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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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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.

This random distribution added to the lack of aggregation became more obvious through the images taken with higher magnification of 25000x (Figure 7-3 b) which shows single rounded AuNPs on and around the curvature of smooth, undamaged cell membrane. The stability of the AuNPs in the bacteria suspension is clearly confirmed. Effect of citrate capped AuNPs on the outer membrane of the bacteria Figure 7-4 below illustrates bacterial samples treated with G2 AuNPs. Different images represent different magnifications. Special attention was paid to detect any structural changes of the outer membrane of the bacterial cells. The smooth, undamaged outer membrane demonstrated by all images indicates that citrate capped gold nanoparticles have no effect on the structure of the membrane. Although it may be expected that due to the electrostatic repulsion between the negatively charged nanoparticles and negatively charged cell membrane (as shown by the zeta potential in Table 7-1 above) close membrane-NP interaction does not occur, Figure 7-4 below shows AuNPs touching on the membrane as indicated by

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molecules on the surface of the nanoparticle were consumed and removed by bacteria, aggregation of AuNPs was inevitable but the very stable nanoparticles in the suspension throughout the exposure period clearly show that they remained coated and stabilised by the citrate.

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.

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The first observation is that PVP capped AuNPs are quite stable in the bacterial growth suspension and there were neither aggregation nor shape transformations observable (Figure 7-5). Furthermore, as in the case of citrate capped particles, the PVP capped particles are randomly distributed on and around the outer membrane of the bacteria cells. Again here there are no localised accumulations of the NPs on specific areas of the bacteria body.

Another important observation is that these cells treated with PVP capped NPs (Figure 7-5 below) have produced more extracellular polysaccharides (EPS) pointed to with arrows between and around cells than the cells growing in blank media (see Figure 7-9 below for comparison).

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bacterial cells, the effect of the PVP capped gold nanoparticles on the surface of the bacteria cells was apparent as can be seen in the following sections. Holes on the surface of the bacteria The severe damage on the outer membrane of the bacteria cells caused by the PVP capped AuNPs was manifested by the formation of wide holes on the surface of the membrane as presented with the TEM images in fFigure 7-6igure 6.6 below which are clearly showing this effect. Those holes which were absent in the images taken after the treatment of citrate capped AuNPs are much wider than the NPs (10 nm core size) treated. These wide holes may possibly facilitate the subsequent internalisation of the PVP capped gold nanoparticles in the cell. Similar effects caused by silver NPs on Escherichia coli were reported previously (Li et al., 2010b). Though the PVP molecules are slightly negatively charged, the electrostatic repulsion forces between the PVP capped gold nanoparticles and the outer membrane of the bacterial cells were not strong enough to prevent the adhesion of the PVP coated NPs on the outer-membrane facilitating the bacteria NPs interaction and the subsequent damage of the cell membrane.

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Figure 7-6: Apparent pits and wide holes on the surface of the outer membrane of the Pseudomonas fluorescens caused by PVP capped AuNPs. Images a and b are 8000x times magnified. c and d are 25000x magnified. Blebbing on the surface of the bacteria Another apparent effect on the surface of the outer- membrane of the bacteria cells caused by PVP capped AuNPs is the formation of blebbings which are abnormal vesicular outgrowths of the outer-membrane of the bacterial cells (indicated with arrows in Figure 7-7 below).

Blebbings are clearly wider than NPs and some are around 100 nm in diameter (Figure 7-7 e.).

Most Blebbings are more electron-dense then the rest of the bacteria cells which may indicate accumulation of NPs and the damaged material of the cell membrane.

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A more rigorous cell architecture damages than the previously mentioned blebbing formations are the complete bursting of some bacterial cells and the formation of long tubular structures on the surface of the outer membrane of the bacterial cells as shown in Figure 7-8 below. Both of these damages were manifested by the bacterial cells treated with PVP capped gold NPs unlike the citrate capped and blank samples inFigure 7-9.

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.

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7.2.4 Internalisation of the NPs in the bacteria cells The toxicity of the NPs on the bacteria was many times attributed to the dissolved ions which easily follow through the cell membrane and manifest their toxicity in the cytoplasm (Ratte, 1999, Sambhy et al., 2006). This toxicity of the ions was supported by the data presented in chapter 5 section which shows complete inhibition of the bacteria growth after the

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the actual internalisation of the NPs in the bacterial cells and the presence of both Ag-NPs (Morones et al., 2005b) and metaloxides ( ZnO and MgO) (Sinha et al., 2011, Stoimenov et al., 2002) inside bacterial cells were confirmed.

The wide holes on the outer membrane of the bacteria caused by the PVP capped NPs described above in section may form easy pathways for the NPs to get inside the bacteria cells. To study these effect, bacterial cells in their exponential growth phase were treated with PVP capped NPs. After an 8-hour exposure bacterial cells were fixed using phosphate buffered non-coagulant dialdehyde glutaraldehyde fixative which stops all biochemical reaction through forming a covalent bonding bridge between proteins in the cells and making them insoluble. In this way, all biological activities were frozen and the structure of the samples was preserved. The so prepared samples were then sectioned into ultra-thinsections using ultra-microtone and stained with uranylacetate to provide contrast for the followed TEM imaging. Sections were placed on TEM copper grid and analysed using jeol2100 operating with 200KV (see section 3.1.2 in the methodology chapter for detailed description of the TEM microscope). Results were presented in Figure 7-10 below. 8 hours was chosen as exposure time since bacteria cells may die naturally if much longer time is waited and their membrane may get damaged if the time allocated for exposure is longer.

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cells were sectioned randomly in different positions. Some of them longitudinally in the whole length of the rode shape bacterial cells while others were sectioned halfway through or just the top of the cell was trimmed. The presence of stable gold NPs in extracellular media between sections of the bacterial cells can easily be seen in Figure 7-10 c and d as indicated with arrows. What is more interesting to observe are the darker electro-dense areas in some cells as shown in Figure 7-10 c, e and f and indicated with arrows. These can be aggregates of the NPs in the cells. To confirm whether or not the more electron-dense dots inside the sections of the bacterial cells are aggregates of AuNPs, EDX spectrum of a normal point in the cell was recorded (Figure 7-11 below) and compared with EDX spectra of the some black dots (Figure 7-12 below).

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

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aggregations of AuNPs, have not shown the presence of gold element. Similarly, the EDX spectrum taken outside the black more electron-dense areas did not show any gold NPs. Since the main element present in the EDX analysis and shown in the spectra is copper, it is probably copper from the TEM grid that presents the black dots. Therefore, it can be concluded that internalisation of gold NPs in the Pseudomonas flourescens bacteria cells did not take place.

7.3 Conclusion Finally, this study has shown that both citrate capped AuNPs and PVP capped AuNPs are very stable in the bacteria growth suspension. There were no aggregation or shape changes observable. Furthermore, unlike citrate capped AuNPs which have not affected the membrane of the bacteria PVP capped AuNPs have a significant effect on the morphological structure of the outer membrane leading to the formation of wide holes, blebbing, tubular formation on the surface of bacteria membrane and complete bursting of the affected bacterial cells. Since citrate capped AuNPs are much more negatively charged than the PVP capped ones previous studies (Stoimenov et al., 2002, Hamouda and Baker, 2000) speculated the importance of electrostatic interaction for the bacteria-NPs interaction but in this study the actual contact of citrate capped NPs on the surface of the cell membrane is visualised.

The two types of NPs used here were both spherical in shape and had similar sizes they do only differ in the coating agents. The environmental implication of this finding is that the effect of environmental NPs on microorganism is mainly determined by the type and surface chemistry of the coating agents such as organic humid substances.

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Nanotechnology is a quite recent and promising field due to the novel and useful applications of the nanomaterials (NM)) which form the basis for many technological fields and scientific areas. This field has opened the door for the production of uncountable new materials due to the discovery of fascinating and unique properties of the matter in the nanoscale. The influence of the nanotechnology on modern society is both explained and justified by the scale of the variety of scientific and technological applications using nano-based ideas. It is very clear that these products will sooner or later reach the environment. Although engineered nanoparticles are designed to fulfil a special purpose and we may have an idea about their original properties during their production, the fate and properties of the NPs, once released in the environment, may differ partially or completely from their original physicochemical properties due to a number of possible reasons: they may form aggregates of macroscale, get coated with organic matter in the environment or interact with other inorganic nanosized particles in nature.

One of the most noteworthy facts to consider regarding nanoparticles is their impact on the natural environment both in short and long term exposures. It is interesting to note that the scientific community is more than ever ready and willing to know and assess the possible adverse effects of nanotechnology on the environment in order not to commit the past mistakes of producing, using and releasing new products (such as asbestos, plastic) into the environment without knowing their impact on different compartments of our planet. In fact, among all environmental compartments, the biomass population is by far the most vulnerable to the effects of the released products into the environment.

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this study was set out to explore the effects of the NPs on planktonic bacteria abundant in the environment. The overall aim of the study was to investigate both the bacterial growth and the inhibition effect of the NP and the interaction of NPs on lab - grown bacteria and interpret this interaction in terms of property-response relationship. Special attention was paid to the effect of both core size and surface chemistry of the NPs on the bacteria by producing well constrained nanomaterials in terms of size, shape, dispersion and stability. Nanoparticles with core sizes varying from 5 nm to 85 nm and with two different coating agents were synthesised using wet chemistry bottom up synthesis methods. A comprehensive list of analytical and imaging techniques was used to fully characterise these freshly - synthesised NPs. Since the size, shape and monodispersity of gold NPs are fundamental features of their effect on the living organisms, these fully - characterised gold NPs can form a reliable basis for a potential reference material which helps the identification of the ecotoxicological effects caused by certain nano-property. The range of sizes and coating agents will provide more versatility for the study of property – effect relationship in (eco) toxicology studies. The stability of the AuNPs for a period of at least six months was monitored. The NPs showed no change in size and shape. The need for testing the stability of the NPs in the bacterial growth media and for fully characterising them in the media in terms of their original physicochemical properties - such as size and shape prior to the exposure process - is profoundly important since the media conditions may alter the intrinsic properties of the NPs and, therefore, make it difficult to interpret any possible effect.

After extensive characterisation of the NPs in the coating agent media and bacterial growth media (Minimal Davis Media), the effect of the size and coating agents on environmental abundant bacteria Pseudomonas fluorescens were investigated. Nanoparticles of comparable

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