1

Photo catalytic Degradation of Ampicillin by

Silver Nanoparticles

Dissertation Report

Submitted in fulfillment of the

Requirement for the award of the

Degree of

Bachelors of Technology in Biotechnology

By

Sonal (Regn No - 11200154)

Pritam (Regn No-11202477)

Sumit (Regn No- 11206616)

Johnson (Regn No-11201990)

Under the guidance of

Mr. Prabhjot Singh Jassal

Assistant Professor

SCHOOL OF BIOTECHNOLOGY AND BIOSCIENCES

LOVELY PROFESSIONAL UNIVERSITY, PHAGWARA

(2012-2016)

2

CERTIFICATE

This is to certify that Pritam, Sumit, Johnson and Sonal bearing registration no. 11202477,

11206616, 11201990 and 11200154 respectively have completed dissertation project report,

entitled “Photo catalytic degradation of ampicillin by silver nanoparticles” under my

guidance and supervision. To the best of knowledge, the present work is the result of their

original investigation and study. No part of the report has ever been submitted for any other

degree at any university. The report is fit for submission and partial fulfillment of the conditions

for the award of B.Tech, Biotechnology.

Mr. Prabhjot Singh Jassal,

Assistant Professor,

School of Biosciences and Biotechnology,

Lovely Professional University

Phagwara, (Punjab)

3

DECLARATION

We hereby declare that the project work entitled “Photocatalytic degradation of ampicillin by

silver nanoparticles” is an authentic record of our own work carried out at ‘Lovely

Professional University’as requirement of dissertation work project for the award of degree of

“B.Tech in Biotechnology” at Lovely Professional University, Phagwara under the guidance

of “Mr. Prabhjot Singh Jassal, Assistant Professor, L.P.U” during January, 2016 to April,

2016. We hereby declare that no previously published document has resemblance with our work

to be identified as under the act of plagiarism. All of the writing and project work in this report

is our own. Whenever we have borrowed material from other sources, we have diligently

acknowledged the sources of borrowed material.

Sonal (Regn No - 11200154)

Pritam (Regn No- 11202477)

Sumit (Regn No- 11206616)

Johnson (Regn No- 11201990)

Date: 29-04-2016

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ACKNOWLEDGEMENT

As we begin to write these lines after completion of our thesis, our heart is filled with deepest

sense of gratitude. We shall ever remain thankfully indebted to all those who have encouraged

us to achieve our goal and enlightened us with the tough of their encouragement.

First and foremost we would like to thanks to our beloved Mother, Father, Sister and

Brother for their affection, blessing, emotional support and love which has actually inspired us

throughout our dissertation work. Without them we would have never come to this proliferative

stage and engaged ourselves in career building. Having such a wonderful family who supported

us whole heartily no matter what the situation was. They have listened our every problems

patiently, suggested us the possible solutions. We have shared each and every moment of

excitation after getting positive results and disappointment after not getting success in some

experiments during my dissertation work. We have no words to pay regards to this moral

support.

We consider, it as a blessing to pursue my dissertation under the guidance of

Mr. Prabhjot Singh Jassal, Assistant Professor, L.P.U, Phagwara. We are grateful to our

mentor for being a great support and a true guide throughout our dissertation work. No words

are enough to express our gratitude for his whole hearted encouragement, supervision and

support. He would take a great concern in troubleshooting problems and was always available

to discuss the problems even in his busy schedules. We heartily thanks to him for being a

patient listener to our problems that we have came across during our dissertation work and for

providing us the useful guidance.

We take privilege to express our sincere thanks and gratitude to our incomparable

guide Miss Robinka Khajuria, Assistant Professor of Department of Biosciences and

Biotechnology, L.P.U, Phagwara able guidance, constructive criticism, valuable suggestions

and her longstanding efforts which brought this report in its present form. She has also provided

us insightful discussions about the research. We feel honored to have her as our guide. This

works bears at every stage the impression of her sagacious guidance.

We are heartily obliged and deeply indebted to Dr. Neeta Raj, Head of School of

Biotechnology and Biosciences for her splendid guidance and Dr. Himanshu Singh, Head of

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Department of Biotechnology and Biosciences for providing us a wonderful opportunity that

has brought a revolutionary change in our life. We would like to thank the whole Department of

Biotechnology and Biosciences, Lovely Professional University, Phagwara for accepting and

allowing us to conduct the experiment regarding the dissertation.

At last we would like to thank almighty GOD for supporting us spiritually throughout our

life and providing us everything that we needed. With all these people it would have possible

for us to successfully complete our project.

Sonal (Regn No - 11200154)

Pritam (Regn No- 11202477)

Sumit (Regn No- 11206616)

Johnson (Regn No- 11201990)

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TABLE OF CONTENTS

Contents Page No.

Certificate 2

Declaration 3

Acknowledgement 4-5

Abstract 10

1.Introduction 11

1.1 Pleurotus ostreatus 13

1.2 Instruments for characterization 14-16

2. Review of literature 17

2.1 Need for antibiotic degradation 18-21

2.2 Cases of antibiotic resistance in India 21-24

2.3 Conventional techniques for antibiotic degradation 24-26

2.4 Chemical method for nanoparticle synthesis 26- 34

2.5 Physical method for nanoparticle synthesis 34-39

2.6 Green synthesis of nanoparticles 39-41

3. Methodology 42

3.1Procurement and maintenance of culture 43

3.2 Preparation of submerged culture 43

3.3 Preparation of cell free extract 43

3.4 Nanoparticles synthesis 43

3.5 Techniques used for nanoparticles characterization 44

3.6 Photocatalytic degradation of antibiotic 44

3.7 Effect of nanoparticles on antibiotic degradation 44

3.8 Effect of antibiotic concentration 45

3.9 Effect of contact time 45

3.10 Effect of pH 45-46

7

4. Results 47

4.1 Procurement and maintenance of culture 48

4.2 Synthesis of nanoparticles 48

4.3 Effect of nanoparticles on ampicillin degradation 49-50

4.4 Effect of antibiotic 50-52

4.5 Effect of contact time 52-53

4.5 Effect of pH 53-54

4.6 Techniques used for nanoparticles characterization 54-56

5 References 57-62

8

LIST OF FIGURES

Sr. No. Title Page No.

1. Growth of Pleurotus ostreatus on malt extract media and

slants

48

2. Synthesis of nanoparticles 49

3. UV-Visible spectrophotometer graph 54

4. Fourier transform infrared spectroscopy Graph 55

5. Size of nanoparticles by transmission electron microscope 56

6.

Shape of nanoparticles by transmission electron

microscope

56

9

LIST OF TABLES

Sr. No. TITLE Page No.

1. Percent degradation of ampicillin by varying nanoparticles

concentration

51

2. Percent degradation of ampicillin by varying antibiotic

concentration

52

3. Percent degradation of ampicillin by varying time 53

4. Effect of pH on ampicillin degradation 54

10

ABSTRACT

We report extracellular biosynthesis of silver nanoparticles by Pleurotus ostreatus. Fourier

transforms infrared and ultraviolet–visible (UV–Vis) spectroscopy, X-ray diffraction patterns,

and transmission electron microscopy were used to characterize the composition and structure

of the nanoparticles. The nanoparticles were used as an active photocatalyst for

photodegradation and removal of ampicillin in aqueous solution. UV–Vis spectroscopy studies

showed that silver nanoparticles absorbs visible light irradiation as well as ultraviolet spectrum,

and therefore, it can be photoactivated under visible and ultraviolet lights. The photocatalytic

activity of silver nanoparticles in degradation of ampicillin molecules in aqueous solution under

natural sunlight irradiation was evaluated and compared with that of silver nanoparticles.. The

effect of operating conditions (pH, Ag nanoparticles dosage, and ampicillin concentration) in

the photocatalytic degradation of ampicillin using silver nanoparticles was investigated. The

optimum conditions for maximum efficiency of ampicillin degradation under 180 minutes

sunlight irradiation were found as 5ppm concentration of nanoparticles, ampicillin

concentration of 10ppm, and pH at 6.

11

Chapter 1

Introduction

12

Chapter 1

Introduction

Untreated disposal of unused or unwanted pharmaceutical products (antibiotics in this case) is

an emerging and complex environmental issue. Recently the occurrence of antibiotics in the

environment has highlighted concerns regarding potential threats of antibiotics to humans and

wildlife and that of losing its potential to kill pathogens or to cure diseases. Antibiotics are

commonly used to prevent diseases and promote growth, making livestock agriculture a major

source of antibiotic pollution. The purpose of our study was to examine the degradation of these

antibiotics by various means. The antibiotics found in the nature are due to the untreated

disposal of swine-water, wastewater and other untreated industrial disposal. Ammonia present

in these waters play a crucial role in the decomposition of the antibiotic and bacterial

inactivation, monochloramine is formed due to its rapid competition for free chlorine. Chlorine

dose close to breakpoint is generally required to achieve the complete removal of antibiotics,

which leads to high consumption of free chlorine. This wastewater is not easily treated in the

biological wastewater treatment plants. One reason being, some of these antibiotics are not

easily degradable in the normal treatment system. They inhibit the biological organisms in the

treatment system. Especially tetracycline, sulfathiazole, and ampicillin are representative

antibiotics found in this wastewater. The pharmaceuticals wastes include a wide variety of

items, like over the counter and prescription medications. The wastes are in the form of solid

pills and capsules, creams, liquids and aerosols. Many pharmaceuticals (antibiotics) intended for

pets are similar or closely identical to those which are prescribed to humans.If this problem is

not taken care, then, these antibiotics may become ineffective against the microbes or pathogens

for which these pharmaceutical (antibiotics) are made, these pathogens or microbes may

become resistive to these antibiotics.

13

Pleutrotusostreatus:

Thespecies of Pleurotus are characterized by a white spore print, attached to decurrent gills,

often with an eccentric (off-centre) stipe, or no stipe at all. They are found to grow on woods,

usually on dead standing trees or on fallen logs. Due to the white shell-like appearance of the

fruiting body it’s commonly known as "oyster mushroom.

Ecology: They are shelf-like clusters growing on living trees (primarily hardwoods, but

sometimes on conifers) and dead logs, causing white rots. Found during various seasons

like winter, spring, and summer. With impunity oyster mushrooms can kill nematodes

and bacteria

Appearance: The cap size of these mushrooms varies from 4-15 cm, they are convex

shaped and later may become flat. The can be of various shapes from kidney-shaped to

fan-shaped or maybe nearly circular if they are growing on top of logs. They are greasy

when fresh and young. They are smooth and can be pale brown to dark brown in colour.

The margins are rolled in when young and later on get wavy or maybe never lined.

Gills: The gills run down the stem, they are close whitish or with a grey tinge,

sometimes yellowish in age; often filled with black beetles, in my collecting areas.

Stem: They are usually rudimentary and lateral or maybe absent when they are growing

from the side of a logs or trees. It may develop a substantial and thick stem which is dry

and slightly hairy near the base.

Flesh: The flesh of these mushrooms is thick and is basically white in colour.

14

1.2 Instruments for characterization

XRD (X-Ray powder Diffraction):

It’s a rapid analytical technique which is used primarily for the identification purpose of the

phases of a crystalline material and it can also provide information about unit cell dimensions.

The material analysed is finely grounded, homogenized and then the average bulk composition

is determined.All the diffraction methods work on the basis

of ‘Generation of X-rays” in an X-ray tube. These X-rays are directed towards the sample and

later the diffracted rays are collected. An important factor for all diffractions is the angle

between the incident ray and diffracted rays. Diffraction is of two types Powder and Single

Crystal Diffraction. They vary in instrumentation by great extent. The X-ray powder diffraction

is the more used of the two for the identification of unknown crystalline materials like minerals,

inorganic compounds.

Applications:

For the identification of fine-grained minerals which are difficult to be determined

optically.

For determining the dimensions of unit cells.

For measuring sample purity.

To determine crystal structures using Rietveld refinement.

To determine modal amounts of minerals (quantitative analysis).

Characterization of thin films samples by determining the thickness, roughness and

density of the film.

For crystalline materials characterization.

15

TEM (Transmission Electron Microscopy):

It is a microscopy techniques were through an ultra-thin specimen, a beam of electron is

transmitted and the beam interacts with the specimen as it passes through it. From the

interaction of the electrons transmitted through the specimen, an image is formed. The image is

magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of

photographic film, so as to be detected by a sensor such as a CCD camera. Due to the small de

Broglie wavelength of electrons, TEMs are capable of imaging at a significantly

higher resolution than light microscopes. This allows the user to examine fine detail even as

small as a single column of atoms. It is thousand times smaller than the smallest resolvable

object in a light microscope.

Applications:

Image morphology of samples, e.g. view sections of material, fine powders suspended

on a thin film, small whole organisms such as viruses or bacteria, and frozen solutions.

Collects a series of images to construct a 3-dimensional image by tilting the

sample.

Helps to analyse the composition and bonding differences.

Physically manipulate samples to measure mechanical properties.

Helps to generate characteristic X-rays from samples for microanalysis.

Helps to know the electron diffraction patterns.

Helps in determining the sample composition or the bonding states of atoms

in the sample.

16

FTIR (Fourier Transformation Infrared Spectroscopy):

It is a highly diverse molecular spectroscopy technique and is also used for chemical analysis. It

provides qualitative and quantitative analysis for various organic and inorganic samples. It is

used to obtain an infrared spectrum of absorption or emission of any phase. Here the (IR)

Infrared Radiation is passed through the sample, some of these radiations are absorbed by the

sample and some are transmitted. Later, over a wide spectral range the spectrometer collects

high spectral resolution data. This technique is used for testing polymers and also used for

pharmaceutical analysis of their products.

Applications:

Spectra can be measured in situation where less energy.

Helps in microscopy and imaging.

Examining homogeneity of pharmaceutical products.

17

Chapter 2

Review of Literature

18

Chapter 2

Review of Literature

2.1 Need for antibiotic degradation

Antibiotics now not are confined to their standard use in drugs. they're employed in

recombinant DNA technology, analysis experiments, animal breeding, crop production, fish

farming and cultivation (Dietze et, 2005 ; Prescott et al, 1999; Yanong, 2006).Pharmaceutical

industries concerned within the production of antibiotics discharge their waste overtly that

contains some amount of the active compounds. Antibiotics administered to humans and

animals don't seem to be 100 percent metabolized by the body (Rang et al., 2003). Some active

amount is excreted once body metabolism and will notice their thanks to municipal waste matter

treatment plants from the excretions. Leaky town sewer systems will so give avenues for the

migration of effluents containing antibiotics to underlying aquifers. Pharmaceutical medicine

detected in a very highschool tank effluent was additionally detected within the underlying sand

and gravel formation. Antibiotics are probably to be free into the aquatic atmosphere via waste

material effluent as a result of ineffective treatment, or improper disposal. sulfa drug, sulpha,

antibacterial drug, antibiotic drug, Ethril and trimethoprim are detected in many waste material

treatment facilities discharging their treated effluents to each surface and ground waters. This

wide unfold and sometimes rampant and unregulated use of antibiotics and improper disposal of

waste accumulating from has become a significant challenge.

Exposure of organisms to the antibiotics within the setting, at inappropriate concentrations,

results in build from ensuant resistance to the antibiotics. Increase within the variety of

antibiotic resistant bacterium strain within the setting could occur and multiple antibiotic

resistant bacteria could also be generated as results of selective adaptation and transfer of

antibiotic resistance-encoding cellular inclusion (Rhodes et al., 2000;Aminov et al. 2001).

Environmental concentrations of antibiotics could also be below acutely unhealthful levels.

However, the chronic and/or synergistic result of the range of antibiotics on humans within the

setting is of nice concern. Some microorganisms within the aquatic environments could have

developed the power to breakdown antibiotics discharged to the aquatic environments, as they

need finished alternative substances like oil merchandise (Leahy and Colwel, 1990).

19

Antibiotics are used since the Forties and have light-emitting diode to a dramatic reduction in

health problem and death from infectious diseases. However in step with the federal Interagency

Task Force on Antimicrobial Resistance, “the in depth use of antimicrobial medicine has

resulted in drug resistance that threatens to reverse the medical advances of the last seventy

years. Since antibiotics are used thus wide and for therefore long, antibiotic resistance has

become a significant public health threat.

Soil, water and food will be contaminated by antibiotic resistant microorganisms as a result of

excessive and/or inappropriate use of antibiotics in placental mammal production. the issues

associated with antibiotic use in placental mammal production are recognized for many years,

however additional recently, in 1997 the globe Health Organization did a serious study and

printed recommendations for countries to implement so as to guard public health and therefore

the surroundings.

A new interactive map has been free on twenty seventh of September by the middle for

unwellness Dynamics, Economics, and Policy (CDDEP), a Washington primarily based

analysis work, show resistance trends by country and a policy document that tracks the

utilization of antibiotics and offers steps for curb their overuse. The information shows that in

low and middle financial gain countries particularly China, India, Brazil and South Africa there

is an frightening antibiotic resistance and therefore the maps demonstrate that human and

animal use of the medication is rising dramatically, and therefore the group's analysis suggests

that efforts to battle resistance ought to focus totally on conserving antibiotics already in use—

not on developing new ones. The newest version shows drug resistance trends in thirty-nine

countries and antibiotic use in sixty nine nations. They track infections caused by twelve

common and generally deadly microorganisms, together with Escherichia coli, Salmonella, and

methicillin-resistant cocciaureus (MRSA). Evidence from round the world indicates associate

degree overall decline within the total stock of antibiotic effectiveness: resistance to all or any

first-line and expedient antibiotics is rising.

20

Some of the trends and patterns of antibiotic consumption altogether over the globe area

unit

Human consumption

Demand for antibiotics continues to rise, notably totreat youngsters with probably fatal infection

and pneumonia: in 2013, respiratory illness was accountable for associate degree

estimated935,000 deaths in youngsters beneath 5 worldwide (Liu et al., 2015). Between 2000

and 2010, total international antibiotic consumption grew by quite thirty %, from close to fifty

billion to seventy billion normal units, supported information from seventy one

countries,including most high population countries (Van Boeckel et a.,2014.).

Resistant microorganism in food animals and also the atmosphere

21

Poultry, cattle, and artiodactyl raised with antibiotics harbor important populations of antibiotic-

resistant microorganism, that square measure transmitted to humans through direct contact with

the animals and thru their meat, eggs, and milk (Marshall and Levy 2011).

Agricultural consumption

Antibiotics are used not solely to treat individual animals with microorganism infections and

stop infections in herds or flocks, however additionally to market growth—a debatable and

high-use application. Worldwide, in 2010, a minimum of sixty three,200 plenty of antibiotics

were consumed by eutherian mammal, probably to be over all human consumption (Van

Boeckel et al2015.). By 2030, this figure is projected to rise by simple fraction, to 105,600 tons,

to fulfill the stress of a projected eight.5 billion human population (United Nations).

2. 2 Cases of antibiotic resistance in india

New metropolis metallo-beta-lactamase one

New Delhi metallo-beta-lactamase one (NDM-1) may be a genetic component with multiple

resistance genes that may be harbored by and transmitted between gram-negative

Bacterium, originally known in an exceedingly Swedish patient arrived from capital of India, in

2008. NDM-1 is a suitable proof against most antibiotics except polymyxins (Moellering,

22

2010). E. coli and enteric bacteria spp. carrying NDM-1 currently account for the bulk of

carbapenem resistance in some countries (Pillai et al. 2011). From their original detection in

2008, NDM-1– carrying Enterobacteriaceae are known in additional than seventy countries all

told regions (Johnson and Woodford, 2013) at the start, abundant of the world unfold was

attributed to travelers exposed through medical treatment or hospital stays within the Indian

landmass and probably the Balkans, but now, NDM-1–carrying organisms square measure

being more and more detected worldwide in cases unrelated to travel, suggesting native

transmission.

NDM-1 has conjointly been known in environmental samples from water sources in India and

Vietnam, indicating that the cistron is gift in each community and hospital settings (Johnson and

Woodford, 2013).

Current methods for swiftness resistance and maintaining the effectiveness of current

drug

Global Action arrange on Antimicrobial Resistance (GARP) has worked with eight countries to

determine the capability and strategies for developing antibiotic resistance policies. Six methods

can contribute to swiftness resistance and maintaining the effectiveness of current medicine.

23

cut back the requirement for antibiotics through improved water, sanitation, and

immunisation.improving coverage for existing vaccines and adding new ones, rising

access to wash water and sewerage systems, and guaranteeing a secure and healthful

food provide all cut back the requirement for antibiotics, thereby reducing antibiotic

resistance rates.

Improve hospital infection management and antibiotic situation, better hygiene, notably

hand laundry with soap or victimization alcohol disinfectant between patients, and

antibiotic situation programs cut back infection rates. Police investigation of resistance

and hospital non-heritable infections provides directors data for management and policy

selections.

Amendment incentives that encourage antibiotic overuse and misuse to incentives that

encourage antibiotic situation.Eliminating economic incentives that encourage the

overuse of antibiotics right along the availability chain—in hospitals, in communities,

and in agriculture—can conserve antibiotic effectiveness.

Cut back and eventually terminate antibiotic use in agriculture.Eliminating antibiotic use

for growth promotion and minimizing use for malady bar needn't jeopardize animal or

human health.

Educate and inform health professionals, policymakers, and therefore the public on

property antibiotic use.Education and tips for care professionals, engagement with

policymakers, and national awareness campaigns for the general public can begin ever-

changing the norms in antibiotic use and promote conservation.

24

Guarantee political commitment to satisfy the threat of antibiotic resistance.Presenting

the case to policymakers and gaining their political and support ar essential to success.

2.3 Conventional technique for antibiotic degradation

In this study, photo-Fenton reaction was applied to degradation of sulfamonomethoxine metallic

element (SMMS) in solution. The operation parameters of pH scale, temperature, and

concentrations of H2O2, Fe2+and SMMS were investigated. The optimum conditions for the

photo-Fenton method were determined as follows: [SMMS]=4.53 mg/L, pH 4.0, [H2O2]=0.49

mmol/L, [Fe2+]= 19.51 mol/L and T=25°C underneath these conditions ninety eight.5% of

the SMMS degraded. The mechanics was conjointly studied, and degradation of SMMS by the

photo-Fenton method can be delineated by first-order mechanics. The apparent energy of

activation was calculated as twenty three.95 kJ/mol. Mineralization of the method was

investigated by menstruation the chemical atomic number 8 demand (COD), and therefore the

COD shriveled by ninety nine once one hundred twenty min. This method can be used as a

pretreatment technique for waste containing sulfamonomethoxine metallic element.

All experiments were dispensed in a very two hundred millilitre double glass cylindrical jacket

reactor that allowed athletics of water to take care of the temperature of the reaction system. The

temperature (20–40) ±0.5°C was adjusted by a thermostat and a magnetic stirrer was

accustomed combine the reaction solutions at two hundred r/min. to begin every check, 100mL

of SMMS resolution was placed within the double glass cylindrical jacket reactor. The pH scale

of every reaction resolution was adjusted to the required level exploitation one.0 mol/L vitriol

or one.0 mol/L hydrated oxide. the desired amounts of Fe2+and H2O2 were then additional to the

reactor and therefore the resolution was stirred magnetically for the latent period within the

photo-Fenton method, a twelve W ultraviolet lamp (ZF-2, Shanghai AnTing lepton Instrument

plant, China) with associate degree irradiation wavelength of 365 nm was turned on once the

H2O2 was additional. The latent period was recorded from once the H2O2was additional to the

answer. Samples were sporadically off from the reactor employing a measuring device. All

samples were filtered through 0.45 micron paper before analysis.

25

River water assortment and analysis

River water was obtained from the new Calabar stream placed regarding one klick from the

University of Port Harcourt Teaching Hospital. Concentration of elect ions within the stream

water was determined. Microorganism enumeration of the stream water was meted out from

exploitation medium. Totally different microorganism isolates were characterised and known

supported colonial morphology was represented by (Cheesbrough, 2006). Pure colonies of those

were developed and preserved. Sabouraud’s grape sugar agar was employed in the isolation of

fungi. Pure colonies of the various flora isolates, known supported macroscopical characteristic

were developed.

Determination of the concentration of the antibiotics to be used

The microorganism isolates were subjected to antibiotic and Erythrocin sensitivity testing

mistreatment the disc methodology. The minimum repressive concentration (MIC) of those

antibiotics makes up my mind for his or her most sensitive isolates. A modification of the broth

dilution methodology as delineated by (Andrews, 2001) was used for the MIC determination.

The broth of every check bacteria was ready by transferring 3 colonies of bacteria to a hundred

cubic centimetre of nutrient broth then incubated for 6 hours. Afterwards, nine cubic centimetre

of the culture was transferred to 10 sterile check tubes the foremost sensitive isolate to antibiotic

and Erythrocin were used because the check bacteria for antibiotic and Erythrocin MIC

determination severally.

An antibiotic resolution of one mg.ml-1 was ready then ten cubic centimetre of this resolution

transferred to ninety cubic centimetre H2O to get an answer of a hundred μg.ml-1. One cubic

centimetre of the a hundred μg/ml resolution was then transferred to nine cubic centimetre

culture of the check bacteria to get a ten μg.ml-1 antibiotic resolution, 0.9 cubic centimetre

resolution + zero.1 cubic centimetre sterile H2O to nine cubic centimetre culture to get nine

μg.ml-1 resolution, 0.8 cubic centimetre resolution and 0.2 cubic centimetre sterile H2O to nine

cubic centimetre culture to get eight μg.ml-1 resolution, and lower dilutions. An Erythrocin

resolution of fifteen μg.ml-1was ready then ten cubic centimetre of this resolution transferred to

ninety cubic centimetre H2O to get an answer of 1500 μg.ml-1.Transfers were created kind of

like that of antibiotic to get the varied Erythrocin concentrations . A tube of the broth culture of

every check bacteria containing no antibiotic was enclosed as growth management. Stock

26

solutions of the antibiotics were ready to achieve the MIC of the antibiotics once supplemental

to the biodegradation media. The stock solutions were sterilized with the help of a membrane

filtration unit, and hold on at 4°C.

2.4 Chemical synthesis of nanoparticles

Chemical reduction

Silver nanoparticles square measure enticing inorganic material; its heap of applications in

photography, catalysis, biosensor, biomolecular detection, diagnostic and antimicrobial

activities. Variety of ways has been used for the synthesis of silver nanoparticles like reduction

in resolution, radiation motor-assisted, chemical and photoreduction in reverse micelles, thermal

decomposition of silver compounds or inexperienced synthesis route (Javed IjazHussain et al,

20011).

These silver nanoparticles are used as a medicine agent to cut back infection furthermore as stop

microorganism growth in binary compound and solid media. Silver nanoparticles are wont to

eliminate microorganisms on textile materials and for water treatment. The antimicrobial

activity depends upon the scale of nano particle. This man of science forever centered on its size

whereas synthesis as a result of little size has nice antimicrobial activity (Maribel G. Gujman et

al, 2008).

Solution of polyvinyl pyrrolidone (PVP) and nitrate was reduced by the aldohexose, and silver

nano-particles were generated to boost reaction speed hydrated oxide was used. The molar

magnitude relation of NaOH to AgNO3 was ranged from one.4 to 1.6 throughout this mixture

unbroken stable and no Ag+ was derived. The nanoparticles were analyzed by X-ray diffraction,

transmission microscope and UV-visible photometer (H wang et al, 2005).

Microemulsion technique

Using small emulsion technique, uniform and size governable NPs may be synthesized

(S.Iravani, 2014). Micro emulsions area unit identical, microscopically same and

thermodynamically stable solutions containing a minimum of 3 elements, specifically a polar

part (usually water), a nonionic part (usually oil) and chemical agent.

27

Preparation of nanoparticles of metal salts: Silver salt, metal sulfate, metal carbonate area unit

metal salts that possess distinctive properties. Synthesis of silver salt nanoparticles (AgBr) was

done by reacting Aerosol OT n-hexane and and solution of eitherAgNO3 or metal halides. Size

of spherical AgBr particles varies from five to 10nm as there was a rise in chain length of

aliphatic compound from n-hexane to n-octane. AgBr particles that were shaped during this

small emulsion technique were studied by UV-VIS spectrophotometry, stopped-flow

spectrophotometry and transmission microscopy (chew et al, 1990).

BaCo3, CaCo3, and SrCO3 area unit metal carbonate nanoparticles synthesized by effervescent

greenhouse gas through the reverse particle resolution containing the corresponding liquid

metal hydroxides. Victimization reverse particle technique AgCl and AgBr nanoparticles were

synthesized (Bagwe and khilar, a,b; Monnoyer et al., 1997).

Particles from fifty to two hundred Å in diameter of bromide were ready by compounding 2

small emulsions containing the precursor salts AgNO3 and KBr. Aerosol OT, n-heptane and

water area unit elements of small emulsions. The particle diameters area unit measured on

photomicrographs obtained by transmission microscopy. The scale of the water cores is usually

smaller than that of the particles. The influence of each theconcentration of precursor salts

within the water cores of the small emulsion and also the size of those water cores on the scale

of the particles has been studied. (PhMonnoyer et al, 1995).

UV initiated photo reduction

To funtionalize MWNTs (Multi well Carbon nanotubes) by use of polyacrylic acid (PAA) with

high density and uniformity a non-oxidative approach was adopted throughout the synthesis of

silver nanoparticles they were anchored into the MWNTs by irradiating the mixture of the PAA

functionalized MWNTs and caustic with 2 ultraviolet lamps. Exploitation Fourier rework

infrared prism spectroscope (FT-IR) qualitative analysis, transmission microscope (TEM), X-

ray powder optical phenomenon (XRD), and X-ray negatron qualitative analysis (XPS) obtained

MWNTs/Ag composites were analyzed. Conformation was done through Associate in nursing

choration of silver nanoparticles with diameters in a very region of 5–10 nm on the surface of

MWNTs by an interaction of silver and gas within the chemical group (Y Lei et al, 2010).

28

Photo induced reduction

For the synthesis of silver nanoparticles ion evoked reduction is one of the popular technique, it

merges the photolysis and chemical reduction of light-sensitive silver salts. Exploitation hardly

soluble silver salt like AgBr is incredibly advantageous as a result of they'll maintain a set

nevertheless around low concentration of silver ions in answer. Chemical agent utilized in this

technique was L- vitamin C. in an exceedingly molar quantitative relation of 2:1 bromide was

reduced with vitamin C. In water answer of photographic gelatin-a natural protecting mixture

close at hand non structural silver formation, silver nanoparticles were ready.

For the synthesis of silver nanoparticles ultrafine crystalline gelatin- stabilised liquid

suspensions of bromide were used as a substrate. By the utilization of latest changed technique

of preparation Lippmann’s suspension bromide crystals suspension was obtained.

The time of exposure to the economical production of the noble metal NPs, the influences of the

reducer to substrate molar quantitative relation, the kind of the supply of non particulate

radiation and therefore the medium’s pH scale were studied. Photo- evoked reduction of

bromide nanocrystals reaction was there in presence of vitamin C underneath given

physiochemical conditions. UV-Vis spectrometry and Dynamic light-weight scattering were

used for examining the properties of resultant silver particles. Transmission microscopy was

used for imaging the silver nanoparticles suspensions (A Dyonizy et al, 2011).

Irradiation

Silver nanoparticles have potential application in varied fields like aerophilic chemical action,

surface en-hanced Raman scattering, nanoelectronics (single-electrontransistors, electrical

connects) (Sato et al.), semiconducting coatings, biomedicine activity etc. ethane have attracted

respectable attention. Even by victimization natural compound synthesis of single-wall carbon

nanotubes is additionally doable.

For reduction of silver ions Gamma-radiolysis route was used as a result of it's terribly

advantageous (i) controlled reduction of metal ions may be performed while not victimization

Associate in Nursing way over a chemical agent or manufacturing any unwanted oxidization

29

merchandise from the chemical agent, (ii) chemical agent is generated uniformly within the

resolution, (iii) the strategy provides metal nanoparticles in totally reduced, pure and extremely

stable state, (iv) no intrusive impurities like metal oxides square measure introduced, (v) the

strategy is extremely reproducible and (vi) synthesis may be administrated at close

conditions.In water nitrate and gum Arabic were ready at four completely different

concentrations. 2ml of gum resolution and .2ml of liquid AgNo3 resolution was mixed and

within the ensuing mixture zero.25ml of 2-propanol was intercalary.

To remove gas the complete resolution was then deaerated by effervescent for 15min with pure

gas gas irradiated with vi0Co gamma at a dose 6 kGy/h for various amount. To avoid

oxidisation because of oxidizing species, namely, radical made in lysis of water 2- alcohol was

intercalary in every sample ( Y.N.Rao et al, 2010).

Microwave assisted synthesis

One of the new promising strategies that have gained sizeable attention for the one-pot synthesis

of aluminous nanostructures in solutions is microwave fast heating. This methodology has

numerous blessings for the preparation of silver, Au, Pt, and AuPd nanostructures. Not solely

spherical nanoparticles, however conjointly single crystalline plane figure plates, sheets, rods,

wires, tubes, and dendrites were ready inside a couple of minutes beneath MW heating.

By reduction of Au salts in numerous solvents beneath oil-bath heating for several hours Au

nanoparticles are synthesized. within the presence of PVP beneath MW heating (480–1100 W),

HAuCl4 was reduced in methyl alcohol, ethanol, or DMF for zero.5–5 min to synthesize mono

distributed, tiny spherical nanoparticles, with diameters below eleven nm inside a couple of

minutes. Recently by employing a closed chamber of associate degree MW system with precise

temperature operation spherical Au particles were ready. In associate degree liquid substance on

containing change state as a stabilizer HAuCl4 was reduced for 15-30 min, size ofAu

nanoparticles was reduced from eighty five to 13nm with increasing reaction temperature,

heating time, and rate of temperature increase.

30

Single crystalline plane figure plates, sheets, rods, wires, tubes, and dendrites were conjointly

ready in conjunction with the spherical nanoparticles inside a couple of minutes beneath MW

heating. By ever-changing numerous experimental parameters, like the concentration of

aluminous salt and surface-active agent compound, the chain length of the surface-active agent

compound, the solvent, and therefore the reaction temperature morphologies and sizes of

nanostructures may well be controlled. In general, nanostructures with smaller sizes, narrower

size distributions, and the next degree of crystallization were obtained beneath MW heating than

those in standard oil-bath heating ( M. Tsuji et al, 2005).

Electrochemical synthetic method

Electrochemical technique for the synthesis of nanoparticles were delineate 1st by Reetz and

Helbig thoroughly that involves a metal sheet, was anodically dissolved and therefore the

intermediate metal salt fashioned was reduced at the cathode giving rise to metallic particles

stabilized tetraalkylamonium salts For the chemical science synthesis of silver nanoparticles in

acetonitrile containing tetrabutyl- ammonia salts this work was with success adopted.

The advantage of this methodology lies within the high purity of particles and while not a desire

for dear instrumentation and vacuum nanoparticles size will be controlled by adjusting a current

density.

It has bound limitations like throughout the chemical science method throughout the chemical

science method there was a deposition of silver on the cathode that diminishes the effective

surface on the market for particle production. The particle productions return to halt along once

the complete cathode surface gets lined with the silver electrodeposits.

By the utilization of a cheap 2 conductor setup silver nanoparticles will be obtained during

which anode and cathode is created in bulk. In atomic number 47 mixture particles atomic

number 47 metals was remodeled. As an anode and cathode being vertically placed face-face

ten metric linear units apart 2 polished silver plates were used because the anode and cathode,

being vertically placed face-face ten metric linear units apart.

31

In Associate in nursing chemical science cell stuffed with five hundred millilitre H2O,

electrodes were immersed. At a temperature vary of 20-95 ºC and at a relentless voltage of 20V

electrolysis was performed beneath close conditions in glass containers made silver

nanoparticles were kept. By use of transmission microscopy (TEM), atomic force research and

dynamic lightweight scattering measurements, morphology of obtained silver nanoparticles

were studied. (R.Khaydarov et.al, 2009).

Thermal decomposition

To produce metal nanostructures thermal decomposition of metal complexes is one in every of

the potential ways in which. For the synthesis of nanoparticles the reaction is used if the

decomposition temperature of metal complexes is low and products is metal, silver salt

(Ag2C2O4) was rotten at around 140°C and yields bimetal silver and CO2. During this noble

metal nanoparticles was synthesized by decomposition of(Ag2C2O4) in water and ethanediol

media has been explored.

Silver salt was ready by admixture fifty millilitre of zero.5 M AgNO3 resolution with thirty

millilitre of zero.5 M acid. The white precipitate shaped was filtered, washed with water, dried

at 60°C and keep in a very dark bottle. Formation of (Ag2C2O4) was confirmed by TGA. To

forty millilitre of water, needed quantity (for totally different ratios) of polyvinyl alcohol (PVA)

(M.W = 125,000) was superimposed and stirred. When the entire dissolution of vinyl polymer,

0.05 g of Ag2C2O4 was superimposed, stirred for ten min and purged with N2. This mixture was

refluxed, in a very flow of N2 gas, at 100°C for three h in AN oil bathtub. The formation of

yellow color mixture was discovered within the reaction mixture. The N2 gas from the outlet

was more responsible a tenth Ba resolution to verify the evolution of any CO2 throughout the

formation of the nanoparticles. Then it had been cooled to temperature beneath N2 atmosphere.

The resultant resolution was centrifuged for five min at 1000 revolutions per minute to separate

the yellow noble metal nano powder. Experiments were dispensed with 1:1, 1:2, 1:5 and 1:10

weight ratios of Ag2C2O4 and vinyl polymer. a similar procedure was used in ethanediol

medium with 1:5 weight ratios of Ag2C2O4 and vinyl polymer.

32

The silver mixture was characterised by UV–visible spectrometry, Transmission microscopy

(TEM), SAED patterns and EDAX spectrum. The medicine activity of the noble metal

nanoparticles was conjointly studied (S. Navaladian et al, 2007).

Sol gel method

The effective method to provide Ag-TiO2 nanoparticles is that the sol-gel technique that is wide

used as a result of it's some blessings like energy potency; low process price, high production

rate, and speedy productivity of fine uniform powder (S. Ramesh, 2013).

Preparation of magnetic nanoparticles: By victimisation sol-gel technique magnetic (Fe3O4)

nanoparticles are with success synthesized combined with hardening underneath vacuum

victimisation cheap, nontoxic metallic element nitrate and ethanediol as beginning materials. At

a comparatively longer temperature ranges of a minimum of 200–400 ºC. Magnetic

nanoparticles are often obtained.

In one hundred cc ethanediol 0.2 gram molecule metallic element nitrates was foremost

dissolved with vigorous stirring a pair for two hour at forty ºC. to get brown gel the sol was

heated to eighty ºC and unbroken at the temperature for two h the gel was aged and for

concerning four h it had been dried at a hundred and twenty ºC. At a temperature vary 200–400

ºC xerosols was tempered underneath vacuum when drying. Finally there was a synthesis of

various size magnetic iron-ore nanoparticles.

X-ray optical phenomenon, emission scanning microscopy, energy-dispersive X-ray prism

spectroscope and moving sample gaussmeter (VSM) were used for the characterization of the

section structures, morphologies, particle sizes, chemical composition, and magnetic properties

of Fe3O4 nanoparticles. Within the result it had been confirmed that as there's a rise in

synthesized temperature size, the corresponding saturation magnetization price and coercivity

price of Fe3O4 nanoparticles conjointly will increase (Jing Xu et al, 2007).

Precipitation method

Zinc nanoparticles have varied applications in numerous areas as catalyst, biosensor, electron

devices, extremely purposeful and effective devices. ZnO could be a band gap semiconductor

33

that is significantly used for its chemical change, electrical, optoelectronic and chemical science

properties. Varied strategies area unit used for the assembly of ZnOnanoparticles like optical

device ablation, hydrothermal strategies, chemical science depositions, sol-gel methodology,

thermal decomposition, combustion methodology etc.

Easy precipitation methodology has been represented to synthesize ZnO nanoparticles by

victimization zinc sulfate and hydrated oxide as a beginning material. Hydrated oxide was

accessorial slowly drop wise solution of zinc sulfate in an exceedingly molar magnitude relation

of 1:2 underneath vigorous stirring.

Stirring ought to be continuing for 12h and resultant precipitate is filtered and washed with

deionized water. it had been dried in associate kitchen appliance at a hundred degree Celsius

and grounded to fine powder victimization chalcedony mortar. Then the powder was calcined at

completely different temperatures like 300ºC, 500 ºC, 700 ºC, 900 ºC for two hour.

Then the synthesized nanoparticles area unit characterised by completely different techniques

like X- ray optical phenomenon, scanning microscope, UV-vis diffuse reflection factor

spectroscopic analysis, nucleon induced X-ray emission (S S Kumar et al., 2013).

Hydrothermal method

It is terribly straightforward to manage particle size in one in every of the promising different

artificial methodology i.e hydrothermal technique. Use of easy instrumentation, catalyst-free

growth, low cost, massive space uniform production, environmental friendliness and fewer

unsafe square measure the benefits of this method. For electronics and plastic physics this

methodology is a gorgeous one due to low reaction temperature. This methodology is incredibly

helpful for the preparation of nano-scale ZnO and alternative light materials over alternative

ways.The hydrothermal synthesis of ZnO powders has heap of benefits like (1) by this

methodology we will acquire powders with micromillimeter size.(2) underneath moderate

conditions the reaction is administrated (3) by adjusting the reaction conditions powders with

totally different|completely different} morphologies and (4) the majority have different

properties from that of the as-prepared powders.

34

Under stirring in 50ml of alcohol stock answer of Zn(CH3COO) a pair of. 2H20(0.1M) was

ready. In alcohol 25ml of NaOH was conjointly ready was additional to stock answer

underneath continuous stirring. Between eight and eleven hydrogen ion concentration price of

reactants was set. In Teflon lined sealed chrome steel autoclaves these solutions were remodeled

and maintained at varied worker. Within vary of 100- two hundred C for six and twelve h

underneath self-produced pressure.

By victimisation diffraction, transmission microscope and hand-picked space negatron optical

phenomenon, synthesized nanoparticles were confirmed (P. M. Aneesh et al., 2007).

2.5 Physical synthesis of nanoparticles

Evaporation-condensation and optical device ablation square measure most typically used

physical ways. It’s heap of benefits over chemical technique like formation of tiny, stable

nanoparticles in high concentration. These square measure stable as a result of the temperature

of heater surface doesn't fluctuate with time. Physical technique is additionally helpful for future

experiments like inhalation toxicity studies (S. Iravani et al., 2014)

Laser ablation

Laser ablation is that the most promising technique manufactures pure and uncontaminated

nanoparticles. Another advantage is that nanoparticles square measure ready within the absence

of chemical reagent(R Bola Sampol, 2014)

In this technique 532 and 1064 nm optical maser were used for the formation of silver

nanoparticles. During this, silver colloids were ready in water and that we processed the relation

between particle size and optical maser wavelength.

YAG optical maser was used for ablation a complete of 355 and 532 nm light-weight were

generated by exploitation KDP crystals. During a glass cell silver targets were settled that

conjointly contained 5ml deionized water. We tend to targeted optical maser light-weight on

surface of silver targets with the assistance of quartz lens. Magnetic stirrer was used for the

stirring of answer throughout ablation (T TSUJI et al, 2002).

35

The particles was characterised by transmission microscope (TEM) operated at 200KV touch of

Na dodecyl salt (SDS) answer was conjointly accessorial to colloid whereas making ready TEM

samples.

One of the drawbacks of optical maser ablation is that wetting agent coating terminated

the formation of nanoparticles. The nanoparticles that square measure fashioned within

the answer of low wetting agent concentration square measurelarger than those

fashioned in high wetting agent concentration (KMMA El-Nour et al.).

Evaporation-condensation

Physical synthesis of silver nanoparticles employing a tube chamber at associate degree air

pressure has some disadvantages like tube chamber occupies an oversized house, consumes an

excellent quantity of energy whereas raising the environmental temperature round the supply

material, and needs lots of your time to realize thermal stability. Moreover, a typical tube

chamber needs power consumption of over many kilowatts and a preheating time of many tens

of minutes to succeed in a stable operative temperature.

It had been incontestable that silver NPs can be synthesized via alittle ceramic heater with

space|a neighborhood} heating area. The little ceramic heater was wont to evaporate source.

The gaseous vapor will cool at an appropriate speedy rate, as a result of the gradient within the

neck of the woods of the heater surface is incredibly steep compared thereupon of a tube

chamber.

This makes potential the formation of tiny NPs in high concentration. The particle generation is

incredibly stable, as a result of the temperature of the heater surface doesn't fluctuate with time.

This physical technique will be helpful as a nanoparticle generator for long experiments for

inhalation toxicity studies, and as a activity device for nanoparticle mensuration

instrumentation. The results showed that the mean diameter, the geometric variance and

therefore the total variety concentration of NPs increase with heater surface temperature.

Spherical NPs while not agglomeration were determined, even at high concentration with high

36

heater surface temperature. The mean diameter and therefore the geometric variance of silver

NPs were within vary of half-dozen.2-21.5 nm and 1.23-1.88 nm, severally (S.Iravaniet at,

2014).

High energy ball milling technique

Nanoparticles have attracted abundant attention attributable to their distinct characteristics, that

square measure unavailable in standard macroscopic materials as an example, the reactions of

nanoparticles with different materials will be additional economical attributable to their high

surface-to-volume ratios, additionally to the high proportion of atoms at the grain boundaries.

Recently, mechanical edge has well-tried to be an efficient and easy technique while not

involving extreme temperature treatment for the assembly of nanocrystalline powders, with the

chance of getting massive quantities of materials with changed properties. during this technique,

beginning powder particles square measure at bay between extremely kinetic colliding balls and

also the inner surface of the bottle, that causes perennial deformation, rewelding, and

fragmentation of premixed powders leading to the formation of fine, dispersed phase within the

grain-refined matrix throughout the edge operation, 2 essential processes have an effect on the

particle characteristics. First, the cold attachment method ends up in a rise in average particle

size of the composite. The second, fragmentation, method causes the breaking apart of

composite particles. Steady-state equilibrium is earned once a balance is achieved between these

processes when a particular amount of edge.

Commercially offered ZnO powder was processed in steel cells (250 mL) victimization

hardened steel balls (diameter fifteen millimeter, weight thirty two gm) in close atmosphere for

various times starting from two to fifty hours. The mechanical edge was performed during a

horizontal periodical mill (Retsch, PM 400) in operation at twenty five cycles. The mixture

magnitude relation of steel balls and ZnO powders was around 15:1 by weight %. The

processed materials were used directly with no added edge media. 5 balls were unbroken in

every cell alongside ten g of the sample powder. 2 parallel cells were employed in this

experiment (the total weight for the sample powder was twenty g).

37

The structural and optical modifications induced within the ‘as synthesized’ nanomaterials were

determined by X-ray diffraction (XRD), scanning microscopy (SEM), and transmission

microscope (TEM), and photoluminescence emission spectra (PL). SEM and TEM results show

a gradual decrease in particle size from around 600 to ∼30 nm, with exaggerated edge time. The

initial microstructures had random shapes, whereas the ultimate form became quite spherical.

XRD analysis showed ZnO during a hexangular structure, broadening within the diffracted

peaks (N. Salah et al, 2011).

Sputter deposition

Sputter deposition has higher capability to fabricate refractory metals and intermetallic

compounds than evaporation and optical device ablation. The sputtering instrumentation is a

smaller amount expensive than electron-beam lithography systems as a result of the atom vapor

is usually generated from targets of pure materials, nanoparticles created by sputtering

sometimes contain fewer impurities within the composition than those created by chemical

strategies.

And since the various target materials is sputtered at the same time, it's able to manufacture

alloy nanoparticles with easier management on composition than the corresponding chemical

reduction strategies, within which metal ions area unit reduced in multiple steps for his or her

completely different reduction potentials. Therefore, sputtering has been wide studied and

applied as a primary physical technique for the assembly of metal nanoparticles.

In the technique, metallike nanoparticles were deposited on completely different substrates a

really reactive particle etching (RIE) chamber by keeping the target and also the substrate at a

very short distance, that is well but the mean free path of the target atoms. The experiments

were distributed during a March Jupiter II RIE system. The first target used was a 15*15 mm2

brass foil (alloy 260, Cu: Zn=70:30, exactness complete merchandise, Inc.) with parallel

openings of 50*1000m2 figured by optical device cutting. For comparison, many alternative

styles of targets consisting of copper, Al, and metallic element were also used. Glass slides from

Fisher Scientific were used because the substrates. The substrate and target were positioned on

prime of all-time low conductor of the RIE chamber, that was connected to the rfpower. The

38

target and also the glass substrate were either cumulous along or unbroken at a distance that

may be adjusted by a spacer. Besides glass substrate, metal and plastic foils were conjointly

used because the substrate materials for comparison. The sputtering method was performed with

SF6 gas at a flow of twenty SCCM (SCCM denotes cc per minute) at standard pressure beneath

the pressure of zero.130 Torr. A fifty W rf power at thirteen.56 MHz was wont to ionize the gas

for varied sputtering times. The generated negative F−ions can then hit the metal target for

instance, brass, Cu, Al, or metallic element to make atoms vapor. As a result, deposition of

metal nanoparticles on the glass substrate can occur later on.

A Hitachi S-4700 field-emission scanning magnifier |microscope FE-SEM and a Veeco

Dimension 3000 atomic force microscope AFM were wont to study the microstructures of the

deposited materials. Compositions associated chemical states of the Cu–Zn nanoparticles were

analyzed by a Kratos radical Axis DLD x-ray negatron chemical analysis (XPS) system

employing a monochromated Al supply with an energy resolution of concerning zero.5 work

unit (M Nie et al, 2009).

Electric arc deposition

It is simplest and most helpful methodology for the synthesis of nanoparticles that embrace

mass scale production of fullerences, carbon nanotubes etc. Inert or reactive gas introduction is

critical during this method; conjointly pressure level is maintained in vacuum system. Once

Associate in nursing arc is ready up, anode material evaporates. This is often attainable as long

because the discharge can be maintained (R S Tomar et al, 2015).This methodology has been

developed to supply Au nanoparticles by direct-current (DC) arc discharge in water throughout

the arc discharge method, once the temperature between electrodes reaches many thousand ºC,

Au wires ar engraved within the water medium. The vaporised metal is condensed a lot of

expeditiously within the insulator liquid than in a very gas part. Au vapor condensed in water

creates a stable Au binary compound suspension. Well-separated nanosize Au clusters in pure

water is thermodynamically stable for a protracted time.

Silver and Au wires used as processed materials ar submerged in deionized water and used

because the electrodes in getting ready the atomic number 47 and Au nanoparticle suspension.

39

DC arc discharge system, consisting of 4 main elements (i) 2 atomic number 47 or Au

conductors (ii) a servo system wont to maintain the space between electrodes to be constant (iii)

an influence offer system used to regulate the DC arc discharge parameters (iv) a glass tank for

holding the deionized water and a Teflon electrode holder wont to collect the atomic number 47

or Au mixture.

The preparation method of the atomic number 47 and Au nanoparticle suspension by

victimization ADM (Arc Discharge Method) in the main bases on a number of parameters, like

initial voltage, peak current, on- and off-pulse length, pressure, the gap between electrodes,

temperature and volume of deionized water, and fabrication time.

During the arc discharge, each ionic silver atomic number 47+ and gilded Ag nanoparticle Agº

is generated. There ar varied apparatuses wont to establish the gilded atomic number 47

nanoparticle and ionic atomic number 47.

The gilded atomic number 47 nanoparticle and ionic atomic number 47 have competed a vital

role within the battle against germs that are getting a lot of drug-resistant once a year. This

methodology indicates that atomic number 47 nanoparticle suspension (SNPS) made-up by

victimization ADM while not value-added surfactants solely contains the gilded atomic number

47 nanoparticle and ionic atomic number 47. Besides that, the ADM in deionized water has

conjointly been used for the fabrication method of Au nanoparticles. The experimental results

indicate that the ready atomic number 47 nanoparticles will react with the dissolved H2CO3 in

deionized water, resulting in the formation of Ag2CO3 considerably completely different to

atomic number 47, the ready Au nanoparticles with their surfaces guaranteed by O ar suspended

in deionized water by the formation of element guaranteed with the neighboring water

molecules (D Tien et al.).

Drawbacks of chemical method

Chemical ways typically involve poisonous chemicals which might be harmful to the

environment though these ways with success manufacture silver nanoparticles. These methods

are usually expensive (H.R Ghorbani et al. 2011).

40

2.6 Green synthesis of nanoparticles

Nanoparticles are the very small particles with size of 100nm or less (Abou El-Nour et

al).nanoparticles have a wide range of application in several areas like cosmetics, feed and food,

health chemical industries, electronics, space industries, drug-gene delivery, single electron

transistors, light emitters, energy applications the use of environmentally material such as plant

leaf, fungus bacteria for the synthesis of nanoparticles like silver nanoparticles or zinc. They

have many benefit of eco-friendly, minimum period, low cost, compatible for biomedical and

pharmaceutical application ((Otsuka et al.2003)

During synthesis of nanoparticles by chemical method having many toxic effect because toxic

solution and non polar solvent are present on surface of nanoparticles therefore need to develop

a clean, biocompatible safe cost-effective, sustainable, non-toxic, area in biological method of

synthesis of nanoparticles have a safe procedure .in which fungus are use to synthesis of nano

particle by the use of fungus because they release a large number of enzyme and biomass which

are easy to manage. They don’t require chemical for the synthesis of nanoparticles.

Many fungi hydrogenise extracellular in the broth when it present in medium. Mushroom is

known for its anti-cancer, anti tumor antibacterial hypertensive activites. Mushroom contain

many different bioactive compounds with different biological activities, the bioactivity depend

on how to mushroom prepared or consume.

During this time need eco-friendly method which don’t need the chemicals in synthesis of

nanoparticles green synthesis approach biological material to synthesis the nanoparticles.

Silver nanoparticles are of interactive because they use wide range in antimicrobial applications,

biosensor materials, composite fibers, cryogenic superconducting materials, cosmetic products,

and electronic components. Some important applications of silver NPs are in pharmaceutics,

medicine, and dentistry. (Bansal et al.2011)

Material and method:

Fungi:

Pleurotutsostreatus belong to class basidomycetes and maintain in potato dextrose sugar at 4

degree celcius biomass use for biosynthesis experiments was grown in liquid media which

41

composition is 5.0g malt extract and 10.0g glucose in 1litre.after that media were inoculated and

kept in rotary Shaker for 3days at 90degree celcius after 3 days filter out the medium.keep the

supernatant and discard the medium in supernatant add agNO3 and again put in rotary shaker

.after .that centrifuge the medium and take out the pallets.this pallets a sthe silver nanoparticles

which was conform by uv-spectrophotometery ,TEM, XRD etc. the glucose is necessary for the

reduction of silver nanoparticles and protein play a important role in bioreduction ( Sanghi and

Verma 2009).

Biosynthesis of silver nanoparticles:-

Using fungus white rod was grown 7days after growth the biomass was filtrated and

resuspended in water .the biomass is filtrated and AgNo3 was added in the fungal liquid t

the white rod fugus biomass were a pale yellow color before the addition of Ag+ ions and this

changed to brownish color on completion of the reaction with Ag+ ions for 28hr.

Mechanism of formation of silver nanoparticle:-

Silver ions form silver salt silver trapped in the surface of fungal cell due to interaction between

the positively silver ions and negatively charged carboxylic group in the enzyme in cell wall of

mycelia

Table 1 Important applications of silver nanoparticles.

Important applications of silver NPs

Treatment of ulcerative colitis & acne

Treatment of dermatitis

Inhibition of HIV-1 replication

Enhanced Raman Scattering Spectroscopy (SERS)

Detection of viral structures (SERS & silver nanorods)

Antimicrobial effects against infectious organisms

Remote laser light-induced opening of microcapsules

42

Silver/dendrimernanocomposite for cell labeling

Molecular imaging of cancer cells

Coating of hospital textile (e.g., surgical gowns & face mask)

Coating of catheter for cerebrospinal fluid drainage

Coating of surgical mesh for pelvic reconstruction

Coating of breathing mask patent

Coating of endotracheal tube for mechanical ventilatory support

Coating of driveline for ventricular assist devices

Coating of central venous catheter for monitoring

Coating of intramedullary nail for long bone fractures

Coating of implant for joint replacement

Orthopedic stockings/ Additive in bone cement

Implantable material using clay-layers with starch-stabilized silver NPsSuperabsorbent

hydrogel for incontinence material/ Hydrogel for wound dressing

Additive in polymerizable dental materials patent

Silver-loaded SiO2 nano composite resin filler (Dental resin composite)

Polyethylene tubes filled with fibrin sponge embedded with silver NPs dispersion

43

Chapter 3

Material and Method

44

Chapter 3

Material & Method

3. METHOD

3.1Procurement and maintenance of culture

Pleurotusostreatuswas procured from Directorate of Mushroom Research, Solan (Himachal

Pradesh). Cultures were maintained on malt extract agar slants and petriplates at 29ºCin

incubator till confluent growth was observed.

3.2 Preparation of submerged culture.

Mycelium Plugs (6mm) of P.ostreatus were excised using sterile plug borer from the

petriplateand inoculated in malt media. The culture was incubated at 25 ºC at 100rpm for 72

hours.

3.3 Preparation of cell free extract

After confluent growth of mycelia, the broth was centrifuged at 3000rpm for 5 minutes and

acquired pellet was inoculated in sterile distilled water followed by incubation in orbital shaker

at 25 ºC, 100rpm for 72 hours.

3.4 Nanoparticles synthesis

Nanoparticles synthesis was carried out according to the method of (R.S Yahia et al, 2014).

Silver nitrate was added to 100ml of cell free extract to achieve final concentration of 4mM

followed by incubation on an oribtal shaker at 25 ºC, 100rpm for 72 hours under dark condition.

100ml solution of 4mM silver nitrate was kept as blank and subjected to similar treatment.

Nanoparticles were harvested by centrifugation at 5000rpm for 15minutes and were dried.

45

3.5 Instruments Used for nanoparticles characterization

(a) UV-Visible spectroscopy

UV- visible spectral analysis was done by UV-Visible spectrophotometer. The reduction of pure

Ag+ was monitored by measuring the UV-visible spectra of the reaction medium at 72hours

after diluting a small aliquot of the sample into distilled water (R.Devika et al, 2012).

(b)FTIR-

FTIR was performed on FTIR SHIMADZU- 8400s manufactured by Shimadzu, japan. For

FTIR analysis, AgNo3cells/pellet were used as a base on which a thin layer of samples were

dispersed. This solid base was kept on wedge for FTIR under infra red rays. Peaks were

observed on display screen for each sample (R.Devika et al, 2012)

(c) TEM-

Sample of Tem analysis was prepared, as mentioned in IR sample preparation. The sample was

first sonicated for 15minutes. Silver nanoparticles were loaded on carbon coated copper grids

and solvent was allowed to evaporate under infra light for 30 minutes. The TEM measurements

were performed at an accelerating voltage at 200Kv (R. Devika et al, 2012).

3.6Photocatalytic Degradation of Antibiotic

Ampicillin stocks were prepared by dissolving 10mg in 1 litre sterile distilled water so as to

achieve a concentration of 10ppm and stored at 4ºC till further use.

3.7 Effect of nanoparticles concentration

Ampicillin Solution was treated with different concentrations of nanoparticles viz. 1ppm, 3ppm,

7ppm, 9ppm, 11ppm, 13ppm, 15ppm, 17ppm and subjected to photocatalytic degradation for a

period of 3 hours. Readings were taken after 3 hours. Antibiotic solution without nanoparticles

served as control and was subjected to similar treatments. Antibiotic degradation was evaluated

according to method reported by (Ahmed et al, 2004). 3ml of Folin Ciocalteu phenol reagent

was added to 5ml of sample and incubated in water bath at 95 ºC for 20 minutes. The mixture

was allowed to cool down and absorbance was measured at 750nm. All experiments were

carried out in triplicates. Percent degradation was calculated by the formula(Co-c)/Co*100

46

given by (R. Nosrati et al, 2012). Co is concentration of degraded antibiotic at zero hour and C

is the concentration of degraded antibiotic at time t.

3.8Effect of antibiotic concentration

Different concentrations of antibiotic viz. 10ppm, 20ppm, 30ppm, 40ppm, 50ppm, 60ppm was

mixed with nanoparticles concentration that exhibited highest photocatalytic degradation in 3.7

and exposed to sunlight for 3hours. Readings were taken after 3 hours. Antibiotic solution

without nanoparticles served as control and was subjected to similar treatments. Antibiotic

degradation was evaluated according to method reported by (Ahmed et al, 2004). 3ml of Folin

reagent was added to 5ml of sample and incubated in water bath at 95 ºC for 20 minutes. The

mixture was allowed to cool down and absorbance was measured at 750nm. All experiments

were carried out in triplicates. Percentage degradation was calculated by the formula (Co-

c)/Co*100 given by (R. Nosrati et al, 2012). Co is concentration of degraded antibiotic at zero

hour and C is the concentration of degraded antibiotic at time t.

3.9 Effect of contact time

The concentration of nanoparticles and antibiotic that exhibited highest photocatalytic

degradation in 3.7 and 3.8 respectively were mixed and exposed to sunlight for 6 hours.

Readings were taken at an interval of 1 hour. Antibiotic solution without nanoparticles served as

control and was subjected to similar treatments. Antibiotic degradation was evaluated according

to method reported by (Ahmed et al, 2004). 3ml of Folinciocalteu phenol reagent was added to

5ml of sample and incubated in water bath at 95ºC for 20 minutes. The mixture was allowed to

cool down and absorbance was measured at 750nm. All experiments were carried out in

triplicates. Percentage degradation was calculated by the formula (Co-c)/Co*100 given by (R.

Nosrati et al, 2012). Co is concentration of degraded antibiotic at zero hour and C is the

concentration of degraded antibiotic at time t.

3.10 Effect of pH

10ppm concentration of antibiotic solution was taken and pH was set before addition of

nanoparticles from a range of 4 to 9. Nanoparticles was added according to 3.7 and exposed to

sunlight for a time at which best photocatalytic degradation occurred in 3.9. Readings were

taken after the time exhibited best degradation. Antibiotic solution without nanoparticles served

47

as control and was subjected to similar treatments.Antibiotic degradation was evaluated

according to method reported by (Ahmed et al, 2004). 3ml of Folinciocalteu phenol reagent

was added to 5ml of sample and incubated in water bath at 95ºC for 20 minutes. The mixture

was allowed to cool down and absorbance was measured at 750nm. All experiments were

carried out in triplicates. Percentage degradation was calculated by the formula (Co-c)/Co*100

given by (R. Nosrati et al, 2012).Co is concentration of degraded antibiotic at zero hour and C is

the concentration of degraded antibiotic at time t.

48

Chapter 4

Results & Observations

49

Chapter 4

Results & Observations

4 .RESULTS AND DISCUSSIONS

4.1 Procurement and maintenance of culture

Pure culture of Pleurotusostreatus was obtained from Directorate of Mushroom Research, Solan

(Himachal Pradesh). Cultures were maintained on malt extract agar slants and petriplates at

29ºC till confluent growth was shown as in Fig 1

Fig 1 (A) (B)

Pleurotusostreatus on malt extract media Pleurotusostreatuson malt extract slant

4.2 Synthesis of nanoparticles

Silver nanoparticles were synthesized using cell free extractas it was shown in Fig

2.Nanoparticles were synthesized using a reduction of aqueous Ag+ with cell free extract of

ofP.ostreatus at 25ºC. Preliminary indications of silver nanoparticles synthesis is given by

change in color of suspension to dark brown color (R.S Yahia et al, 2014; Z.A Ali et al, 2015).

Silver nanoparticles in solution show brown/amber due to surface Plasmon resonance (SPR).

On drying the color is transformed into black color due to absence of SPR. The nanoparticles

are easily aggregated and this aggregation will occur the change in color from brown to black

(B.J Wiley et al, 2006).

50

.

(a) (b)

Fig 2 change in color of reaction mixture indicating silver nanoparticles synthesis

(a) Before synthesis (b) After synthesis

4.3 Effect of nanoparticles on ampicillin degradation

Solution of 10ppm of ampicillin was mixed with different concentrations of silver nanoparticles

i.e. 1ppm, 3ppm, 5ppm, 7ppm, 9ppm, 11ppm and 13ppm and subjected to photocatalytic

degradation for a period of 3 hours. Readings were taken after 3 hours. Absorbance of samples

was measured at 750nm. Antibiotic solution without nanoparticles served as control and was

subjected to similar treatments.Solution of ampicillin containing nanoparticles was highly

degraded as compared to solution of ampicillin without nanoparticles. Nanoparticles act as a

photocatalyst and this feature is advantageous in the photodegradation and removal of

ampicillin under directsvisible and ultraviolet’s light.

Degradation efficiency was calculated by using formula (co-c)/co*100 (R. Nosrati et al, 2012)

Co- concentration of degraded ampicillin at zero hour

C- Concentration of degraded ampicillin at 3rd hour

51

Table 1 Percent Degradation of ampicillin on different concentration of nanoparticles

Concentration

Percent Degradation

Antibiotic solution NP+ Antibiotic sol.

1ppm 64 ± 0.032 70± 0.67

2ppm 72 ± 0.029 80 ±0.045

3ppm 76.3 ± 0.045 88±0.34

5ppm 82.4 ± 0.39 94±0.39

7ppm 81.3 ± 0.06 92.0±0.63

9ppm 74 ± 0.54 86.5±0.051

11ppm 67 ± 0.037 77±0.049

13ppm 65 ±0.70 66.9±0.056

Ab- antibiotic

NP- Nanoparticles

The percentPhotocatalytic degradation was increased from 64% to 82% in case of solution

containing only antibiotic and from 70 to 94 in case of solution containing antibiotic and

nanoparticles by increasing the concentration of nanoparticles as shown in table 1. After that

increasing the concentration of nanoparticles caused a decreasing in removal efficiency of

ampicillin. This is due to because there is an increase in the surface area of catalyst available for

absorption and degradation. However increase in the concentration of nanoparticles increases

the solution opacity leading to decrease in penetration of photon flux in the reactor thereby

decreasing the Photocatlaytic degradation ( R Shetty et al, 2016; R. Nosrati et al, 2012; Kaur at

al, 2007)

4.4 Effect of antibiotic concentration

The concentration of nanoparticles exhibited highest degradation in 4.3 was mixed with

antibiotic concentration viz 10ppm, 20ppm, 30ppm, 40ppm, 50ppm, 60ppm and exposed to

sunlight for 3 hours. Readings were taken after 3 hours. Antibiotic solution without

52

nanoparticles served as control and was subjected to similar treatments.Antibiotic degradation

was evaluated according to method reported by (Ahmed et al, 2004).

Degradation efficiency was calculated by using formula (co-c)/co*100 (R. Nosrati et al, 2012)

Co- concentration of degraded ampicillin at zero hour

C- Concentration of degraded ampicillin at 3rd hour

Table 2 Percent Degradation of ampicillin on different concentration of antibiotic

Concentration Percent Degradation

Ab solution Ab solution+NP

10ppm 92±0.023 98±0.025

20ppm 84±0.035 92.8±0.038

30ppm 75.4±0.039 86.3±0.042

40ppm 67±0.043 74.2±0.045

50ppm 59.9±0.048 69.9±0.050

60ppm 33±0.053 58.6±0.056

Ab- antibiotic

NP- Nanoparticle

Percent degradation decreases from 92 to 33 in case of solution containing only antibiotic and

from 98 to 56 in case of solution containing antibiotic and nanoparticles with increase in

concentration of antibiotic from 10pp to 60ppm shown in Table 2. This is due to because at a

particular concentration of nanoparticles, the amount of active centers on photocatalyst is finite

therefore the rate of Photocatalytic degradation is fast at lower concentration of ampicillin. The

rate decreases because the molecules of ampicillin are excessive in comparison to the amount of

active sites on photocatalyst.

Amother reason is that as the initial concentration of antibiotic increases, C/Co increases. A

description is that with increasing concentration of antibiotic, while the concentration of

nanoparticles and light irradiation is stable, active sites for the generation of reactive radicals are

53

steady and definite radicals consume for the degradation of increased organic molecules. This

leaded to reduction in decomposition efficiency of ampicillin ( R Shetty et al, 2016; R. Nosrati

et al, 2012).

4.5Effect of contact time

The concentration of nanoparticles and antibiotic that exhibited highest photocatalytic

degradation in 4.3 and 4.4 respectively were mixed and exposed to sunlight for 6 hour.

Readings were taken after an interval of 1 hour. Antibiotic solution without nanoparticles served

as control and was subjected to similar treatments.Antibiotic degradation was evaluated

according to method reported by (Ahmed et al, 2004).

Degradation efficiency by varying time was calculated by using the formula (co-c)/co*100 (R.

Nosrati et al, 2012).

Co- concentration of degraded ampicillin at zero hour

C- Concentration of ampicillin at 1st, 2nd…. 6th hour

Table 3 Percent Degradation of ampicillin by varying the time

Time Percent Degradation

Ab solution Ab solution+ NP

11am 78±0.061 81±0.032

12pm 80.4±0.072 88.6±0.082

1pm 86.4±0.085 90.7±0.087

2pm 90.9±0.090 93.2±0.092

3pm 94.7±0.095 96±0.097

4pm 95±0.100 95.5±0.125

Ab- antibiotic

NP- nanoparticles

As the light intensity increases the percent Photocatalytic degradation was increased from 78 to

95 in case of solution containing only antibiotic and from 81 95 in case of solution containing

54

antibiotic and nanoparticles. This is because with increasing light power, the production of

hydroxyl radicals through photodissociation of water is more.Also with the production of

photons by UV light, electrons transferred from the valence band to the conduction band of

silver nanoparticles (M Farzadika et al, 2014).

4.6Effect of pH

10ppm concentration of antibiotic solution was taken and pH was set before addition of

nanoparticles from a range of 4 to 9. Nanoparticles was added according to 4.3 and exposed to

sunlight for a time at which best photocatalytic degradation occurred in 4.5. Readings were

taken after the time exhibited best degradation. Antibiotic degradation was evaluated according

to method reported by (Ahmed et al, 2004).Antibiotic solution without nanoparticles served as

control and was subjected to similar treatments. Ampicillin is stable from pH 5 to 7. After 7 it

will not degrade. We performed this assay by varying pH from 4 to 9 before the addition of

nanoparticles.

Degradation efficiency by varying time was calculated by using the formula (co-c)/co*100 (R.

Nosrati et al, 2012).

Co- concentration of degraded ampicillin at zero hour

C- Concentration of ampicillin at 5th hour

Table 4 Effect of pH on ampicillin degradation

pH PercentDegradation

Ab solution Ab solution +NP

4 84±0.012 89±0.015

5 89.7±0.020 92±0.025

6 93.5±0.026 95.5±0.028

7 90.1±0.030 93±0.032

8 75±0.055 83.3±0.058

9 63± 0.034 77± 0.091

Ab- antibiotic

NP- nanoparticles

55

As shown in Table no.4 degradation was increased from the range of pH 4 to 7 while the

concentration of antibiotic and nanoparticles is stable and light irradiation is also stable. After

pH of 7, degradation was slow because ampicillin is not stable after Ph 7. Ampicillin is

negatively charged at basic pH and positively charged at acidic pH. At pH 5, an acidic solution

ampicillin has a positive charge and photocatalyst has a negative charge that favors the

absorption of ampicillin molecules on photocatalyst surface and it shows high degradation

efficiency. In alkaline solutions at neutral pH both catalyst and ampicillin are negatively

charged so there is limited degradation(R. Nosrati et al, 2012; Elmolla ES et al, 2010).

4.7 Techniques used for the characterization of nanoparticles

UV-VISIBLE SPECTROSCOPY

The preliminary conformation of formation and stability of silver nanoparticles in colloidal

solution was monitored by UV-Visible spectral analysis. Fungal cell filtrate treated with silver

nitrate showed the sharp peak at around 435nm with high absorbance which is very specific for

nanoparticles. The wavelength scanning was done from the range from 300nm to 600nm as

shown in Fig 4 (A. Ingle et al, 2009; R. Devika et al, 2012)

Fig 3 Uv-visible graph showing best peak at 435nm

56

FTIR

FTIR (Fourier transform infrared spectroscopy) study was done to characterize the functional

group attached with our nanoparticles. FTIR measurements were carried out to indentify

molecules responsible for the reduction of Ag+ions. Spectra of nanoparticles manifests

absorption peak located at 515.01, 588.38, 648.1, 699.32, 796.63, 1037.74, 1116.82, 1265.35,

1317.43, 1342.5, 1561.1, 1546.96, 1562.39, 1645.33, 1676.2, 1741.78, 1770.71, 2854.74,

2926.11, and 3441.2 in the region 500- 4000/cm. In this present study the spectra reveals the

presence of different functional groups like alkyl halide C-Br, alkyl halide C-Cl, nitro N-O

group, amide N-H, alkyl halide C-F, nitro N-O,Anhydride c=o, Ketone alpha, beta- unsaturated,

Amide C=O, Anhydride c=o, Alkyl C-H , Alcohol/phenol O-H , Carboxylic acid O-H stretch as

shown in Fig 5 (A Ingle et al, 2009; k.VSelvi et al, 2012).

Fig 4. FTIR graph showing different functional groups attached with silver nanoparticles

57

TEM

Transmission Electron Microscopy (TEM) is a vital characterization tool for directly imaging

nonmaterial’s to obtain quantitative measures of particle and/or grain size, size distribution and

morphology. The size of our nanoparticles varies from 12.9nm to 18.5nm and is of spherical

shape as shown in Fig 6.A. Ingle et al, 2009; R. Devika et al, 2012)

(A) (B)

Fig 6 (A) Size of nanoparticles (B) Shape of nanoparticles

58

Chapter 5

References

59

Chapter 5

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