Growth Optimization And ?-carotene Accumulation In Two Industrial Strains Of Dunaliella Salina

Carotenoids are important natural pigments produced by many microorganisms such as microalgae,fungi and bacteria.Among them,?-carotene and lycopene are the two most widely used pigments widely used in cosmetic,pharmaceutical and nutritional health products Dunaliella salina is an eukaryotic microalga that can reproduce and grow in a high-salt environment and able to produce pigment mainly consisting of ?-carotene.Dunaliella salina can survive in high-salinity and high-light conditions by producing glycerol under osmotic stresses and ?-carotene for protection from the light.?-carotene can reach about 14%of dry weight in Duanliella salina.Environmental stresses such as high salinity,high light intensity,nutrogen deprivation are most potent environmental factors inducing ?-carotene accumulation in D.salina.The synthesis of ?-carotene carotene is regulated by the expression and activity level of carotenoid biosynthesis enzyme.Dunaliella salina produces less carotene under growth conditions.Diphenylamine was known to inhibit ?-carotene hydroxylase,which controls the key step in the conversion of ?-carotene to zeaxanthinIn this study,two industrial strains of Dunaliella salina CCAP19/18 and Dunaliella salina B01 were used as experimental materials.Dunaliella salina CCAP 19/18 and B01 were both wild type strains isolated from sea water in Australia and a saline lake in Inner Mongolia plateau,respectively.The two strains were both used for local industrial production of ?-carotene.This study optimized the growth conditions of Dunaliella salina to achieve maximal volumetric cellular density and maximal growth rate.Moreover,the two algal strains were subjected to environmental stress including nitrogen deficiency,biological inhibitors and ultraviolet irradiation to investigate their growth and ?-carotene accumulation,providing a new way to obtain more natural ?-carotene.The major results are as follows:(1)Dunaliella salina at the same cell concentration was inoculated into ATCC medium,vitamin B12-deficient ATCC medium,and ATCC medium chelated iron solution.It was found that vitamin B12 is not an essential component for the growth of Dunaliella salina CCAP 19/18 and Dunaliella salina B01,but the chelated iron solution is an essential component for the growth of Dunaliella salina CCAP 19/18 and B01.The orthogonal composition was used to optimize the other components of the ATCC medium.The optimal growth ATCC meidum for Dunaliella salina CCAP19/18 contains 1.5 g/LNa2CO3,70 g/LNaCl and the optimal pH was 8.For strain Dunaliella salina B01,the optimal growth ATCC medium contains 2 g/L Na2CO3,90 g/L NaCl and the optimal pH was 7.5.(2)The effects of nitrogen on cell growth and ?-carotene accumulation was investigated in Dunaliella salina by growing them in ATCC complete medium followed by nitrogen-deficientmediuma,the results showed that the metabolism of Dunaliella salina changed from cell growth to ?-carotene accumulation under the nitrogen deficient condition.The accumulation of ?-carotene in Dunaliella salina CCAP19/18 increased from 2.17 ?g/108 cells to 3.29 ?g/108 cells,while those in Dunaliella salina B01 increased from 2.19 ?g/108 cells to 3.25 ?g108 cells.(3)The effect of diphenylamine on the growth and accumulation of ?-carotene in Dunaliella salina.The results showed that diphenylamine at a concentration of 20 g/mL inhibited the growth of Dunaliella salina CCAP19/18 while 50 ?g/mL diphenylamine leads to highest ?-carotene content up to 5.0 ?g/108 cells.For strain Dunaliella salina B01,the growth of algal cells was also inhibited by 20 ?g/mL while 100 ?g/mL diphenylamine leads to highest ?-carotene content up to 26 ?g/108 cells.(4)To explore the effects of UV mutagenesis on Dunaliella salina.The results showed that Dunaliella salina CCAP19/18 exposed to ultraviolet light for 2 min can survive in 20?g/mL diphenylamine as compared with wild-type strain.The accumulated ?-carotene content of the irradiated algal cell population could reach 1.383 ?g/106cells.Based on the above research results and the requirements of industrial production,this study firstly selected the optimal culture medium and culture conditions suitable for industrial production of Dunaliella salina CCAP 19/18 and Dunaliella salina B01.Secondly,the two-step production method is recommended in industrial production,the optimal culture conditions suitable for the growth of Dunaliella salina cells are adopted at first,and when the concentration of algal cells reaches the maximum,the two-step nitrogen-deficient culture or the method of adding diphenylamine are used to induce Dunaliella salina Promote the accumulation of ?-carotene to achieve the maximum yield of ?-carotene in a single cell.In order to screen out mutant microalgae superior to wild algae strains,follow-up experiments should further screen the algae strains under the above mutagenesis and screening conditions and explore the mechanism of ?-carotene accumulation to find algae strains with a dominant phenotype and confirm its mutation genes and mutation sites.