Riddin, Tamsyn Louise (2009) Investigating the enzymatic mechanism of platinum nanoparticle synthesis in sulfate-reducing bacteria. Masters thesis, Rhodes University.
Efforts to discover an efficient yet environmentally friendly mode of metal nanoparticle (NP) synthesis are increasing rapidly. A ‘green’ route that avoids the high costs, toxic wastes and complicated protocols associated with chemical synthesis methods is therefore highly sought after. A biologically based protocol will provide the possibility of gaining control over the mechanism merely by manipulating the experimental conditions of the system. Given that the properties of nanoparticles are highly dependant on the morphology of the particles themselves, this mechanistic control will provide significant industrial advantages with regards to tailoring specific properties of the nanoparticles produced. The key objectives of this study were to: a) determine whether a consortium of sulfate-reducing bacteria was capable of platinum nanoparticle synthesis, b) elucidate the bioreductive, enzymatic mechanism responsible, and c) attempt to control the morphologies of the particles produced. A consortium of sulfate-reducing bacteria (SRB), isolated from sewage sludge, was used in these investigations due to the advantages a consortium provides in comparison to pure cultures. The syntrophic relationships established within the constituent species not only prevent the growth of contaminant microbes, but increases the oxygen-tolerance of the system as a whole. The sulfate-reducing consortium was shown to possess an aerobic mechanism for Pt(IV) reduction which, though different from the anaerobic bioreductive mechanism previously identified in literature, did not require an exogenous electron donor. It was demonstrated that the Pt(IV) ion becomes reduced to Pt(0) via a two-cycle mechanism involving Pt(II) as the intermediate. Further investigation elucidated the reduction of Pt(IV) to Pt(II) to be dependant on a novel Pt(IV) reductase which becomes upregulated in the presence of Cu(II), while the reduction of Pt(II) to Pt(0) occurred by means of a periplasmic hydrogenase. To our knowledge, this is the first time a coupled mechanism for Pt(IV) reduction by micro-organisms has been proposed. A cell-free, crude protein solution from the consortium produced both geometric and irregular platinum nanoparticles. The wavelength of 334 nm was chosen as a nonquantitative indicator of Pt(0) nanoparticle formation over time. The optimum conditions for nanoparticle synthesis were pH 9.0, 65 ˚C and 0.75 mM Pt(IV) as H2PtCl6 salt. In the absence of a buffer a Pt(IV) concentration > 1 mM resulted in the precipitation of protein-nanoparticle bioconjugates, due to unfavourable acidic conditions. This demonstrated that the nanoparticles were binding to and becoming stabilised by general protein in the cell-free solution. Upon addition of a sodium-bicarbonate buffer, a general increase in Pt(IV) reduction to Pt(II) was observed. The addition of the buffer also resulted in an unexplained change in particle morphology and for this reason was not used in subsequent investigations. Polyvinylpyrrolidone (PVP) was shown to compromise the reduction rate of the Pt(IV) ion by SRB cells. The presence of extracellular NP’s was suggested by the colour of the supernatant turning brown and the A334 increasing over time. Attempts to visualise the particles by transmission electron microscopy (TEM) resulted in an unexpected phenomenon where nanoparticles could be observed to form dynamically upon irradiation by the electron beam. Extended irradiation by the electron beam also resulted in structural changes of the particles occurring during observation. An increase in temperature was shown to increase the reduction rate which in turn resulted in particles decreasing in size. The starting pH was shown to have a significant effect on the reduction rate and particle morphology although specific trends could not be identified. In conclusion, the cell-soluble extract from the sulfate-reducing consortium investigated, is capable of Pt(0) nanoparticle synthesis. Precise control over the particle morphology was not attained although the mechanism was further clarified and optimal conditions for nanoparticle synthesis were determined.
|Item Type:||Thesis (Masters)|
|Uncontrolled Keywords:||Platinum, Pt(IV) Reductase, Sulfate-reducing bacteria Nanoparticles|
|Subjects:||Q Science > QR Microbiology|
|Divisions:||Faculty > Faculty of Science > Biochemistry, Microbiology & Biotechnology|
|Deposited By:||Mrs Carol Perold|
|Deposited On:||15 Mar 2012 12:57|
|Last Modified:||15 Mar 2012 12:57|
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