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«By Yusuf Nur A thesis submitted to The University of Birmingham for the Degree of DOCTOR OF PHILOSOPHY School of Geography, Earth and Environmental ...»

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Gold Nanoparticles: Synthesis, Characterisation

and their Effect on Pseudomonas Flourescens


Yusuf Nur

A thesis submitted to

The University of Birmingham

for the Degree of


School of Geography, Earth and Environmental Sciences

The University of Birmingham

April 2013


University of Birmingham Research Archive

e-theses repository

This unpublished thesis/dissertation is copyright of the author and/or third

parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation.

Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.

Abstract As recently developed products with many unknown aspects, the effect of nanoparticles on the natural environment is of growing concerns among environmental scientists and the wider community. Both the fate and behaviour of the nanoparticles in the environment and their effects on the living organisms need to be better understood in order to maintain environmental health and ensure the sustainability of the important nanotechnology industry.

This dissertation focused on the effects of gold nanoparticles on the environmentally relevant bacteria, Pseudomonas fluorescens, as a model of the planktonic bacterial biomass in the environment.

A bottom - up chemical method was deployed to synthesise constrained gold nanoparticles of a variety of sizes and with two different coating agents (citrate and polyvinylpyrrolidone (PVP)) followed by a series of characterisation steps both pre and post bacterial exposure.

Citrate capped gold NPs were synthesized by citrate reduction of HAuCl 4.3H2O at 100 oC.

The synthesis of AuNPs capped with PVP was carried out at both room temperature and 70 o C to compare the effect of the temperature on the quality of the final NPs. Nanoparticles were characterised by measuring their relevant physicochemical properties. Among the determined properties are size, shape, zeta potential, surface charge and stability in environmentally relevant ionic strength and in bacterial growth media.

The freshly synthesized gold nanoparticles remained stable for at least six months. The citrate capped gold nanoparticles (AuNPs) were less stable in environmentally relevant ionic strength solutions and in bacterial growth media than the PVP capped gold nanoparticles. The citrate capped gold NPs formed aggregates which are much bigger than 100 nm in the solutions of ionic strength as low as 16 mM and in full strength of bacterial growth Media

–  –  –

in the above mentioned solutions and in solutions of ionic strength as high as 80 mM.

Fully characterised gold NPs were incubated in the bacterial exposure media minimal Davis media (MDM). The effects of the gold nanoparticles on the bacteria were investigated both via measurements of bacterial growth inhibition (optical density at 595 nm) and by visual examination of the structural damage of the bacterial membrane using transmission electron microscope (TEM).

The citrate capped AuNPs of (14 nm core size) of 10 ppm final concentration in the exposure media had no measureable effect on the bacterial growth as no inhibition was recorded when compared with control samples. Similarly no membrane damages were shown by the TEM images of the bacterial cells incubated with citrate capped NPs, while similar concentration and comparable sizes of PVP capped gold NPs have affected the bacterial growth. This effect was manifested through the reduction of optical density and was demonstrated by the transmission electron microscope (TEM) images in the form of membrane damage including blebbing formation, tubular structures on the surface of the outer membrane of the bacterial cells and, in severe cases, the complete bursting of bacterial cells. It was found out that gold ions inhibit completely the bacterial growth as shown by optical density measurements.

In conclusion, this research has confirmed the effect of gold nanoparticles on the planktonic bacteria Pseudomonas fluorescens. The importance of the surface chemistry of the nanoparticles on the bacteria was clearly shown since NPs with similar concentration and core size but with different coating agents showed different effect on the bacteria.

–  –  –

There are lots of people whom I want to take this opportunity to thank for their valuable support in the course of my PhD project. My first and foremost gratitude goes to my supervisor, Professor Jamie Lead for his honest and friendly guidance, his extremely useful ideas and endless professional support throughout the project. Thank you very much for your valuable time and immeasurable patience. My special thanks also go to my co-supervisors Dr Jerome Duvel and Professor Herman van Leeuwen for their excellent ideas and support. I am extremely thankful to all of my colleagues in the Environmental Health Research group in the School of Geography, Earth and Environmental Science at the University of Birmingham, especially, to Mrs Milla Tajamaya for her useful discussions and mutual moral support needed for the continuation of the PhD project, Dr Mohammed Baalousha for his support with atomic force microscope (AFM) images and Dr Ruth Merrifield for her initial training with the analytical equipments in the laboratory. I owe special thanks to the group of Microscopic Centre at the University of Birmingham for both the training and technical support of the imaging of my samples. The data of this project will be incomplete without the support of Dr Stephen Baker for the ICPM-MS measurements, thank you infinitely for your help. I am also very grateful to my friend and previous colleague, Mr Khaled Ghazel, for his continuous encouragement and support with the structure of the dissertation.

I would particularly like to thank the Natural Environmental Research Council (NERC) for the funding of my project.

Finally, I am extremely grateful to my family for their patience, compromise, continuous moral support and understanding during the last three and a half years in which most of my time and energy was devoted to the completion of this project.

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Table of Contents

List of abbreviations

List of figures

List of tables


1.1 Aims and Objectives

1.2 Outline of the dissertation


2.1 Types and the origin of the nanoparticles (NPs)

2.1.1 Natural nanoparticles

2.1.2 Accidental nanoparticles

2.1.3 Engineered nanoparticles Production of engineered nanoparticles Examples of engineered nanoparticles Fullerenes and Carbon nanotubes

–  –  – Quantum dots: Metals Silver nanoparticles. Gold nanoparticles

2.2 Fate and behaviour of the manufactured NPs in the environment................ 27 2.2.1 Nanoparticles in soil

2.2.2 Nanoparticles in water: Fate and behaviour of NPs in freshwater Fate and behaviour of NPs in Marine water

2.2.3 Ecotoxicology and the effect of nanoparticles on bacteria population

2.3 Bacterial population in the environment

2.3.1 Biofilm

2.3.2 Role of bacterial population in the environment

2.3.3 Pseudomonas fluorescens

2.4 Pseudomonas flourescens and nanoparticles.



3.1.1 Centrifugation and ultracentrifugation

3.1.2 Transmission Electron Microscopy (TEM)

3.1.3 Atomic Force Microscopy (AFM)

3.1.4 Dynamic Light Scattering (DLS)

–  –  –

3.1.6 Surface Plasmon Resonance (SPR) spectroscopy.

3.1.7 Field Flow Fractionation (FFF)

3.1.8 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

3.1.9 Potentiometric Titrations Method


4.1 Introduction

4.2 Synthesis of gold nanoparticles

4.2.1 Materials

4.2.2 Synthesis of AuNPs capped with Citrate

4.2.3 Synthesis of AuNPs capped with PVP Cold process Hot process.

4.3 Sample Preparation

4.3.1 Glassware Cleaning

4.3.2 Ultrafiltration

4.4 Characterisation of the synthesised gold Nanoparticles (AuNPs).................. 77 4.4.1 Centrifugation and ultracentrifugation

4.4.2 Imaging NPs with Transmission Electron Microscopy (TEM)

4.4.3 Topography of the NPs measured with atomic force microscopy (AFM)............... 79 4.4.4 Hydrodynamic and zeta potential measurement with dynamic light scattering..... 80 4.4.5 Surface Plasmon Resonance (SPR)

–  –  –

4.4.7 Concentration of gold with Inductively Coupled Plasma Mass Spectrometry....... 82 4.4.8 Surface charge of the particles measured with potentiometric titrations method.. 83 4.4.9 Studying the aggregation of AuNPs in solutions of different ionic strength.......... 83 4.5 Bacterial growth and media preparation techniques

4.5.1 Media Preparation

4.5.2 pH-measurement

4.5.3 Sterilisation Techniques

4.5.4 Routine preparation of bacterial specimen for examination in a Transmission Electron Microscope (T.E.M.) : Chemical Fixation Dehydration Embedding Ultra Thin Sections

4.5.5 Quantification of Bacterial Growth

4.6 Statistical analysis of the samples.


5.2 Results and discussion.

5.2.1 Synthesis and growth of AuNPs

5.2.2 Characterisation of AuNPs Presence of gold nanoparticles

–  –  – DLS and FFF measured hydrodynamic sizes Colour change analysis. TEM and AFM measured core sizes. Comparison of the sizes measured with different techniques Shape of the AuNPs Surface chemistry Dissolved and nanoparticles fractions of gold

5.2.3 Stability of the AuNPs

5.2.4 Effect of ionic strength on the stability of the NPs

5.3 Conclusion


–  –  –

6.1 Introduction

6.2 Stability of the gold nanoparticles in Minimal Davis Media

6.2.1 Stability of citrate capped NPs in undiluted MDM media AuNPs capped with citrate in undiluted MDM media. AuNPs capped with citrate in 4x diluted MDM media AuNPs capped with citrate in 10x diluted MDM media

6.2.2 Stability of gold NPS capped with PVP in MDM media AuNPs capped with PVP in undiluted MDM media

6.3 Growth curve of Pseudomonas fluorescens

–  –  –

6.4.1 Effect of AuNPs on the growth of the Pseudomonas fluorescens Interaction of 14 nm citrate capped on Pseudomonas fluorescens Interaction of 5 nm citrate capped on Pseudomonas fluorescens Interaction of PVP capped NPs of different sizes on Pseudomonas fluorescens...... Interaction of gold ions on Pseudomonas fluorescens Citrate and PVP as carbon source for Pseudomonas fluorescens Comparison between citrate NPs, PVP NPs and gold ions

6.4.2 Characterisation of AuNPs after exposure on bacteria Characterisation of citrate capped AuNPs after exposure to bacteria Characterisation of PVP capped AuNPs after exposure to bacteria

6.5 Conclusion



7.1 Introduction

7.2 Results and discussion

7.2.1 Zetapotential measurement of the AuNPs and pseudomonas flouresencs......... 161 7.2.2 Interaction of citrate capped AuNPs with the Pseudomonas flourescens............ 163 Distribution of Citrate capped AuNPs on the surface body of the bacteria.......... 163 Effect of citrate capped AuNPs on the outer membrane of the bacteria................ 164 7.2.3 Interaction of PVP capped NPs with the bacteria

–  –  – Bursting bacterial cells and tubular formations

7.2.4 Internalisation of the NPs in the bacteria cells

7.3 Conclusion





Appendix A1: TEM images of G2 (citrate capped)

Appendix A2: TEM images of G5 (PVP capped)

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OECD Organization for Economic Cooperation and Development PEG polyethylene glycol PGPR plant growth-promoting rhizobacteria PGPR plant growth-promoting rhizobacteria

–  –  –

SRHA Suwannee River humic acid STM scanning tunneling microscopy SWCNTs single-walled carbon nanotubes TEM transmission electron microscope

–  –  –

Figure 1-1: Schematic diagram illustrating source, release and the presence of the nanoparticles in the environment.

Figure 1-2: Different fields of application for nanaoparticles

Figure 2-1: Schematic representation of the size of some objects in nanometers scale..... 10

–  –  –


Figure 2-3: Structure of Fullerene C60 molecule. Purple balls represent the places of carbon atoms.

Figure 2-4: Representation of SWCNT and MWCN at the top and their TEM images at the bottom

Figure 2-5: Schematic illustration for the deduced process of gold nanaoparticles formation.

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