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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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Both the intensity and the place of the DLS peak at the start of the experiment (just after the addition of the NPs to the solution) were compared with its intensity and place at the end of the experiment. The two measurements show fairly comparable results for both the intensity and place of the peaks (see Figure 6-24 and Figure 6-25). The place of the peaks indicates that the size of the NPs did not change during the exposure period while the unchanged intensity implies that no NPs were lost or absorbed by the bacteria. In the case of the unfiltered suspension, the huge peak around 800 nm (Figure 6-23) represents the average hydrodynamic

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bacterial cells the AuNP intensity has sharply increased.

To confirm the stability of the NPs indicated by the DLS results, TEM samples were prepared at the end of the exposure period and images were taken directly after. The presence of stable gold NPs can be seen on and around the bacterial cells in the suspension in Figure 6-26. There was no sign of the NPs aggregation in the suspension. The extracellular polysaccharides (EPS) produced by the bacteria cells may have a similar stabilising effect on the NPs as natural organic matter. The effect of different natural humic substances on different NPs was studied previously and was reported that they stabilise NPs by coating them (Deonarine et al., 2011, Akaighe et al., 2011).(Baalousha, 2009, Chen and Elimelech, 2007).

Figure 6-26: TEM images showing stable citrate capped 14.7 nm core size AuNPs randomly distributed on and around the bacteria body. Images were taken after 9 days of exposure.

SPR spectra of the filtered bacteria suspension were recorded both at the start and after two days of exposure of gold NPs. The spectra have shown the characteristic absorption peak of

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bacteria) and the results are summarised in Figure 6-27 below.

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. Characterisation of PVP capped AuNPs after exposure to bacteria Similarly, the PVP capped AuNPs was characterised after exposure time of two days in bacterial suspension. The characterisation techniques used for this purpose were DLS to measure the hydrodynamic diameter and to detect any aggregation that took place during the exposure. TEM was used to image the NPs in the suspension media and Uv-vis to record the surface Plasmon resonance (SPR) of the NPs. The measurement of the SPR serves to confirm

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0.4 0.3 0.2 0.1 0 450 500 550 600 650 700

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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 TEM images of both the NPs and bacteria cells, c) is the SPR of the NPs.

Figure 6-28.a shows two peaks for the size distribution by intensity measured with DLS. The more intensity peak above 10 nm corresponds to the scattering of the NPs while the less intensity peak could be caused by loose PVP molecules which can pass through the pore size

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show the presence of stable, spherical NPs in the suspension media. The images show both the NPs and bacteria cells. The presence of gold NPs at the end of the growth period is confirmed by the surface Plasmon resonance absorbance peak around 520 nm. MDM blank show no absorbance around 500 nm.

6.5 Conclusion The main purpose of this chapter is to investigate the effect of the both the core size and the surface chemistry of NPs on environmental bacteria. Prior to the exposure to the bacteria, NPs were fully characterised and their stability in the bacterial growth media was investigated. The characterisation of the NPs in media is essential so that their effect on the bacteria cells can be compared and related to their properties in the media. Here, the behaviour of the AuNPs in different dilution of Minimal Davis media (MDM) was studied.

The full strength media caused citrate capped NPs to aggregate immediately as confirmed by instantaneous colour change followed by disappearing surface Plasmon which completely vanished within 24 hours. TEM images have also showed big aggregates of the particles while the PVP capped NPs are relatively more stable in the undiluted media. This media has high ionic strength. Therefore, charge stabilised citrate capped NPs normally aggregate in high ionic media. When the media was diluted 4 times or more and the ionic strength was reduced both types of particles (citrate capped and PVP capped) were stable in the diluted media for a period of 2 weeks as measured with DLS and supported by SPR spectra. The stability of the NPs in the diluted MDM media is further confirmed by the TEM images of the samples which clearly showed the presence of single spherical AuNPs in the bacterial suspension media. After completing the stability test investigations, 4x diluted MDM media

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different coating agents on the Pseudomonas fluorescens since this dilution has guaranteed both the stability of the NPs and the bacterial growth. Different types of the NPs have manifested different effects on the bacteria growth. While both sizes (5 and 14.8 nm) citrate capped AuNPs have slightly increased the bacteria growth, the 10 nm PVP capped AuNPs slowed down the growth slightly. This emphasises the importance of the coating agents around the NPs for the bacteria-NPs interaction. Apart from the NP-bacteria interaction, the effect of gold ions on the bacteria was investigated. Gold ions of similar concentration as NPs have completely inhibited the bacteria growth and caused the immediate death of the cells.

The important implication for future practice which has been highlighted in this research is the importance of studying the stability of the NPs in the growth media prior to their exposure to the bacteria. Taken together, The results from this research has shown that AuNPs of similar core size may affect differently on the growth of bacterial cultures due to their different surface chemistry which is determined by the type of coating agents.

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7.1 Introduction Bacterial cells are delimited by their cell wall and membrane which consists of outer and inner cytoplasm membrane. The outer membrane is the barrier which normally has a direct contact with the environmental surrounding wherein the bacteria are living (Figure 7-1). The physicochemical properties of the wall and outer membrane are crucial and determine how substrates including NPs interact with bacterial cells. Equally important are the nature and properties of the surface chemistry of the NPs presented by the coating agents though the importance of this outer layer is many times ignored (Luo et al., 2010).

Figure 7-1: Schematic diagram showing details of gram negative bacterial cell wall, outer and inner membrane.(Dahl, 2008)

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membrane and the capping agents (Luo et al., 2010, Kang et al., 2007, Thill et al., 2006). This interaction may be the result of electrostatic forces based on the charge of the approaching surfaces (Stoimenov et al., 2002). The overall surface charge of the two types of bacteria gram positive and gram negative is negative charge due to the surface chemistry of the outer membrane. The outer membrane of the gram positive bacteria has carboxyl groups which by deprotonation form a negatively charged surface (Yee et al., 2004). Similarly the charge of the gram negative is originated by lipopolysaccharides which together with phospholipids are the main constituents of the outer cell membrane (De Castro et al., 2008, Raetz, 1990). This means that NPs with similar core sizes and shape may have different effects on the bacteria due to their different surface chemistry caused by their coating agents which is the main means of keeping NPs dispersed and preventing them from aggregation.

Depending on the type and surface structure of the NPs on one hand and on the type of bacterial cell membrane on the other hand the interaction effect may be manifested in different structural changes of the membrane including blebbing and tubule formation (Stachowiak et al., 2010) and enlargements of the membrane defects (Leroueil et al., 2007).

Because of their long known antimicrobial effect, silver nanoparticles (Ag-NP) have been widely studied and previous studies on their interaction with the different bacteria have shown different effects on the surface of the bacteria. Bovine serum stabilised silver nanoparticles (Ag-NP) have repeatedly disrupted and damaged the outer cell membrane of E.coli bacteria as studied by Pal et al (Pal et al., 2007). Membrane damages and formation of pits on the bacteria cell membrane caused by Ag-NP were reported by Sondi et al (Sondi and Salopek-Sondi, 2004).

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nanoparticles were studied. The very abundant soil and water bacteria pseudomonas aurigenosa has developed a rigid cellular outer membrane on hydrophilic surfaces while the membrane become relatively soft in hydrophobic substrates (Luo et al., 2010). The disruption of the cell membrane of the pseudomonas aurigenosa and the subsequent uncontrolled leakage of cell DNA was reported after interacting with diaminopyrimidineethiolfunctionalised, cationic 3 nm AuNPs (Zhao et al., 2010). Other studies (Stoimenov et al., 2002, Zhang et al., 2007) on ZnO have revealed similar effects on the bacterial cell membrane where alterations of membrane architecture and its permeability were reported.

For a more complete list of the effect of nanoparticles on different types of bacteria can be referred to the Table 2-3 and Table 2-4 in chapter 2.

Despite the above mentioned isolated findings the exact mechanisms of how both natural and engineered NPs interact with bacterial outer membrane still remain a matter of investigation and are not yet well understood (Hayden et al., 2012). In this study gold nanoparticles with two different capping agents (PVP and citrate) were exposed on Pseudomonas flourescens bacteria growing in a liquid Minimal Davis Media (MDM). The bacteria-AuNPs interaction was investigated using TEM (see sections 3.1.2 for a detailed theoretical background of these techniques). The overall aims of this research were threefold.

1) To study the distribution of the NPs on the surface of the bacteria,

2) To observe any changes on the bacterial outer membrane

3) To investigate whether NPs are internalised in the bacteria cells.

Furthermore, any observed effect of the different surface chemistry of AuNPs on the bacteria membrane will be discussed and compared.

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Prior to any effect caused by nanoparticles on the bacteria, the interaction between the outer cell membrane and the coating agents of the nanoparticls needs to take place. This bacteriaNPs interaction is mediated by the properties of the outer membrane and the surface chemistry of the nanoparticls. The overall charge of the approaching surfaces may play a crucial role in this interaction. To confirm the charge of both AuNPs and Pseudomonas flourescens cells, the first measurement carried out prior to the bacteria- AuNPs interaction investigation was the measurement of the zeta potential as described in the following paragraphs.

7.2.1 Zetapotential measurement of the AuNPs and pseudomonas flouresencs Charged particles in a solution are surrounded by ions of opposite charge which form a fixed layer around the charged particles. Beyond this fixed layer, there is an electrically neutral diffusive layer. When charged particles are placed in an electric field they move toward the oppositely charged electrode together with the fixed layer and small portion of the diffusive layer called sliding surface. Zeta potential is the electrical potential on the sliding surface and it is a measurement of the stability of the charged particles in the sense that the higher the zeta potential is the more stable the particles in the solution are. Aggregation of the particles occurs when the value of the zeta potential tends to zero(Elimelech et al., 1995). The zeta potential of the freshly synthesised AuNPs, the bacterial growth media and the purified bacteria cells through washing with milli-Q and centrifugating three times with 5000 rpm for 10 minutes were measured and summarised in Table 7-1 below.

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The data in Table 7-1 shows that the zeta potential of citrate capped AuNPs are more negatively charged than the PVP capped AuNPs. This difference can be associated with the nature of the capping agents. Citrate is negatively charged ion dissociates from the three sodium citrate salt while PVP is long polymer with a limited dissociable functional groups (see Figure 7-2 below for the structure of citrate and PVP molecules).

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bacteria cells presented by their zeta potential is highly negatively charged more than both types of the NPs used.

7.2.2 Interaction of citrate capped AuNPs with the Pseudomonas flourescens Citrate capped gold nanoparticles (G2 see Table 4-1 for the experimental conditions and Table 5-1 for detailed characterisation results of the sample) were added to the bacterial growth suspension in its exponential phase. After an incubation period of 2 days TEM samples were prepared through the drop method described in section 4.4.2 of the materials and methodology chapter and TEM images were recorded using both TEM JEOL 2100 and Jeol

1200. The findings were summarised in the following two sections. Distribution of Citrate capped AuNPs on the surface body of the bacteria It is worth value to know that in contrast to recently published findings where the accumulation of cationic monolayer protected AuNP on specific location on the membrane of Escherichia coli (Hayden et al., 2012) here the distribution of the AuNPs is completely random on and around the surface of the body of the bacteria. There were no localised accumulation of the NPs nor were there any signs of aggregation observed as can be seen by 8000x magnified images in Figure 7-3. This lack of self-organisation distribution of AuNP on the surface of bacteria (Staphylococus aureus) was previously reported by Chwalibog (Chwalibog, 2010).

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