Roy, Uttam Kumer (2018) Culturing and processing of Dunaliella for enrichment of enzymatic antioxidants. PhD thesis, University of Greenwich.

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Abstract

Dunaliella, a singled cell microalga, accumulates antioxidants as the main line of defence against reactive oxygen species to protect the cell from oxidative stress-induced damage. The high adaptability of this halotolerant Dunaliella makes this alga a promising and a sustainable natural source of antioxidant enzymes (catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD)). To enhance enzymatic activity in Dunaliella, and increase its commercial value, the combined effect of exogenous abiotic stressors [light (15, 145, 550 µmol photons m-2 s-1), temperature (10, 20, 30°C), nitrogen level (0.05, 0.5, 5 mM KNO3)] on cell density, the accumulation of antioxidant molecules, antioxidant enzyme activity and protein content were investigated. To evaluate the post-harvest storage stability of antioxidant enzymes, the effect of storage time (0 - 247 days) and temperature (4, -20, -80°C) on enzyme activity in four types of biomass (wet whole cells, freeze-dried whole cells, crude extract, freeze-dried crude extract) were examined. Short-term cultivation (>2 days) of D. tertiolecta at >20°C under low to medium light irradiation and with nitrogen concentration >0.5 mM KNO3 stimulated CAT activity, as did prolonged cultivation (>6 days) with high nitrogen levels which was found to increase CAT activity when light irradiation was kept at lowest level. Prolonged cultivation promoted POD activity when cells were grown with medium nitrogen levels under medium light irradiation and at low temperature, or when cells were grown with the same nitrogen levels at 20°C under high light. SOD activity was stimulated in cells when cultured with nitrogen 0.5 mM at >20°C under high light irradiation for moderate time (up to 6 days) or grown with low nitrogen at 20°C under medium light irradiation. Optimal culturing conditions were also identified for ascorbate, glutathione, phenolic, chlorophyll, carotenoid and protein content.

Response Surface Methodology (RSM) was applied to determine the optimal values of light, temperature, nitrogen level, and cultivation time for enhanced enzyme activity, which indicated that the highest CAT (18.39 U mg-1 protein) and SOD activity (14.57 U mg-1 protein) can be obtained in D. tertiolecta when cells are cultured with 4.8 mM KNO3 at 19°C under light irradiation of 331 µmol photon m-2 s-1 for 5.6 days. Maximum POD activity (0.93 U mg-1 protein) will be achieved at 10°C after 7.5 days of cultivation under light irradiation of 64 µmol photons m-2 s-1 and with 1.7 mM KNO3 in the medium. To our knowledge, this is the first study using RMS to optimise antioxidant production in microalgae.

After cultivation, harvested whole cells retain antioxidant enzyme activity for up to five months when stored at -80°C. In freeze-dried whole cells, CAT and SOD activity remain unchanged for at least eight months at -20°C. Wet cells or crude extract stored at -20°C retain antioxidant activity for up to one month only.

A simple alkaline (0.1 M NaOH) extraction method and protein assay were optimised to determine the total protein content accumulated in Dunaliella. The extraction procedure was reproducible (<3.6% CV) and suitable for quantifying protein in fresh and freeze-dried Dunaliella biomass. Also, a ‘nitrogen-to-protein’ conversion factor of 4.25 for commercial grown Dunaliella was suggested.

This research demonstrates a novel stress strategy (interactive effect of light, temperature and nitrogen concentration) to stimulate antioxidant responses in D. tertiolecta, and the employment of RSM to optimise these factors to achieve enhanced antioxidant enzyme activity. In addition, optimal post harvesting storages conditions are identified. The findings of this thesis will greatly benefit the commercial exploitation of Dunaliella as a source of antioxidants.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Microalage; Dunaliella; antioxidants;
Subjects:	Q Science > QK Botany
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	11 Aug 2022 08:41
URI:	http://gala.gre.ac.uk/id/eprint/24802

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