3. Electronic Theses and Dissertations (ETDs) - All submissions

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    Genomic and transcriptomic analyses of epsilon-poly-L-lysine producing Streptomyces albulus
    (2018) Crosse, Amanda Jane
    Biodiesel production has increased significantly in the past 15 years since it offers a renewable solution to fossil fuels. This production increased the availability of the process’s by-product, crude glycerol. Crude glycerol contains harmful contaminants and with expensive disposal options, often ends in landfill sites where it causes environmental problems. Crude glycerol conversion to valuable products enhances value and aides with disposal problems. Streptomyces albulus was shown to utilize biodiesel-derived crude glycerol as a sole carbon source. Commercially the bacterium is used to synthesize useful secondary metabolites from glucose, e.g. ε-poly-L-lysine (ε-PL). This work aimed to provide a genomic platform for genetic engineering to optimize the production of key secondary metabolites. The first draft genome of S. albulus was sequenced and annotated, together with several relevant transcriptomes. Bio-informatic assessment identified potential genes and pathways involved in glycerol, glucose and ε-PL metabolism. Additionally, 56 silent secondary metabolite gene clusters not previously known, were also identified. Transcriptionally active gene candidates were confirmed by sequencing transcriptomes for different carbon sources (glucose and pure glycerol) over the course of exponential to stationary growth phases – phases linked to ε-PL metabolism. Analyses revealed that the glucose transporter and glycerol uptake facilitator proteins’ differential expressions halved with a pH drop. Glycerol kinase’s and glycerol-3-phosphate dehydrogenase’s (aerobic isoenzyme) relative expression levels decreased with a similar factor, though glycerol-3-phosphate dehydrogenase (anaerobic isoenzyme) showed an increase. Aspartokinase relative expression levels during ε-PL synthesis remained high for both carbon sources, thereby providing S. albulus with sufficient L-lysine monomers during production. Interestingly, ε-PL synthase and both ε-PL degrading enzymes were expressed throughout the growth cycle of S. albulus with both carbon sources, but with ε-PL synthase levels higher at pH 3 than pH 5. This infers that the activity of these enzymes may be more reliant on pH, or substrate availability, than gene expression levels. Genomics, transcriptomics and culturing analyses revealed S. albulus as a halotolerant microorganism. Indeed, three of the four genes of the ectoine pathway, a hydroprotectant, were upregulated with increased salt concentrations. Interestingly the glycerol operon is also upregulated under the high salt concentrations.
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    Biodiesel production with waste product cycling
    (2018) Makhale, Itumeleng
    Bio-derived fuels such as biodiesel and bioethanol have gained considerable attention as liquid transport fuels. Economic viability for large-scale bio-based fuel production in South Africa remains a contentious issue. This study aims to describe an enzymatic biodiesel production process, with a greater goal to stimulate small-scale biodiesel production in and around the country. The possibility of using an E. coli host strain to develop a bacterial-biodiesel generating system was considered utilising a synthetic lipase for the trans-esterification of sunflower oil. The conditions for expression were evaluated with induction trials at 30 and 37 °C respectively; the concentration of IPTG was adjusted in 0.25 mM increments from 0 to 1 mM. The ideal concentration of IPTG was 0.75 mM, and the ideal temperature was 37 °C. The lipase's hydrolytic capacity was further evaluated on para-Nitrophenol palmitate, and the catalytic amount necessary was determined to be below 0.00729 µg for the substrate concentrations employed.
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    Biosynthesis and characterization of metallic nanoparticles produced by paenibacillus castaneae
    (2017) Hiebner, Dishon Wayne
    Nanomaterials (NMs) have been shown to exhibit unique physical and chemical properties that are highly size and shape-dependent. The ability to control synthesis of nanoparticles (NPs) with particular shapes and sizes can lead to exciting new applications or enhancements of current systems in the fields of optics, electronics, catalytics, biomedicine and biotechnology. Due to increased chemical pollution as well as health concerns, biological synthesis of NMs has quickly emerged as potentially being an eco-friendly, scalable, and clean alternative to chemical and physical synthesis. In this study, the inference that the heavy metal-resistant bacteria, Paenibacillus castaneae, has the propensity to synthesize metal NPs was validated. NP formation was achieved after the exposure of bacterial cell biomass or cell-free extracts (CFE) to excess metal ion precursors in solution. These include lead nitrate and calcium sulphate dehydrate, gold (III) chloride trihydrate and silver nitrate, respectively. All reactions were incubated at 37 °C for 72 h at 200 rpm and observed for a colour change. UV–visible (UV-Vis) spectral scans (200 nm – 900 nm) were measured on a Jasco V-630 UV-Vis spectrophotometer. For scanning electron microscopy (SEM), samples were fixed, dehydrated and loaded onto carbon-coated aluminium stubs. The stubs were then sputter-coated with either Au/Pd or Cr and analysed on the FEI Nova Nanolab 600 FEG-SEM/FIB. Size distribution analysis was done using transmission electron microscopy (TEM) using the FEI Tecnai T12 TEM and Image J software. Powder X-ray diffraction measurements were carried out on a Rigaku Miniflex-II X-ray diffractrometer. Colour changes indicative of the synthesis of PbS, Au and Ag NPs were observed as a white precipitate (PbS), purple (Au) and yellow-brown (Ag) colour, respectively. This was confirmed by absorbance peaks at 325 nm and 550 nm (PbS), 595 nm (Au) and 440 nm (Ag) from UV-Vis analyses. Exposure of P. castaneae biomass and CFE to PbS ions in solution resulted in the production of nanospheres, irregularly-shaped NPs, nanorods, nanowires as well as large nanoflowers. Exposure of P. castaneae biomass to Au3+ ions in solution produced Au nanospheres, nanotriangles, nanohexagons, nanopentagons and nanopolyhedrons. Ag/AgCl NP production occurred using both the P. castaneae biomass and CFE, and resulted in the synthesis of nanospheres only. This is the first report of the biosynthesis of such a diverse set of anisotropic NPs by P. castaneae. It is also the first instance in which anisotropic PbS nanorods and nanowires, 3-D Au nanoprisms as well as “rough” Ag/AgCl nanospheres were bacterially produced. This study serves as an eco-friendly approach for the synthesis of NPs that is a simple yet amenable method for the large-scale commercial production of nanoparticles with technical relevance. This in turn expands the limited knowledge surrounding the biological synthesis of heavy metal NMs.
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