Submitted in fulfilment of the requirements of the degree of Master of Technology: Biotechnology, Durban University of Technology, 2014. / Cyanobacteria contain phycocyanin a light harvesting pigment found to have numerous biotechnological applications, such as: a natural colorant in food and cosmetics, fluorescent tags employed in clinical and immunological research and also in therapeutic processes. Successful phycocyanin production depends on growth characteristics, ability to accumulate high quantities of the pigment, and an effective downstream process. Therefore, the aim of this research was to optimize the extraction method and production by determining the optimal cultivation conditions for phycocyanin producing cyanobacterium. This cyanobacterium was isolated from a hypersaline water body in Kwa-Zulu Natal, and subsequently purified using traditional streak and spread plate techniques. Different cell disruption techniques and a range of buffers were evaluated for the extraction of phycocyanin. The buffer concentrations and pH was subsequently optimized. Results showed that maximum phycocyanin was extracted when cells were suspended in 50mM sodium phosphate buffer (pH-7.5) supplemented with 10 % lysozyme and then disrupted using the freeze–thaw method at -20 & 4°C. The UV-Vis absorption spectral scan of the crude extracted pigments showed a peak at 620 nm. This corresponds to phycocyanin production. Unwanted proteins were removed using a 25and 50% saturated ammonium sulphate precipitation, followed by dialysis. SDS-PAGE showed two subunits with molecular masses of 19 and 20 kDa. These masses corresponded to phycocyanin α and β subunits. Furthermore, a food grade purity ratio (A615/A280) of 1.20 was achieved. The effects of various abiotic factors (temperature, light and pH) on growth and phycocyanin production of the Cyanothece sp. was investigated. Temperature ranging from 20-45°C and pH (5-10) was evaluated for 2 weeks. Cultures were then subjected to four photoperiods (24:0, 18:06 12:12 and 8:16 h light: dark) three light intensities (25, 75 and 125 µmol photons per m2 per –s) at varying wavelengths i.e. blue, red and green and Grolux light. Ideal conditions were observed at 35°C, 125 µmol photons.m2.s-1 of Grolux light for a 16:8 light and dark photoperiod. It was observed that the highest biomass and phycocyanin production was found to be at 35°C, temperatures below or above resulted in a decrease in both growth and pigment synthesis. Phycocyanin concentration changed in response to light quality and intensity. A significantly higher (p<0.05) phycocyanin yield was found when the culture was exposed to 125 µmol photons.m2.s-1 of Grolux light compared with the other three light conditions. Using Design of experiments, a series of fractional factorial experiments were carried out to optimize media components for pigment production. The final optimized growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 2g/L NaNO3, 0.06g/L K2HPO4, 0.12 g/L MgSO4.7H2O, 0.033 g/L CaCl2.2H2O, 100g/L NaCl, 12mL minor nutrients and 0.5 trace metal. A 72 % increase in phycocyanin was observed. This research revealed that this particular Cyanothece sp. shows great potential as a reliable source of phycocyanin.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:dut/oai:localhost:10321/1117 |
| Date | 08 August 2014 |
| Creators | Mogany, Trisha |
| Contributors | Bux, Faizal |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 179 p |
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