Strain selection and culture of Dunaliella salina (Chlorophyceae) for beta-carotene production

Abstract

The strain selection and culture of Dunaliella salina yielding high beta-carotene were carried out in four steps, i.e. (1) field survey ;and water sampling in Chon Buri, Chachoengsao, Chanthaburi and Samut-Songkhram province for the clone selection, (2) selection of the clones yielding high beta-carotene, (3) optimizing the culture condition for beta-carotene production in laboratory and (4) outdoor mass culture. It was found that nitrate and phosphate concentrations of salt pond water samples increased with increasing salinity and decreasing pH. The number of D. salina was highest (1.5×104 cell/ml) at 300 pp. Six clones of D. salina were isolated from salt ponds in Samut Songkhram by the single cell isolation technique and clonal culture were made in ESM medium. Thereafter, the selection process was conducted by culturing the cells in three levels of salinity,i.e. 10, 20 and 30% NaCl (w/v) in J/l medium at 20,000 lux light intensity. The isolated clone DS91008 was selected. This clone produced the highest carotenoid content (80.4 pg/cell in 30% NaCl). Growth rates of D. salina were not different at three light intensities (5,000, 10,000 and 15,000 lux). The carotenoid contents increased with increasing light intensity and the cells colour changed from green to orange. The growth rate was reduced by the decreasing nitrated concentration. Increasing the light intensity and reducing nitrate concentration affected the rising carotenoid content which consisted merely beta-carotene (98%). KNO3 0.1 g/l (10% of J/l medium) and 20,000 lux light intensity, D. salina could accumulate carotenoid up to 137.2 pg/cell or 12% beta-carotene (ash free dry weight (AFDW)). The phosphate concentration and initial pH affected growth rate but did not significantly change the carotenoid content. The outdoor mass culture of D. salina was conducted in 9.1 m2 raceway pond with a paddle wheel for the water circulation. The cultivation provided specific growth rate of 0.15 or the doubling time of 4.62 days. The maximum biomass was 12.04 g-AFDW/m2 at day 22. Algal cells were harvested and dried by the freeze dry method and oven drying method (70℃). The freeze dried algal powder contained significantly higher beta-carotene content (5.9% / AFDW) than the oven dried method (1.9% /AFDW).