Application of whey as a medium for cultivation of Donalia salina microalgae

Introduction: Microalgae are considered to be functional foods due to their unique bioactive compounds. Dunaliella salina is a single-celled green alga that lives in coastal waters, brackish waters, and rocky parts of ocean waters. This marine microalga contains beta-carotene pigment, phycocyanin, polysaccharides, iron, and zinc. Dunaliella salina is most important because of its high ability to collect various levels of beta-carotene. Beta-carotene of Dunaliella salina is currently widely used in the food and pharmaceutical industries. Feeding alkaline algae due to the presence of vitamins C and E and large amounts of beta-carotene increase the activity of complement and lysozyme, and ultimately increases the body's immune system. Also, the high level of protein and its usability has made algae a valuable source of protein. The price of the cultivation medium is one of the important factors affecting the final price of the biotechnological product. Whey is used as a culture medium for the cultivation of microorganisms, such as bacteria and algae, due to water-soluble proteins, minerals, organic acids, and vitamins. Regarding the high cost of culture medium used for microalgae cultivation, the aim of this study was to determine the feasibility of Dunaliella salina microalgae cultivation in whey as a cultivation medium and to study its biomass production.
Material and methods: In this research, the effect of different ratios of whey and Walne medium on the biomass production of Dunaliella salina and biochemical properties including total phenolic compounds, total flavonoid, soluble sugars and antioxidant activity were studied. For this purpose, three factors including the concentration of Walne medium (0, 25 and 50 μl), whey (0, 2.5 and 5%), and incubation time (0, 7 and 14 days) were studied using the response surface methodology in a Box-Behnken design with 17 samples. After collecting the data, a second-order model was used for fitting and regression analysis at α = 0.05. Finally, the optimal conditions were determined using numerical optimization and based on the desirability function. Statistical analysis was done with Design-Expert 17. For the cultivation of Dunaliella salina microalgae, 20 ml of algal suspension was added to 100 ml of mixed medium with various concentrations of Walne medium and whey in a sterile condition. The pH of the culture medium was adjusted to 5.7 by 6N HCl and NaOH. In order to create an ideal growth condition, the salinity of the culture medium was constant. The incubation temperature was 25◦C and light intensity was 2500 lux. The samples were kept in the same conditions for 14 days and the air pump was used to prepare aerobic condition for the samples.
Results and discussion: The results showed that with increasing whey content as well as Walne medium, cell density and flavonoids increased, which it was statistically significant (P<0.05). The results showed that the incubation time had a significant effect on cell density, antioxidant activity and soluble sugar (P<0.05), and cell density, antioxidant activity, soluble sugar, and amount of flavonoid decreased by increasing the incubation time. Also by increasing the percentage of whey and incubation time, the amount of soluble sugar decreased significantly at the end of incubation time (P<0.05). With increasing whey, total phenol content was not significantly decreased and soluble sugar content remained constant (P<0.05). By increasing the concentration of Walne medium and the incubation time, the antioxidant activity and soluble sugar decreased significantly (P<0.05). Minerals of whey such as calcium, phosphorus, magnesium, zinc, sodium and potassium are present in some of the chemical compounds of the Walne medium. As well as, whey contains a variety of water-soluble vitamins such as vitamins B₁₂, B₆, B₅, B₂, Acid folic acid and ascorbic acid which are essential to grow of Dunaliella salina and caused to increase its cell density. Sala et al. (2016) investigated the effect of whey protein concentrate on Spirulina platensis algae, and concluded that the use of whey protein concentrates in algae cultivation media increased the cell density (biomass) of Spirulina platensis. Generally, the changes in the antioxidant activity of Dunaliella salina cells depend on the concentration of nitrogen and NaCl sources. Also, the antioxidant level in Dunaliella salina increases with low nitrogen levels and high NaCl concentration and high light intensity. Therefore, due to the low nitrogen sources in this study, the antioxidant activity was decreased. Most algae increase the production of phenolic compounds under the environmental stress. Since this research has tried to minimize the environmental stresses in Dunaliella salina cultivation medium, it has reduced the production of phenolic compounds. Also, because phenolic compounds are produced at low temperatures, and the applied temperature to the growth of Dunaliella salina in this study was high which caused to reduce the amount of phenolic compounds. According to the literature, dilution of nutrients in Zarrouk medium and the addition of whey protein concentrate with high levels of lactose and low level of phosphorus, potassium and iron increase carbohydrate production in Spirulina platensis. So, the nitrogen concentration in the cultivation medium is a main factor that affecting the accumulation of carbohydrates in microalgae.
Conclusion: The optimum conditions were obtained at day 5 of incubation time, 5% whey and 0% of Walne medium. Based on the results of this study, the use of whey as a medium for cultivating Dunaliella salina microalgae has been successful.