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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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Engineered nanoparticles are the backbone of modern nanotechnology where research and development are growing very fast and attract substantial funding both from public and private sectors (Joner et al., 2008). Particles in the nanometer-size ( 10-9 m) range have gained much attention due to their fascinating electrical, optical, magnetic and catalytic properties associated with their nanoscale dimensions (Christof, 2001). Those fascinating, unique and novel properties make nanomaterials pysicochemically different and often superior to both the atomic and bulk materials of the same element. For instance, copper which is opaque at macroscale becomes completely transparent to visible light at the nanoscale (Zong et al., 2005, Shanmin and et al., 2003); stable materials like aluminium turn combustible (Shafirovich et al., 2006, Shafirovich et al., 2007); Gold, which is rarely insoluble in water at the macroscale, becomes more soluble in the nanoscale (Paolo Pengo, 2003). While a known insulator, silicon, becomes a conductor of electrical current at nanoscale (Hu et al., 2003), A material such as platinium, which is chemically inert at normal scales, can serve as a potent chemical catalyst at nanoscales (Luo et al., 2005, Tian et al., 2007). They have many applications varying from communications to catalysts, computing chips, nanomechanical

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ant-aging drugs and they are attracted by a wide range of scientific researches in different fields of science ( Figure 1-2) (Ferrari, 2005, Templeton et al., 1999).

Figure 1-2: Different fields of application for nanaoparticles (Krumov et al., 2009). Reprinted with the permission from copyright 2009 John and Willey Therefore, both the number and type of the manufactured nanoparticles have amplified tremendously with the development of this new technology. Consequently, their concentration in the natural environment has increased proportionally since enormous amounts of products are being commercialised and marketed through the world and finally released directly or indirectly into the environment (Ray et al., 2009, Flahaut, 2010). This undesired presence of manufactured nanoparticles in the environment may augment if the recycling processes, waste management and monitoring activities are not effective enough to relinquish the leakage of nanoparticles into the environment. Although we may have some knowledge about the properties of synthesised nanoparticles during the synthesis process and

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understanding is eminent (Ju-Nam and Lead, 2008). They can interact with other naturally occuring nanosized particles and produce new particles with unknown properties. Their surface chemistry may change due to the relevant environmental conditions.

However, there is growing awareness and increasing interest in the fate, behaviour and environmental impact of the nano-scaled materials. To avoid past mistakes of the chemicals and different products (plastics, asbestos) designed and used without any clear understanding of their possible impact on the environment, researchers of today are now more than ever willing to understand the effects of nanomaterials on the environment before their use becomes widespread (Theodore, 2005). Once NPs are released they may end up in the different compartments of the environment which are: air, water, soil and living organisms.

Since the physicochemical properties of these compartments are quite different, the NPs are more likely to behave differently in each compartment. Information about the NPs behaviour in any compartment will increase our understanding of the nanomaterials in the environment which, in turn, is essential to study any effects caused by the NPs in the environment including living organisms such microorganisms.

1.1 Aims and Objectives The overall aim of this PhD project is to investigate the effect of gold nanoparticles as a model of engineered NPs on environmental abundant planktonic bacteria. The objectives of

this study are:

1. to synthesise and fully characterise well constrained gold NPs.

2. to study the fate and behaviour of gold NPs in bacteria growth media.

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4. to investigate the effect of the coating agents (surface chemistry) of the NPs on the bacteria.

During the first stage of the investigation, gold nanoparticles of different sizes and different coating agents will be synthesised and thoroughly characterised using a variety of different but complementing analytical and imaging techniques. Prior to the exposure of the NPs to the planktonic bacteria, their behaviour in liquid bacteria growth media are studied in terms of size, shape, aggregation, overall charge and surface plasmon resonance. In media characterisation step of the NPs is essential to study whether the NPs keep their original physicochemical properties in the media. Then fully characterised gold nanoparticles were used to test the effect of both size and coating agent on planktonic bacteria. The chosen strain for this study is pseudomonas fluorescens SBW25, a widespread, bacterium which is very helpful for both plants (agriculture) and humans (it produces antibiotics). Special care is taken to the core size of nanoparticles so that the possible effect of coating agents can be determined and compared. Increasing knowledge about these kinds of systems extends our understanding of the effect of the NPs core size and their surface chemistry on bacteria and can facilitate the prediction of the effect of similar nanoparticles on similar bacteria.

1.2 Outline of the dissertation

This PhD thesis consists of seven chapters whose topics are summarised below:

Chapter 1 introduces the concept of nanoparticles and describes both the aims and objectives of the thesis. It also gives clear outline of the thesis chapters as follows.

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the types and origin of the nanoparticles, their fate and behaviour in natural environment is examined. After that, outlined are the possible difficulties imposed by the complex natural system which may make almost impossible to gain accurate understanding of their expected effects on the living organisms. Special attention is devoted to the effect of the nanoparticles on the bacterial biomass which is an integral part of the environment.

Chapter 3 addresses the variety of methods used to perform the project. It gives a short description of gold nanoparticles syntheses methods followed by characterisation techniques.

Metal nanoparticles can be prepared by physical methods like evaporation of a metal in a vacuum or laser ablation, or by chemical methods involving the reduction of metal salts. All nanoparticles used in this project are prepared by the chemical method. After the reduction of gold ions into gold atoms, the resulting gold nanoparticles need to be stabilised. This can be achieved either sterically using relatively big molecules such as polymers or by charge. In this project, Sterically stabilised gold nanoapraticles are synthesised using PVP as coating agents and NaOH as reducing agents in room temperature while charge stabilisation of the gold nanoparticles is achieved by using sodium citrate as capping agents as was introduced by Turkevich (Turkevich, 1951) and modified by Frens (Frens, 1973) and others (Kumar et al., 2006). The synthesised NPs are fully characterised using a variety of analytical and imaging techniques. Among the techniques used to elucidate the physicochemical properties of the nanoparticles are transmission electronic microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS), and ultraviolet-visible spectroscopy (UV_VIS). To study the bacterial growth and to investigate the effect of the NPs on the bacteria, optical densities of bacteria treated with NPs and untreated bacteria are recorded and compared.

TEM and imaging of the outer membrane of the bacteria will help to visualise any membrane

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followed by TEM imaging will serve to observe internalisation of the nanoparticles in the bacterial cells.

Chapter 4 presents the results of the synthesised gold nanoparticles. The focus is to synthesise a wide range of sizes of gold nanoparticles with different coating agents. Two coating agents (PVP and sodium citrate) are used and for each one a range of different sizes are achieved and fully characterised. This chapter investigates the physicochemical properties of the synthesised gold nanoparticles in terms of size, shape, charge and electrophoretic mobility and tries to discover any patterns in the data. Special attention will be devoted to the quality of the data in terms of monodispersity, roundness and stability.

Chapter 5 investigates the stability of goldnanoparticles in the bacteria growth media (Minimal Davis Media) through complete post exposure characterisation in terms of relevant physicochemical properties. The effect of the fully characterised NPs on the bacteria is also studied in this chapter. The growth inhibition of the bacteria caused by the NPs is studied by recording the optical density (OD) of the bacterial population with spectrophotometer. This chapter aims to visualise the distribution and stability of the NPs in the bacteria suspension media using TEM image measurements.

Chapter 6 studies the interaction of the synthesised and characterised gold nanoparticles on the bacterial outer membrane. TEM and images of treated bacteria with different types of gold NPs are used to observe any membrane damage caused by the NPs. Internalisation of the gold NPs in the bacterial body is also studied though fixation and sectioning techniques.

Chapter 7 summarises the general findings of the PhD project and draws analysis of the results followed by some useful suggestions for future work in the field of the fate and

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2.1 Types and the origin of the nanoparticles (NPs) To have an idea of the scale of the nanostructured materials, Figure 2-1 below puts the size of nanoscale regime materials into perspective and compares them with some well-known objects. There are a number of ways, based on different aspects, that NPs can be classified into groups. Considering their origin, they can be divided into natural and anthropogenic (man-made) which, in turn, can be subdivided into accidental and engineered or manufactured NPs (see Table 2-1 below) or they can be further separated, based on their chemical composition, into organic (carbon containing) and inorganic(Nowack and Bucheli, 2007).

As the name clearly implies, natural NPs are a group of nanosized materials which are generated by the action of natural processes and have been present in the natural environment for a very long time since the origin of the planet Earth. In addition to natural NPs, earlier human activities like mining, agriculture, forest burning and constructions have tremendously increased the concentration of accidental nanoparticles into the environment especially into the atmosphere. Despite the fact that NPs have been present in the environment for millions of years as natural and later as accidental NPs, it was not until quite recently that their interest in both pure and applied sciences have been extensively exploited due to the discovery of their promising, novel and applicable properties which make them attractive for a vast number of products in a wide range of scientific researches and for many sectors of the modern technology.

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Nanostructured materials behave differently and are often superior in properties to bulk materaials due to two primary factors: surface effects (enhanced surface phenomena caused by the very high surface area to volume ratio. Most atoms are on or near the surface thus they can be weakly bonded and more reactive) and quantum effects (discontinuity in behaviour caused by the confined delocalised electrons on the surface of the NPs due to their small dimensions) (Roduner, 2006). Those properties affect the overall physicochemical properties of the nanoscale materials in a way that they demonstrate novel and unprecedented properties. Nanotechnology takes the advantage and exploits these aforementioned unique properties of nanoscale matter to design, translate and apply science to plenty of useful products. This new technology forms the basis of a wide interdisciplinary area of research development and industrial activity that has been growing fast throughout the world for the past few decades (Aitken et al., 2006).The importance of nanotechnology in today’s modern society is clearly explained by both the variety of scientific and technological applications using nanoparticles (Fabrega et al., 2009) and by the magnitude of the exponentially growing investments in this field, allocated by public and private sectors worldwide.

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have estimated the worldwide market for nanotechnology related products at around £ 105 billion in 2005 and £ 700 billion in 2010 (Taylor, June 2002,) and about $1 trillion by 2011Roco, 2005).The USA is the leading country in this relatively new field of technology followed by countries like China, Japan, the European Union and South Korea. The above mentioned prediction made by Roco in 2005 for a worldwide nano-products value of $ 1 trillion by 2011-2015 still appears to hold in 2011(see Figure 2-2 ) which shows that the market is doubling every three year as a result of successive introduction of new nanoscale products (Roco, 2011) Figure 2-2: market timeline : projection for the worldwide products that incorporate nanotechnology (Roco, 2011).

Reprinted with the permission of copyright@ 2011, Springer science and business B.V.

Although the term nanoparticle (see chapter 1 for its definition) is a general term used for nanoscale materials, there are a number of different types of nanoparticles. It is the task of the

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engineered nanoparticles in terms of their properties, types and synthesis routes followed by their fate and behavior in the natural environment with special attention being paid to both aquatic environment and biomass population.

Table 2-1: Classification of nanaparticles (Nowack and Bucheli, 2007).

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