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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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2.3 Bacterial population in the environment Bacterial populations are omnipresent living organisms which means that they can be found everywhere in the environment including places with extreme conditions like hot springs, polar ices and even in the radioactive nuclear waste (Dunne, 2002, Allison, 2000). They colonise and grow in all different environmental compartments introduced in the previous sections. Planktonic bacteria exist as unicellular organisms in the environment and every cell behaves as a small independent organism which is invisible with the naked eye. They can only be seen with the aid of microscope. When the cells grow and reach certain size they divide into two identical new cells (see Figure 2-8 below). In this way, the population increases in number as long as there are favourable environmental conditions for the bacteria growth including enough supply of food in the near environment, right temperature and pH.

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To get energy from the organic food, bacterial cells need to decompose the molecules in one of the following two respiration processes. Similar to higher level organisms (plants and animals), Aerobic bacteria need oxygen to extract energy from the available raw organic materials and, for this reason, aerobic bacterial populations colonise and thrive in environmental compartments where there is a good supply of air. In contrast, anaerobic bacteria can survive without oxygen and they live in non-oxygenated areas as under Earth crust but also on the surface of the Earth.

Apart from the unicellular planktonic bacteria described above, another prevailing structure of bacteria survival that forms in almost all environmental compartments is the biofilm structure explained below.

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Biofilms are defined as matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces. This definition includes microbial aggregates and floccules and also adherent populations within the pore spaces of porous media (Costerton et al., 1995). A “film” is a thin coating. “Bio” refers to the living nature of this film. In other words, a biofilm is a thin coating comprised of living material. Unlike the more familiar planktonic lifestyle, in

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grows thicker, the film often includes many bacterial species and the matrix develops a complex structure (Dunne, 2002, Allison, 2000, Callow and Callow, 2006). Different cells in the biofilm have different tasks in the community and, in this way, they support each other to survive in hostile conditions like antibiotics, antiseptics, highly reactive chemical biocides, dehydration and nutrient depletion (Costerton et al., 1999).

Biofilm development is a series of complex but discrete and well-regulated steps. The exact molecular mechanisms differ from organism to organism, but the stages of biofilm development are similar across a wide range of micro-organisms (O'Toole, 2003, Kaplan et al., 2003). The sequential stages of biofilm development on different surfaces can be recognised as consisting of five stages.(Percival, 2000) (See Figure 2-9 below for a simplified

representation of the development stages of bacterial biofilm):

Figure 2-9: Biofilm formation steps (Monroe, 2007). Stage 1, initial attachment; stage 2, irreversible attachment; stage 3, maturation I; stage 4, maturation II; stage 5, dispersion.

Although many people associate bacteria with adverse effects like diseases and infections, most bacterial populations are extremely useful for both our life and the well - being of the

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biofilm on the other side in the last two decades, there are relatively few published materials studying the transport of the nanoparticles through biofilms (Tong et al., 2010). The limited studies currently available fail to identify any significant effects at the microbial level of nanoparticles in more complex systems (Neal, 2008).

2.3.2 Role of bacterial population in the environment Bacterial population in the natural environment, whether it is planktonic unicellular form or more complex biofilm structures, has an important role in the productivity and function of the ecosystem. The most important role of a bacterial population is its ability to decompose organic materials and release useful nutrients for plants. Organic nitrogen in the dead organisms and their excretion are converted into ammonium which is an inorganic form of nitrogen through a process called ammonification in soil living fungi and bacteria called decomposers. Ammonium cannot be used directly by plants unless it is converted into nitrates by nitrifying bacteria (Belser, 1979, Kuenen and Robertson, 1994, Abeliovich, 1992). In this way, many useful plants including crops depend on bacteria and live symbiotically with bacteria in order to gain basic nutrients for their growth. Apart from the important nitrification process explained above, there are some free - living bacterial groups called plant growth-promoting rhizobacteria (PGPR) which protect plants from deadly pathogens (Babalola, 2010, Kloepper et al., 1980, Lugtenberg and Kamilova, 2009). These beneficial bacteria can be used to improve crop production and reduce the consumption of pesticides.

Similarly, bacteria plays an important role for both carbon (Heimann and Reichstein, 2008, Davidson and Janssens, 2006) and phosphorus cycle (Ammerman, 2003b, Ammerman, 2003a) in the ecosystem.





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treatment plants where especially cultured bacteria provide necessary enzymes to reduce waste materials. Since bacteria reproduce in the sewage plant and increases in number, so is the amount of enzymes produced to decompose waste materials. This application is the cheapest available alternative for the abovementioned purpose and can be applied for any process where organic waste causes problems such as food processing systems, remediation of oil contaminated soils, contaminated aquifers, fish ponds and many more.

Both the very important role of bacteria population in the environment and its useful applications mentioned above justify the reason why many environmental scientists are concerned with the effect of human activity including nanotechnology on the environmental bacteria. To increase our knowledge of the environmental problems caused by NPs, this

project aims to study the effect of gold nanoparticles on widespread environmental bacteria:

Pseudomonas flourescens. It is the task of the next two sections to give a detailed introduction of the Pseudomonas flourescens and the summary of the published literature on the effect of the nanoparticles on this bacterial strain.

2.3.3 Pseudomonas fluorescens Pseudomonas flourescens are gram negative bacteria and they belong to RNA- homology group Ι in the genus pseudomonas (Stanier et al., 1966, Palleroni et al., 1973). Other members of this group include Pseudomonas putida, Pseudomonas aureginosa, Pseudomonas syringae, (Palleroni, 1974). One of the main characteristics of the Pseudomonas flourescens is their ability to synthesise a yellow greenish water - soluble fluorescent pigment called pyoverdines (Stanier et al., 1966). These clearly visible fluorescent pigments attracted scientists to use them as taxonomic markers for the classification of different strains of

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available for these mainly aerobic pseudomonas species where the transport of oxygen is essential for their respiration process. Structurally, pyoverdines has three main domains (Meyer, 2000): a quinoline chromophone (responsible for the colour), an acryl chain and peptide chain which are important structures for the taxonomy purpose since different strains of pseudomonas flourescens have different peptidic chains (Kilz et al., 1999). Like other members of the Pseudomonas genus, it is a motile bacterium by means of polar flagella.

Pseudomonas flourescens are widely found in many different habitats in the environment.

They can grow and flourish in water, soil and on plant leaves and roots. The fact that they can colonise a variety of different habitats in the environment indicates that they have simple flexible nutritional requirements. They can metabolise a wide range of different organic compounds as carbon sources (Dooren de Jong, 1926). Many strains of Pseudomonas flourescens are plant growth-promoting rhizobacteria (PGPR). They promote the growth of a different variety of plants by colonising their roots and producing antibacterial substances which stops the effect of the plant pathogens microorganisms. It is recognised that the compound responsible for the abovementioned mutualism is the pyroverdine pigment (Choi et al., 2008). Economically, the application of Pseudomonas flourescens on agricultural crops has increased, offering a green way of controlling deadly plant pathogens which can replace the traditional environmental non-friendly pesticides (Dey et al., 2004).

To gain a basic and preliminary understanding of the general problem of the NPs’ interaction with bacterial population, the abundance of Pseudomonas flourescens in the environment makes them suitable organisms for the study of NPs in the environment since they are more likely to encounter and interact with them than the other less abundant organisms. Despite

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of nanoparticles with Pseudomonas flourescens.

2.4 Pseudomonas flourescens and nanoparticles.

It is important to understand the effect of the NP on the microbiota once released in the environment. Unicellular planktonic bacteria can be used as a model to investigate the bacteria-NPs interaction at cellular level and at molecular level. Researches on planktonic bacteria may provide useful information about structural changes of the bacterial cell membrane and possible internalisation of NPs. Previous studies have reported the interaction in terms of intermolecular forces such as electrostatic forces, van der waals and hydrophopic interaction (McWhirter et al., 2002, Parikh and Chorover, 2006). Due to their antimicrobial activities, the effect of titanium dioxide NPs (Wei et al., 1994, Block et al., 1997) and silverNPs on both gram-positive and gran-negative varieties of bacteria are widely studied.

However, among the wealth of information available in the field of nanoparticles toxicity on the microbiota, only very few studies have dealt with Pseudomonas flourescens despite its abundance in the environment. See Table 2-4 below for summary of the published literature on the effects of engineered nanoparticles on Pseudomonas flourescens).

Table 2-4: Published literature on the effect of engineered NPs on pseudomonas flourescens.

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flourescens were reported ((Jiang et al., 2009). Similarly, the effect of silver nanoparticles on the Pseudomonas flourescens were studied and the sign of toxicity at high concentration (2000 ppm) of the NPs (Fabrega et al., 2009) was found. Copper oxide NPs have caused the production of reactive oxygen species followed by DNA damage of Pseudomonas flourescens. Therefore, further work is needed to understand the effect of the engineered NPs on the environmental relevant Pseudomonas flourescens bacterium. As it was highlighted in chapter1 (introduction), it is the overall aim of this project to investigate the effect and interaction of gold nanoparticles of different sizes and coating agents on Pseudomonas flourescens.

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Aiming to have well defined gold nanoparticles, the first part of this project was assigned to synthesize monodisperse batches of gold nanoparticles with systematically varying sizes and with two coating agents: PVP and citrate. After the synthesis of gold nanoparticles, complementing modern analytical and imaging techniques have been employed for the full characterisation of the nanoparticles as prepared, in bacterial growth media and after exposure to bacteria. Among important pysicochemical properties of the NPs include: the size, shape, charge, solubility, surface Plasmon resonance and surface chemistry (Filella and Buffle, 1993, Elzey and Grassian, 2010). In this project we characterise the freshly synthesised gold NPs by measuring their size, shape, zetapotential, surface Plasmon resonance, surface charge, stability overtime, and stability in a range of ionic strengths.

Due to the complexity and diversity in nature of the aforementioned physicochemical properties, It is obvious that they cannot be determined by using only one analytical technique so a multi-method approach has been chosen (Lead and Wilkinson, 2006). The following sections will be devoted to give a detailed introduction of the theoretical background of the characterization techniques of the NPs operated to elucidate the forgoing physicochemical properties of the gold NPs. Since the second part of the project aims to study the interaction of AuNPs on planktonic bacteria short description of the bacterial growth quantification techniques and sterilization techniques used during this project will be also be provided.

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Where FC is the centrifugal acceleration of the particles which rotate angular speed of ώ (rad/s) in a rotor with radius r (m). Using F = ma and substituting acceleration with Equation 3-1 the force that applies on the particles can be given in Equation 3-2 below

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Where m stands for the mass of the particles and F is the centrifugal forces acting on the particles. Here, the force is directly proportional to both the mass and the square of the angular speed of the rotating particles in the suspension. For the sake of the practicality, the angular speed is, many times, given in terms of rotational speed which is expressed in round per minute (rpm). Since in one minutes there are 60 seconds and in one round there are 2π rads, the angular speed is related to the rotational speed N through Equation 3-3 below

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