Optimisation of noble metal nanoparticle biosynthesis by both a prokaryotic and eukaryotic host

The aims of this work were to explore novel biosynthetic methods to produce metal nanoparticles (MNPs) with short synthesis time and focussed particle size distribution. Current methods for the synthesis of MNPs use potentially hazardous compounds or extreme synthesis conditions, which raises concerns about their environmental impact, in-vivo toxicity and cost of synthesis. MNP biosynthesis in microorganisms have been attempted to lower the environmental impact and cost of nanoparticle and nanomaterials. Although microbial biosynthesis is inexpensive and does not involve the use of exogenous capping ligands/dispersing agents for MNP stabilization, it produces a nanoparticle population with a broad particle size distribution that is unsuitable for practical applications without further refinements. In this thesis, the problems of wide size distribution and slow synthesis kinetics, in bacterial, fungal and mammalian cell hosts are addressed. The introduction (Chapter 1) provides a broad introduction on nanoscience and nanotechnology, including an overview of MNP chemical synthesis methods. Chapter 2 describe the material and experimental methods relevant to this work. Chapter 3 reports MNP biosynthesis in the dissimilatory metal reducing microorganism Shewanella sp. , and discusses the effect of mild electrochemical reducing potential on the biosynthesis rate and silver nanoparticles (AgNPs) size and distribution in Shewanella biofilms grown on carbon electrodes. Results show that the optimal reducing potential is -0.2 V vs. Ag/AgCl. AgNPs synthesis was slower at higher potential (0 V vs. Ag/AgCl), while electroplating was the prevalent process at -0.4 vs. Ag/AgCl. The particle size at -0.2 V vs. Ag/AgCl was 61 ± 1 nm. In Chapter 4, the mechanism of AuNP biosynthesis was studied in the fungi Rhizopus oryzae. Previous studies showed that redox enzymes located on the cell surface reduce Au3+ ions to AuNPs. A purified cell surface protein extract was used to generate small, uniform AuNPs. Since protein extraction may influence protein structure and the resulting AuNP size and shape, the modulatory effects of DTT, SDS and Triton X-100 extraction buffers on protein extraction and AuNP biosynthesis by the protein extracts were examined. Results show that 1% v/v Triton X-100 produces AuNPs with the best size distribution (19 ± 1nm), crystallinity and antimicrobial activity. 7 Finally, the biosynthesis of gold nanoparticles (AuNPs) using mammalian vascular endothelial and smooth muscle cells was examined in vitro in Chapter 5. Cell culture conditions such as phosphate buffer and foetal bovine serum (FBS) concentration were optimised as well as initial Au concentration. The AuNPs produced under optimal conditions were semi crystalline in nature. The average particle sizes were 23 ± 2nm for endothelial cells and 23 ± 4nm for smooth muscle cells, respectively. Results suggest that the production of reactive oxygen species during oxidative stress reduced the Au3+ ions, although there may also be some Au3+ reducing activity in the secretome. Taken together, these studies suggest that MNPs size distribution is an inherent problem of the biosynthetic process, and that MNP mean size is influenced by reducing potential, protein structure, and cell type. Crystallinity of the MNPs is dependent on the temperature of the synthesis, rather than the cell type, reducing potential or protein structure. Further the protein capping ligands uncovered in this study is predominantly proteins, although their identity is not yet clear. The use of fungal protein extracts is the most ideal strategy for MNP biosynthesis, specifically Triton X-100 protein cell extract, due to the low cost of synthesis compared to the mammalian cell culture, smaller size compared to the bioelectrochemical synthesis and repeatability. Bioelectrochemical synthesis is a promising alternative, however further optimisation is required to lower MNP size and size distribution. Bovine aortic smooth muscle cells produced a high concentration of AuNPs, may be an ideal eukaryotic host for the production of biocompatible AuNPs for in-vivo use, however a comprehensive toxicity study of these AuNPs is required.