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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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first, monitoring bacteria growth with UV-vis and second, characterising the NPs after bacteria exposure by DLS, Uv-sis, and TEM.

6.4.1 Effect of AuNPs on the growth of the Pseudomonas fluorescens In the first stage, the bacteria were grown in MDM media for 12 hrs to reach the middle of the exponential growth phase before any treatment with NPs is administered. This is followed by the continuous monitoring stage in which the growth of the bacteria in untreated media and in the samples treated with 10 ppm gold NPs of different sizes and coating agents were

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hours with Uv-vis. The size of the NPs used vary from 100 nm to 5 nm to cover the whole nanoscale range and the inhibition effect of two capping agents citrate and PVP on the bacterial growth were also tested and compared with the untreated samples. The data of this part of the experiment are presented in the following sections starting first with the effect of two separate sizes of citrate capped AuNPs followed by the PVP capped particles and finally the effect caused by the gold ions.

6.4.1.1 Interaction of 14 nm citrate capped on Pseudomonas fluorescens Freshly synthesised citrate capped gold nanoparticles (G2) (see Table 5-1 in Chapter 5:for the physicochemical properties of G2 particles) were exposed on the Pseudomonas fluorescens for a period of two consecutive days to investigate their possible toxicity with bacteria. All experimental handlings were carried out in a clean flow laminar cabinet sterilised with 70% ethanol while the air in the cabinet was kept sterilised by using Bunsen burner. Gold AuNPs were added aseptically to the bacteria suspension by using 0.2 m filter. Since the effect of NPs is most of the time associated with their small size and their large surface area it was hypnotized that gold NPs may affect the growth of bacteria negatively causing bacterial growth inhibition. To test this hypothesis and to make data reliable two independent tests were carried out in two different days. Results are presented in Figure 6-17 below and each point in the graph is the average values of 5 independent replicates.

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0.15 0.14 0.14 0.13 0.12 0.12

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Figure 6-17: Optical density (OD) on 595 nm wavelength measured with Uv-Vis spectrophotometer of the bacteria treated with 14.8 nm AuNPs capped with citrate compared with the OD of the bacteria in the MDM media. Two graphs are for two independent replicates which were recorded in two different days.

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presence of gold NPs capped with citrate. This is manifested by the fact that the OD595 of the bacteria treated with NPs in both tests is slightly higher than the MDM only media especially in the exponential growth phase area of the bacteria. This increase in growth caused by citrate capped NPs can be explained since the citrate is one of the nutrients in the MDM media. The NPs solution provides more citrate available for the bacteria to consume and grow. The data also suggest that this type of NPs have no negative effect on the growth of the bacteria. It is worth knowing though that the experimental errors were calculated and the error bars show that the increase of the growth caused by gold NPs solution is not significant since the error bars are overlapping. Similar investigations were carried out for 5 nm core size citrate capped NPs (hereafter called G3) 10 nm core size PVP capped NPs (G5 and 100 nm hydrodynamic sizes PVP capped NPs (G4). Physicochemical properties of these NPs are summarised in the result Table 5-1 in Chapter 5:. Findings are listed in the following sections.

6.4.1.2 Interaction of 5 nm citrate capped on Pseudomonas fluorescens G3 NPs with final concentration of 10 ppm were exposed on Pseudomonas fluorescens growing in MDM media. The optical density was recorded and compared to the untreated sample. Size based toxicity of gold NPs was reported in some literatures where it was found out that the smaller particles manifest some degree of toxicity unlike the bigger particles(Pan et al., 2007). So, it was expected that smaller particles easily penetrate in the bacteria cells and effect its growth. The results from the G3 AuNPs on the growth of the bacteria are presented in Figure 6-18 below and it can be seen similar effect as G2 NPs where the growth of the bacteria is positively affected. This fact implies that the citrate capped particles of sizes

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bacteria as carbon resource.

.

141 0.24 0.22

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Figure 6-18: Optical density (OD) on 595 nm wavelength measured with Uv-vis spectrophotometer of the bacteria treated with 5 nm citrate capped AuNPs comaped with the OD of the bacteria in the MDM media. The two graphs are for two independent replicates of samples measured in two different days.

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earlier in the literature by several previous studies (Hwang et al., 2008, Amin et al., 2009).

Amin et al have investigated the effect of 15 nm citrate capped AuNPs on the growth of Esherichia coli and pointed out that there was no significant toxic effect caused by gold nanoparticles. In our study we found that both 14.8 nm core size and 5 nm core size (as measured with TEM) citrate capped AuNPs have no significant inhibition effect on the growth of Pseudomonas fluorescens.





6.4.1.3 Interaction of PVP capped NPs of different sizes on Pseudomonas fluorescens.

After preliminary stability investigation of two sizes of PVP capped AuNPs, one from the upper limit of the nanoscale and one near to the lowest limit of the nanoscale were chosen and their interaction with the bacterial samples were tested.

10 nm core size and 100 nm hydrodynamic diameter particles were added to bacteria suspension which is in the exponential phase of its growth phase. The final concentration of the gold in the bacteria suspension was kept at 10 ppm. As measurement of bacterial population, the optical density (OD) of the suspension at a wavelength of 595 nm was continuously recorded in a period of two days. For each size, two independent tests measured in two different days were carried out and each measurement point in the graphs represents the average value of 5 readings. Growth curves normalised on initial readings are illustrated in Figure 6-19 for 10 nm NPs and in Figure 6-20 for the 100 nm PVPAuNPs. The optical density of the bacteria treated with the PVPNPs showed similar growth curve as in the MDM media as illustrated in the Figure 6-19 below.

143 0.21 0.18 0.19 0.16 0.17 0.15 0.14 0.13 0.12

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Figure 6-19: Optical density (OD) on 595 nm wavelength measured with Uv-vis spectrophotometer of the bacteria treated with 10 nm PVP capped AuNPs compared with the OD of the bacteria in the MDM media. Two graphs are for two independent replicates of samples measured in two different days.

144 0.026

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This implies that the PVP may have some toxic effects on the growth of the bacteria as manifested by lower optical density. It is important to know though that the difference is in the experimental error margin presented by the error bars calculated from 5 independent replicates and thus the difference is not significant. Linear trapezoidal method explained in section 4.5.5 was applied to estimate the area under the growth curve of the bacterial culture.

The percentage growth inhibition caused by the PVP capped NPs was quantified using Equation 4-4 and a value of 10% inhibition was calculated for the 10 nm particles.

6.4.1.4 Interaction of gold ions on Pseudomonas fluorescens Because of its antibacterial activity, the toxicity of silver ions attracted lots of consideration and its toxic effects on a range of different bacterial variety was confirmed by different researchers (Matsumura et al., 2003, Sambhy et al., 2006, Ratte, 1999, Feng et al., 2000).

Similar studies for gold ions tested on a different variety of bacteria have indicated the toxicity of gold in the ionic form(Karthikeyan and Beveridge, 2002, Southam and Beveridge, 1994). The experiments described in the previous sections regarding the effect of gold NPs on environmental bacteria pseudomonas fluoresces had not shown any significant change on the growth of the bacteria represented by optical density of the bacteria population. It is worth knowing that the sizes of the AuNPs investigated and represented in the previous sections have almost covered the whole nanoscale range starting from 100 nm all the way down to 5 nm core size. Therefore, one final experiment investigating the effect of gold ions on the growth of bacteria was planned and conducted in a similar way as the nanoparticles experiments. In accordance with previous experiment the final concentration of the gold ions was kept at 10 ppm in the bacterial suspension and ions were added in the media after 12 hrs

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of the two independent tests carried out in two different days were plotted in Figure 6-21 below.

The data in Figure 6-21 below were normalised on the initial reading value, the experimental errors were calculated and presented as error bars in the curves. Although the initial reading of the OD in the MDM alone and in the ions - treated samples started on the same point (0.12 AU) there was an increase of OD in the case of the MDM alone and a decrease of the optical density in the case of the ions - treated samples. The decreasing optical density growth curve of the bacteria indicates that the effect of gold ions on the growth of bacteria can be classified as bactericide effect and total inhibition of the growth has occurred. This effect is clearly comparable to the effect of silver ions reported earlier.

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0.12 0.11 0.09 0.1 0.07 0.08

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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 OD of the bacteria in the MDM media. Two graphs are for two indepenedent replicates of samples measured in two different days.

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The results from citrate and PVP capped NPs on the growth of the bacteria presented in the previous sections indicated that the capping agents affected the bacteria growth in different ways. This led to further investigation of the bacteria growing in PVP and citrate media without any other carbon sources. The overall aim of this experiment was to test whether Pseudomonas fluorescens can grow in media without carbon resources except the capping agents of the NPs used in this project. The bacteria were incubated in citrate and PVP solutions separately. The concentrations of the capping agents in the solutions were comparable as their concentrations in the AuNPs synthesis media. After one week of incubation, the effect observed was presented in Figure 6-22 below. It can be undoubtedly seen that there is growth of the bacteria in citrate media manifested by the cloudy greenish colour of the solution caused by the bacteria cells producing the characteristic yellow-greenish Pyoverdine, pigment for the Pseudomonas fluorescens (Meyer et al., 2002). In the case of the PVP, there is no sign of growth at all which implies that the bacteria cannot utilise the PVP polymer as carbon source or PVP may have toxic affect on the bacteria which may be the reason why the growth of the bacteria in PVP capped NPs is slightly less than in the MDM media alone.

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6.4.1.6 Comparison between citrate NPs, PVP NPs and gold ions In summary, the citrate capped NPs have slightly increased the growth of the bacteria. In contrast, the growth was reduced slightly by PVP capped AuNPs. It is worth noting though that both effects described above were not significant since the differences were in the error margins. The effect of gold ions on the bacteria growth was clearly significant. Gold ions had not only stopped the growth but they had a clearly bactericide effect as shown by the decreasing growth curves (see Figure 6-21 above).

6.4.2 Characterisation of AuNPs after exposure on bacteria Although the AuNPs were stable in the 4x diluted MDM media there is no guarantee that their behaviour will stay the same in the bacterial growth suspension where apart from the

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citrate and PVP capped NPs have been exposed to bacteria suspension for two days and then fully characterised. TEM images of the NPS with bacteria were recorded; DLS measurements of both unfiltered samples and 100 nm filtered samples were taken. The surface Plasmon resonance (SPR) of NPs was measured. Results are presented in the following sections.

6.4.2.1 Characterisation of citrate capped AuNPs after exposure to bacteria Characterisation of citrate capped AuNPs after two days exposure on bacterial suspension was conducted using DLS, Uv-vis and TEM. DLS measurements of unfiltered bacterial suspension are illustrated in Figure 6-23 and compared with the measurement carried out after filtering the suspension with 100 nm sterile filter presented in Figure 6-24 and Figure 6-25.

29

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

151 11 9

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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.

13 11

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Figure 6-25: Size distribution by intensity of bacterial suspension treated with 14.8 nm core size citrate capped AuNPs and filtered with 100 nm pore size filter. The measurement was taken after two days of exposure at the end of the experiment.



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