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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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Reduction and nucleation are faster processes than coalescence of nuclei..... 26 Figure 2-6: Schematic represantation of the key factors and processes that govern the behaviuor of the nanoparticles in the natural enviroement.

Figure 2-7: Schematic diagram outlining the possible fate of nanoparticles (NPs) in the marine environment and the organisms at risk of exposure

Figure 2-8: Visualising bacterial cell division stages using thin-section TEM.................. 40 Figure 2-9: Biofilm formation steps. Stage 1, initial attachment; stage 2, irreversible attachment; stage 3, maturation I; stage 4, maturation II; stage 5, dispersion. 41 Figure 3-1: Schematic diagram of the basics of TEM

Figure 3-2: Block presentation of the internal components of the AFM

Figure 3-3: Interatomic force variation versus distance between AFM tip and sample....... 54

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Figure 3-5: Schematic diagram illustrating the charge distribution around a charged

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charged particle.

Figure 3-6: Localised Surface plasmon resonance (LSPR) of conductive electron density

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spectrophotometer showing the light sources the monochromater, reference and sample holder and the detector.

Figure 3-8: Schematic diagram of field flow fractioning with all main parts illustrated... 65 Figure 3-9: Separation of particles of different sizes with different modes of the FFF 9 (a)

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hyperlayer mode.

Figure 3-10: A diagram showing the cross section of the different components of modern quadrupole ICPMS

Figure 3-11: Titration of acid with strong alkali.

Figure 4-1: Double beam 6800 Uv-vis spectrometer from Jenway

Figure 5-1: Yellow colour of the gold solution changed into ruby red during the formation of AuNPs.

Figure 5-2: The surface plasmon resonance (SPR) of freshly prepared AuNPs and the maximum absorption takes place around 520 nm.

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Figure 5-4: DLS diagrams of eight samples illustrating the sizes of the particles. Samples

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shows that the last two samples are clearly more polydispersity than the other samples.

Figure 5-5: Freshly synthesised AuNPs of different sizes. Ruby red colour at the left is for

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manifested by bigger particles (arounf 45 nm hydrodynamic size)................... 101 Figure 5-6: Graph showing the relationship between the size and the SPR maximum. The

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increasing size. Part b) presents the positive correlation between z-average and the SPR maximum

Figure 5-7: TEM images and size distribution histograms of citrate capped AuNPS of two

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description of the nanoparticles in Table 5-1.

Figure 5-8: TEM images and size distribution histograms of cold method prepared PVP capped AuNPs of different sizes. Right-hand side of the figure is presented the with excel calculated size distribution of the particles,

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corresponding size distribution. AFM images courtesy Dr Mohamed Baalousha.

Figure 5-10: Typical TEM images of freshly synthesised gold nanoparticles showing highly spherical moodispercy AuNPs. The particles have an average core size of 10 nm

Figure 5-11: Shape factor distribution diagrams of AuNPs calculated from TEM images. G1

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physicochemical properties of the Nps presented in this graph are summarised in table 5.1 above.

Figure 5-12: Stability of two samples of AuNPs coated with either citrate or PVP is monitored over a period of six months from the synthesis date.

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citrate stabilised NPs b) is stabilised with PVP and prepared through cold method as explained in section and c) are samples prepared through hot method ( see section and stabilised by the PVP.

Figure 5-14: The correlation curve of the z-average values of citrate capped NPs and the ionic strength of the media. the media used for this purpose was monovalent electrolyte which is NaNO3. Section a represents citrate capped NP. Section b is PVP capped NP

Figure 5-15: DLS diagram of citrate capped NPs in 16 mM NaNO3 solution showing that citrate capped AuNPs particles clearly aggregate at this ionic strength and higher as shown by the mixture of peaks.

Figure 5-16: DLS diagram of PVP capped NPs in 80 mM NaNO3 solution showing that

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flagelles(Silby, 2006).

Figure 6-2: Pseudomonas fluorescens growing on freshly prepared agar base plate.......... 123 Figure 6-3: Surface plasmon resonance (SPR) of 14,8 nm citrate capped gold NPs in full

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intervals of 40 minutes were used throughout the first 5 hrs of the experiment.

Figure 6-4: Graph showing how the maximum SPR of citrate capped AuNPs of 14.8 nm core size in undiluted MDM changes with the time.

Figure 6-5: The characteristic red colour of the freshly synthesised AuNPs has changed

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Media (MDM)

Figure 6-6: DLS graphs showing aggregation of the citrate capped AuNPs (G2) in

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about 30 minutes after exposure

Figure 6-7: TEM images showing aggregates of different sizes of citrate capped AuNPS in undiluted MDM media.

Figure 6-8: Surface plasmon resonance measured with Uv-vis of 14.8 nm core size citrate

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capped AuNPs after 5 days in 4x diluted MDM media

Figure 6-11: Size distribution by intensity measured with DLs of 14.8 nm core size citrate

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the exposure.

Figure 6-12: TEM images showing stabile single particles of citrate capped AuNPs (G2) after 9 days of exposure in 4x diluted MDM media.

Figure 6-13: a) SPR of citrate capped NPs in 10x diluted MDM media monitored ten days.

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Figure 6-14: The SPR measured with uv-vis of 10 nm core size PVP capped NPs in undiluted MDM media monitored in a period of 12 days.

Figure 6-15: TEM images of gold nanoparticles capped with PVP in undiluted MDM media.

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Figure 6-16: Typical growth curve of the psoudomonas flourescencs strain SBW25 in 4x diluted MDM media.

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are for two independent replicates which were recorded in two different days.

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two independent replicates of samples measured in two different days........... 144 Figure 6-20: Optical density of the bacteria treated with 85 nm PVP capped AuNPs compared with the OD of the bacteria in the MDM media.

Figure 6-21: Optical density (OD) on 595 nm wavelength measured with Uv-vis spectrophotometer of the bacteria treated with gold ions compared with the

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replicates of samples measured in two different days

Figure 6-22: Testing the ability of bacteria to consume citrate and PVP as carbon resources.

Green liquid shows bacteria growing in citrate solution while clear liquid shows no growth of bacteria in the PVP solution.

Figure 6-23: Size distribution by intensity measured with DLs of unfiltered bacteria suspension treated with 14.8 nm core size citrate capped AUNPs.

Figure 6-24: Size distribution by intensity measured with DLS filtered with 100 nm filter bacterial suspension treated with 14.8 nm core size citrate capped AuNPs. The measurement was taken view minutes after treating the NPs in the bacterial suspension.

Figure 6-25: Size distribution by intensity of bacterial suspension treated with 14.8 nm core

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randomly distributed on and around the bacteria body. Images were taken after 9 days of exposure.

Figure 6-27: SPR spectra measured with Uv-vis spectrophotometer of the citrate capped

14.8 nm core size AuNPs after exposure to bacteria suspension. All samples were filtered through 0.2 µm filter to remove bacteria cells.

Figure 6-28: Characterisation of 1o nm core size PVP capped AuNP after exposure in bacteria suspension. a) is the DLS size distribution by intensity, b) shows

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Figure 7-1: Schematic diagram showing details of gram negative bacterial cell wall, outer and inner membrane.

Figure 7-2: Structure of the a) PVP and B) citrate showing that citrate has three charged carboxyl functional groups while PVP has no charged functional group....... 162 Figure 7-3: TEM images showing the random distribution of stable citrate capped AuNPs on and around the surface of the un-sectioned whole bacterial body a) 8000x magnified images b) 25000x magnified image.

Figure 7-4: Images of bacterial cells treated with AuNPs capped with citrate. Arrows are indicating the stable AuNPs on the smooth, undamaged curvature of the bacterial cells.

Figure 7-5: PVP capped AuNPs randomly distributed on and around Pseudomonas fluorescens cells. No aggregation of the NPs is visible in the bacteria growth media.

Figure 7-6: Apparent pits and wide holes on the surface of the outer membrane of the

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pointing to the places of the blebbing on the surface of bacterial cells............ 169 Figure 7-8: Bursting bacterial cell top row and tubular structures on the surface of the cell membrane bottom row caused by the AuNPs capped with PVP.

Figure 7-9: TEM images of bacterial cells grown in blank Minimum Davis Media (MDM).

Cell membranes are smooth and undamaged.

Figure 7-10: TEM images showing the sections of bacterial cells. Image c shows stable AuNPs outside the cells. Images c, e and f represent different magnifications

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pointed with arrows. Sections d shows atable AuNPs distributed outside bacteria cells. Sections a and b are lower magnifications and they only show bacterial cell sections.

Figure 7-11: EDX spectrum of a normal point inside the cell but outside the more electrondense black areas in the bacterial cells. EDX spectrum were taken with Jeol 2100 TEM.

Figure 7-12: EDX spectra of black more electron-dense areas inside the bacterial cells sections.

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

Table 2-2: Toxic effects of nanomaterials on bacteria adopted from Klaine et al and updated with more recent references (Klaine et al., 2008).

Table 2-3: Toxicity of AuNPs on different strains of bacteria.

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

Table 4-1: Experimental conditions and the concentrations on the reactant used for the synthesis of AuNPs capped by citrate and PVP10 separately.

Table 4-2: Minimal Davis Media (MDM)

Table 4-3: Concentrations of the alcohol used for dehydration of the bacterial cells....... 87 Table 5-1: Hydrodynamic diameters and core sizes of AuNPs as measured with different techniques.

Table 5-2: Significant test data for the size difference between TEM core sizes and DLS hydrodynamic diameters.

Table 5-3: Surface chemistry properties of freshly synthesised AuNPs with different coating agents in terms of surface charge, pH and zeta potential.................. 113 Table 5-4: Concentration of the total and dissolved gold in the samples as prepared and measured with ICPMS.

Table 7-1: Electrokinetic properties of freshly synthesised AuNPs of different coating agents and bacteria cells both purified and in the growth media.................. 162

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Nanoparticles (NPs) are building blocks of nanotechnology and are referred to a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm – 100 nm (European, 2011). It is important to understand that the abovementioned definition of the nanoparticles is mainly for regulation purpose and there are other definitions given by other institutions (SCENIHR, 2007, Dowling, 2005). Throughout the thesis the European Union definition will be used. The source of NPs can be both natural and anthropogenic (manmade). Natural nanoparticles have been present in the environment for millions of years and they have been generated by a number of natural processes including weathering, erosion, volcanic eruption, hydrolysis and biological activities. Recently, however, several sources have resulted in an increase in anthropogenic nanoparticle formation (Pratim and Chang-Yu, 2005): among the different activities that contributed to the augmentation of the nanoparticles in the environment are: coal fired combustions, transportations, welding processes followed by more recent processes where engineered nanoparticles are designed and produced deliberately (see Figure 1-1).

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