Increasing the Production of Carotenoids in Chlorella sorokiniana IG-W-96 by Changing the Concentration of Nutrients and Phytohormones

Introduction  Carotenoids have many effects on human health. These compounds are produced by plants and microalgae. The extraction of carotenoids from microalgae such as Chlorella has received much attention, since microalgae grow all year round (regardless of the season) and at a much faster rate than plants in non-arable lands. The aim of this research was to optimize the concentrations of nutrients (nitrogen and phosphorous) in the growth medium of microalgae with the objective of maximizing carotenoids content. At the optimized nutrient conditions, the effect of phytohormones on production of carotenoids using Chlorella sorokiniana IG-W-96 was investigated.   Materials and Methods Chlorella sorokiniana IG-W-96 was cultivated in BG11 growth medium with light intensity of 25000 lux and light: dark cycle of 16: 8 supplied with compressed air flow of 0.5 vvm containing 6% vol carbon dioxide. Under three concentrations of nitrate (0.04, 0.25, 1.5 ) and three concentrations of phophate (0.01, 0.04, 0.16 ) and carotenoid concentration was measured. Full factorial experimnetal design was performed and the resuts of the experiments were analyzed using Minitab (ver. 21.01.1). Finally, the best concentrations of nitrate and phosphate were chosen for pigments production, and at that concentration, naphthalene acetic acid (0, 2.5, 5, 7.5, 10 and 12 ppm) was added to the culture medium to check its effect on pigments production. By measuring the dry weight of C. sorokiniana, its growth rate was determined. After extracting the pigments with solvent, the concentration of the pigments was determined by measuring the amount of light absorption.   Results and Discussion Dry weight The results showed that the highest amount of dry weight was related to the treatment with nitrate amount of 0.25 , and nitrate more and less than this amount caused a decrease in growth. This result was not dependent on the amount of phosphate and was true for all phosphate concentrations. Nitrate reduction from 1.5 to 0.25 increased the growth of microalgae up to 81.8%, so that the dry weight of 0.88  reached 1.6 . However,  reduction of nitrate from 0.25 to 0.04  decreased the dry weight by 65.6%. In order to reach the maximum growth rate, it is necessary to determine the appropriate concentration of each nutrient.   Carotenoids Unlike the dry weight, not only the pigment production did not decrease with the excessive of nitrate concentration, but also the maximum amount of pigment production was related to the treatment with the maximum amount of nitrate concentration. Based on the results obtained, the concentration of carotenoids was higher in the concentration of 1.5  of nitrate and 0.04  of phosphate (6.7 ). When the nitrate concentration was very low (0.04 ), changing the phosphate concentration had no significant effect on the production rate of any of the pigments. Only when the nitrate concentration was high (1.5 ), change in phosphate concentration caused a change in pigments concentration. The increase of phosphate concentration from 0.01 to 0.04 increased the carotenoids concentration to 1.65-fold. Of course, increasing phosphate concentration to 0.16 did not affect the pigments concentration.  Based on the statistical analysis, the P-value<0.05 indicated that the effect of the factors and the model was significant. In this situation, in order to increase the production of carotenoids, naphthalene acetic acid was added to the phytohormone culture medium. At the optimal concentration of 2.5 ppm of naphthalene acetic acid, the concentration of carotenoids increased by 26.71% and reached 8.49 . However, phytohormone had no significant effect on dry weight.   Conclusion Carotenoid production using microalgae could be maximized through optimization of nutrients concentrations (nitrate and phosphate) in the growth medium. Phytohormones could further increase the prodcution of carotenoids at optimum concnetrations.