hence the use
of plant extracts for the formation of nano particles is being favoured due its
salubrious nature towards the environment. Even in the industry it produces much less toxic
waste.

 The plants supplement both the reducing as well as stabilizing agents for the nanoparticles
which otherwise have to be externally added in other methods.

 The chemical method is being proven less economically beneficial as compared to the plant
method as the maintenance cost is much less and the waste disposal requires less effort
among other factors.
  This method is even better than using the biological method as the maintenance of whole
plant system is much less than a culture of bacteria which needs a myriad of phenomena to
be taken care of.

 Recent studies have shown that the therapeutic effects of plants , from which the
nanoparticles are being derived, can also be imbued upon the particles hence providing
us with perfect vehicles to the therapeutic materials to act upon the site of action as
well as eliminating the need to artificially develop a drug for that particular ailment.

2.6 Silver Nanoparticles

 Leaf extract of Polyalthia longifolia was used synthesize silver nanoparticles (reported by
Prasad and Elumalai, 2011). The average size of the particle hence formed was 58 nm. The
reduction was ascribed to the phenolics, terpenoids, polysaccharides and flavones
compounds present in the extract. Their bacterial activity peaked at 45 μg/mL (Huang etal.,
2007).

 Stable size of 16-40 nm was acheived using geranium (Pelargoniumgraveolens)

leaf extract. Geranoil, a natural monoterpene alcohol found in some plants, along with
silver nitrate produced nanoparticles of range 1-10 nm.

 Sukirtha et al. (2011) synthesized silver nanoparticles using a leaf extract of Melia
azedarach and showed them to be active against the HeLa cervical cancer cell line.

 Methanolic Extracts of Eucalyptus hybrida leaves have been reportedly used to synthesize
silver nanoparticles. Flavonoid and terpenoid compounds present in the extract acted as
stabilizing agents.
 Fig 2-2 Chemical constituents of plant extract (from reference 9)

2.7 Synthesis of silver nanoparticles using Acalypha indica leaf extracts

 Materials: The healthy leaves of A. indica were collected from campus of University of
Madras, India. AgNO3, MTT (methyl thiozolyl diphenyl-tetrazolium bromide) were
purchased from Himedia Lab-oratories Pvt. Ltd., Mumbai, India. The bacterial cultures of E.
coli (MTCC-443) and V. cholerae (MTCC-3904) were obtained from Microbial Type
Culture Collection, Chandigarh, India.

 Method: Preparation of plant extract Aqueous extract of A. indica was prepared using
freshly
collected leaves (10 g). They were surface cleaned with running tap water, followed by
distilled water and boiled with 100 ml of distilled water at 60 ◦C for 5min. This extract was
filtered through nylon mesh, followed by Millipore filter (0.45_m) and used for further
experiments.

 Synthesis of silver nanoparticles: For synthesis of silver nanoparticles, the Erlenmeyer

flask containing 100 ml of AgNO3 (1mM) was reacted with 12 ml of the aqueous extract of
 A. indica. This setup was incubated in dark (to minimize the photoactivation of silver
nitrate), at 37 ◦C under static condition. A control setup was also maintained without A.
Indica extract.

 Characterization of silver nanoparticles: Synthesized silver nanoparticles was confirmed

by sampling the reaction mixture at regular intervals and the absorption maxima wasscanned
by UV– vis spectra, at the wavelength of 200–700nmin Beckman-DU 20
spectrophotometers. Further, the reaction mixture was subjected to centrifugation at
75,000×g for 30 min, resulting pellet was dissolved in deionized water and filtered through
Millipore filter (0.45_m). An aliquot of this filtrate containing silver nanoparticles was used
for SEM, HRTEM, XRD and EDS studies. For electron microscopic studies, 25_l of sample
was sputter coated on copper stub and the images of nanoparticles were studied using SEM
(JEOL, Model JFC-1600) andHRTEM(JEOL-3010). ForXRDstudies, dried nanoparticles
were coated on XRD grid and the spectra was recorded by using PhilipsPW1830 X-ray
generator operated at a voltage of 40 kV and a current of 30mA with Cu K_1 radiation. In
addition, presence of metal was analysed by energy dispertive spectroscopy.

 Minimal inhibitory concentration of silver nanoparticles: Minimal inhibitory

concentration of AgNO3 was determined by M.I.T. assay by using 96-well microtitre plate.
The mean of live cells of E. coli and V. Cholera was recorded using ELISA reader ( Emax
precision microplate reader). The MIC was based on different concentrations, where there
was no increase in OD595 and was zero
Fig 2-3 UV-vis. Spectra of aqueous silver nitrate with A. Indica leaf extract at different time interval
(from reference 11)

 Changes in membrane permeability of bacteria cells: To study the membrane

permeability of bacteria cells, the viable bacteria cultures of E. coli and V. Cholerae in
nutrient broth were treated with synthesized silver nanoparticles. Ten millilitres of log phase
cultures were centrifuged at 6000 rpm for 10 min and the pellet was suspended in sterile
distilled water. Five millilitres of this solution were exposed to 100 ppb of silver
nanoparticles and the conduction was recorded after incubation of 1, 3, 6 and 24 h using a
conductivity meter. The same procedure was adopted in the control experiments i.e. cultures
treated with AgNO3.

 Determination of respiration activity of bacteria cells: Changes in the respiration of log

phase cultures of E. Coli and V. Cholerae in nutrient broth were studied with biological
Oxygen Monitor (YSI-Model-5300, USA). It provides a measure of oxygen consumption by
bacteria cultures. The changes in oxygen uptake among the untreated and silver nanoparticles
treated cultures were recorded.

 EDAX analysis of silver nanoparticles showed characteristics

 Fig 2-4 EDAX analysis of silver nanoparticles (from reference 11)

2.8 Remarks

The use of the plant extract for the synthesis of nanoparticles has proven to be inexpensive, easily
scalable and most importantly environmentally benign. This aspect makes it indispensable for
therapeutic application. This method also ensures flexibility as manipulation regarding size and
other properties is characteristic implication of this procedure. Their antimicrobial properties have
also been studied as discussed above. In vivo application, however, are still underdevelopment.

CHAPTER 3: SYNTHESIS OF NANOPARTICLES BY BACTERIA

3.1 Abstract

Nanoparticles are the spearheads of the rapidly expanding field of nanotechnology. An array of
physical and chemical methods is used for the synthesis of nanoparticles. The development of
impeccable protocols for the synthesis of highly monodisperse nanoparticles of various sizes,
geometries and chemical composition is one of the most challenging obstacles in the field of
nanotechnology. Ultraviolet irradiation, aerosol technologies, lithography, laser ablation, ultrasonic
fields, and photochemical reduction techniques have been used successfully for nanoparticle
synthesis, but they continue to remain expensive and involve use of hazardous chemicals. There is
growing concern to develop eco-friendly and economically viable methods for synthesis of
nanoparticles. Biological systems, masters of ambient condition chemistry offer an environment-
friendly alternative way to produce nanoparticles than the currently used protocols. Microbial
synthesis of nanoparticles is a green chemistry approach that interconnects the fields of
nanotechnology and microbial biotechnology. Biological synthesis of gold, silver, gold-silver alloy,
platinum, palladium, selenium, tellurium, silica, Titania, zirconia, magnetite and uraninite particles
by bacteria, actinomycetes, fungi and yeasts have been reported worldwide. In spite of the stability
of nanoparticles synthesized this way, the method faces several challenging obstacles like low
monodispersity and production rates and higher production costs. Detailed study of cellular,
biochemical and molecular mechanisms that govern the growth of the nanoparticle in biological
systems is required to overcome these obstacles. In this paper, we describe the current status of
nanoparticle production by microorganisms.

3.2 Introduction

Optoelectronic, physiochemical and all other properties of nanoparticles are determined by the
shape, size and monodispersity of the particle. These characteristics depend upon the method of
synthesis of nanoparticles. Physical and Chemical methods used now for production of nanoparticles
though lead to monodisperse nanoparticles, but they are less stable and various toxic chemicals are
used. The use of toxic chemicals and non-polar solvents in synthesis leads to the inability to use
nanoparticles in clinical fields. Therefore, development of clean, non-toxic, biocompatible and eco-
friendly method for synthesis of nanoparticles deserves recognition. Even though biological
synthesis of nanoparticles is considered cost effective, safe, environment-friendly and sustainable, it
has various drawbacks. The culturing of microorganisms is time-consuming and it is difficult to
have fine control over shape, size and crystallinity. The particles are not monodisperse and the rate
of production is slow. These are the various problems which have vexed the biological synthesis of
nanoparticles. But optimization of factors involved like pH, temperature, metal ion concentration,
and the strain of the microbe used has given hope for large scale application of biological synthesis.
Moreover genetically engineered strains which express the reducing agent maximally can be used in
the future which will provide better control over the shape and size of nanoparticles. Interaction
between microbes and metals has been known for long and is used in bioremediation,
biomineralization, bioleaching and biocorrosion but it its use in the synthesis of nanoparticles is a
recent discovery and lot of study is required before it can be put to practical use.

3.3 Nanoparticles synthesis by bacteria

Microorganisms often produce inorganic materials of nano-size either extarcellularly or

intracellularly. Microbial systems are able to detoxify heavy metals by virtue of their ability to
reduce the metal ions or precipitate the soluble toxic ions into insoluble non-toxic metal
nanoparticles. A great deal of study has been carried out on synthesis of nanoparticles by prokaryotic
bacteria since they are the easiest organisms to handle and can be manipulated most easily. Bacteria
are able to form nanoparticles both intracellularly via bioaccumulation and extarcellularly on the cell
wall using its enzymes. Intracellular nanoparticles are of a fixed size with less monodispersity than
extracellular particles. Hence, extracellular production has more commercial applications in various
fields. Since monodispersity is the major factor in usefulness of nanoparticles, biological processes
must be designed in such a way to ensure maximum monodispersity.

To obtain intracellular particles from bacteria requires further processing steps like ultrasound
treatment or reaction with suitable detergents. This property can be exploited for extraction of
precious metals from mine wastes and the metal nanoparticles can also be used as catalysts. When
cell wall reductive enzymes or secreted enzymes are involved in the reduction of metal ions then it is
logical to find the metal nanoparticles outside the cell. The extracellular nanoparticles have wider
applications in the field of optoelectronics, bioimaging and sensor technology than intracellular
particles.
 Fig 3-1 Crystal topologies: triangular, hexagonal and spheroidal Ag-Nps found at cellular binding
sites (from reference 13)

In one of the earliest study in this field, it was found that a silver resistant bacterial strain isolated
from silver mines, Pseudomonas Stutzeri AG259 was able to accumulate silver nanoparticles in its
periplasmic state with the size ranging from 36-45 nanometres along with some silver sulphide. It
was also noted that when these bacteria are placed in concentrated aqueous solution of silver nitrate
larger nanoparticles up to 200 nanometres with defined morphology were formed. Cell growth and
metal incubation conditions may be the reason for the disparity in size. The exact mechanism of
formation of nanoparticles by the species of bacteria is yet to be understood. The ability of
microorganisms to resist the high concentration of toxic metal ions may result from the specific
reaction mechanisms. They may include the efflux system, extracellular precipitation by cell wall
enzyme, alteration of solubility and toxicity changing the oxidation state of metal or absence of
certain transport system. Nanocrystalline silver can be recovered from bacteria by thermally treating
bacteria to yield a carbonaceous nanomaterial. This material is composed of five percent by weight
silver nanoparticles and rest dry biomass and has found application in thin-film coating materials.
Bacillus subtilis was able to reduce gold to form octahedral gold nanoparticles of size between 5-25
nanometres when incubated along with gold chloride solution. These are the examples of generation
of nanoparticles by bacteria in nature settings. Some bacteria are able to produce the nanoparticles in
presence of concentrated metal solution and appropriate incubation setting even when naturally they
do not face such conditions and do not produce any nanoparticles naturally. The exposure of some
Lactobacillus strains (found in buttermilk), like lactobacillus sp. A09, to silver and gold solution lead
to the formation of nanoparticles. It can also be used for the production of gold-silver alloy.
Similarly, dried cell mass of Corynebacterium sp.SH09 was able to produce silver nanoparticles with
silver diamine complex at 60 degree centigrade after 72 hours. It is believed that organic matrix of
the cell provides peptides that contain amino acid moieties that serve as a nucleation point for the
start of nanoparticle formation. Silver precipitating peptides were found to have capable of reducing
aqueous silver face centered cubic structured silver crystals. The exact mechanism of the formation
is not yet known and further research needs to be carried out.

A prokaryotic bacterium Rhodopseudomonas capsulata, was found to deposit gold nanoparticles of

10-20 nanometers at 7 pH and room temperature extarcellularly. As the pH of the solution was
changed various nanoparticles with different sizes and geometries (like triangular and spherical at
4.0 pH) were formed. It was found experimentally that cell free extract of Rhodopseudomonas
Capsulata can also be used for production of gold nanoparticles. SDS-PAGE analysis of the extract
demonstrated the involvement of one or more proteins (14-98 kDa) in the reduction of gold and
capping of gold nanoparticles. Similarly, silver nanoparticles can be produced extarcellularly using
Enterobacter culture supernatant. These bacteria secrete enzymes in their culture solutions which are
able to reduce silver and assist in the formation silver nanoparticles. UV-visible spectroscopy and
Transmission Electron Microscopy of the solution estimates the size of particles between 28-122
nanometers with the average size of 52.5 nanometers.

In addition to gold and silver much attention has been focused on synthesis protocols of
semiconductors (quantum dots) like cadmium sulfide, zinc sulfide and lead sulfide. These
luminescent quantum dots are emerging as a new set of materials with important applications in cell
imaging and biosensing, based on the conjugation between biorecognition molecules and quantum
dots. On conjugation these can be visualized easily because of their luminescence. Clostridium
thermoaceticum was found to deposit CdS nanoparticles on the cell surface as well as in the solution
in presence of cadmium chloride and cysteine hydrochloride. Possibly, cysteine hydrochloride acts
as the source of sulfur. When Klebsiella aerogenes is exposed cadmium ions in the growth medium
it forms cadmium sulfide nanoparticles of 20-200 nanometers deposited on the cell surface.
Escherichia coli when incubated with cadmium chloride and sodium sulphide forms intracellular
cadmium sulfide nanoparticles in wurtzite crystal phase. Experiments show that the growth phase of
the cells affect the formation rate of nanoparticles and is 20 times more I stationary phase than in
late logarithmic phase. Zinc sulphide nanoparticles are formed by the family of Desulfobacteriaceae
in their natural settings by a complex mechanism. This can be used to bring down the zinc
concentration in drinking water to below acceptable levels. Magnetic iron sulphide nanoparticles can
also be produced by sulphite reacting bacteria.
 Fig 3-2 TEM of negatively stained cells of M. gryphiswaldense displaying the magnetosome

chain and isolated magnetosomes (from reference 13)

(a) Enlarged view of the magnetosome chain. The bar denotes 0.1 μm
(b) Isolated magnetosome particles with intact magnetosome membranes. Magnetosome membrane
is indicated by arrows.

Studies have shown that microaerophilic bacteria aquaspirilium magnetotacticum was able to form
single domain magnetite nanoparticles with octahedral geometry. Marine magnetotatic bacterium
MV-1 isolated from sulphide rich phase sediments was also able to form magnetite nanoparticles
(parallelepiped) of dimension 40*40*60 nanometers. A thermophilic fermentative bacterial strain
TOR-39 was also able to form single domain (<12 nanometers) magnetite octahedral nanoparticles
exclusively outside the cell. Another bacteria magnetospirillium magnetotacticum was also able to
form single domain nanoparticles which are subsequently assembled into folded chain and flux
closure assemblies. Their 2-D arrangement is responsible for the head-tail assembly. Magnetization
studies have shaown that the magnetite nanoparticles are not superparamagnetic. All magnetotactic
bacteria producing magnetic particles intercellulary contain another organelle contain magnetosomes
which are comprised iron minerals crystals protected by a membrane vesicle. This membrane is not
likely the structure that holds the crystal at a particular location in the cell as well as serve the
starting point (nucleation point) for nanoparticle formation. Possibly, biological systems exert
control over growth of the crystal using the same magnetosmal membrane. The bacteria producing
the magnetic nanoparticles can be separated from the culture using micro electromagnets. After they
are separated from the culture the can be subjected to lysis to leave the crystal at desired locations.
Magnetite nanoparticles have also been produced by non magnetotactic bacteria Geobacter
metallireducens GS-15 isolated from the sediments of Potomac river. The amorphous ferric oxide
acts as a terminal electron accepter during organic matter oxidation and magnetite crystals are found.
 Fig 3-3 TEM image of flocculated UO2 nanoparticles associated with Desulfosporosinus spp.
Bacteria (from reference 13)

Stenotrophomonas maltophilia SELTE02, a strain isolated from soil near selenium accumulator
legume was capable of transforming selenite to elemental selenium and accumulate it inside the cells
as well as deposit it extracellularly. A facultative anaerobe Enterobacter cloacea SLD1a-1 and
Desulfovibrio desulfricans have also been capable to reduce selenite to selenium. Desulfovibrio
desulfricans NCIMB 8307 was able to generate palladium and platinum nanoparticles.
Chapter 4 : SYNTHESIS OF NANOPARTICLES BY FUNGI, YEAST

4.1 By Fungi

Fungi are a relatively recent addition to the list of microorganisms used for nanoparticle synthesis.
Fungi are more beneficial than other microorganisms in many ways. They grow in the form of
mycelial mesh which helps them to bear flow pressure and agitation and other conditions to which
microbes are subjected to in a bioreactor used for large scale production. Though they require
better precision and care to grow but they are easier to handle and manipulate. They secrete
enzymes in large amounts. The nanoparticles are generally generated extarcellularly (sometimes
intracellular production does occur) they are devoid of various impurities from the cell and can be
used directly.

The use of eukaryotic organisms for nanoparticle synthesis was first demonstrated by use of
Verticillium sp. for the synthesis of gold nanoparticles. In this experiment, gold nanoparticles were
reported on the surface and cytoplasmic membrane of the fungal mycelia. Due to the formation of
gold nanoparticles the mycelial mass attains a typical purple color demonstrating intracellular
generation. TEM analysis shows that particles of well-defined geometry like triangular, hexagonal
or spherical shape were formed on the cell wall and quasi-hexagonal morphology were formed of
the cytoplasmic membrane. The fungal biomass on exposure to silver nitrate solution was also
found generate silver nanoparticles intracellularly. The powder diffraction indicates the crystal
nature of both the nanoparticles. The exact mechanism for the synthesis of nanoparticles by
Verticillium is not yet known. It is thought that the first step is the interaction between positively
charged metal ions and negatively charged carboxylates on the enzymes present in fungal cell wall
and adhesion of the metal ions to the surface as a result of this interaction. The enzymes reduce
these metal ions to elemental metal which serve as nucleation sites and further growth is carried out
by subsequent reduction and accumulation. The ability of Verticillium to grow and replicate even
after exposure to metal ions demonstrate their ability to be used commercially for production of
nanoparticles.
A plant pathogenic fungus, Fusarium oxysporum has also been studied extensively. It was found that
it was able to generate gold and silver nanoparticles extarcellularly rapidly. This was observed from
the fact that the supernatant changed its color but the mycelial mass retained its original color.
Moreover the fungal extract was also able to generate gold and silver nanoparticles. It is believed
that the fungus releases reductases in the solution which are responsible for the reduction of metal
ions. This makes in-vitro generation of nanoparticles using an enzyme/cell extract-based process
possible. It was recently discovered that when F. oxysporum is exposed to equimolar solutions of
hydrogen tetrachloroaurate (III) and silver nitrate led to the production of gold-silver alloy. The
presence of only one plasmon resonance, shifting gradually from gold to silver and back indicates
the formation of homogeneous alloy rather than segregated metal or core/shell type structure. The
fungus on exposure to cadmium sulfate solution was found to yield cadmium sulfide quantum dots
(5-20 nanometers) with hexagonal morphology. Long term incubation of the fungus with cadmium
nitrate does not yield cadmium sulfide nanoparticles which indicate the action of sulfate reducing
enzyme. Polyacrylamide gel electrophoresis of the extract led to four different protein bands. These
proteins were extracted using dialysis and addition of ions to this solution does not yield cadmium
sulfide which attests to the presence of some other factor. Addition of ATP and NADH restored the
capability to produce quantum dots.

4.2 By yeast

Yeasts are most useful in the synthesis of semiconductor nanoparticles like cadmium sulfide, lead
sulfide, antimony oxide, etc. Candida glubrata is the yeast which can intracellularly synthesize
uniform spherically shaped peptide-bound CdS nano-crystals of size about 20 Å. They tend to
form metal–thiolate complex with phytochelatins which neutralize of metal ions
Schizosaccharomyces pombe can also synthesize CdS nano-crystals with hexagonal crystal
structure of particle size 1-1.5 nm. Torulopsis sp. was the first yeast in which synthesis of face
centered cubic structured PbS nano-crystals, showing semiconductor properties, which was
intracellularly produced in the vacuoles having a dimension of 2–5 nm in spherical structure when
incubated with Pb2+ reflects λmax of 330 nm in UV–Vis spectrophotometer. Diode junction can be
formed using these nanoparticles. S. cerevisiae (baker’s yeast) can reduce Au+ 3to give gold
nanoparticles. Reduction happens in the peptidoglycan layer of the cell wall by the aldehyde group
present in reducing sugars. Pichia jadinii is another yeast which can intracellularly synthesize the
gold nanoparticles of different morphologies like spherical, triangular, hexagonal etc. of the size
less than 100 nm in the cytoplasm of the cell within a day. Gold nanoparticles are also synthesized
by the tropical marine yeast Yarrowia lipolytica by reducing the gold ions using pH control
regulations. It synthesizes nanoparticles of various morphologies when subjected to different pH
cultures. At pH 2.0 it produce hexagonal and triangular gold crystals because of the nucleation of
the nanoparticles on the cell surfaces giving rise to golden

color which falls in the visible range spectrum having wavelength 540 nm and at pH 7.0 and pH 9.0
gold nanoparticles gives pink and purple colors with an average size of ∼ 15 nm. S. cerevisiae can
also synthesize face-centered cubic unit cell antimony oxide (Sb2O3) nanoparticles with spherical
morphology of size 2-10 nm at room temperature conditions. This was possibly due to the radial
tautomerization of membrane-bound quinines or by membrane- bound or cytosolic pH-dependent
oxidoreductases. Antimony oxides are an important ingredient for semiconductor industry. MKY3 is
the only yeast so far discovered which is capable of extracellular silver nanoparticles synthesis of
hexagonal crystal structure of size 2-5 nm in log phase growth of the yeast. These silver
nanoparticles are used to make silver tolerant strain.