Cell Culture of Spirulina Microalgae (Spirulina platensis) and Comparison the Efficiency of Enzymatic, Ultrasound, Freeze-defrosting and Mineral Solvent Methods in Extraction of Phycocyanin Pigment

 Phycocyanin is one of the pigments used in the food industry due to its antioxidant and antibacterial as well as coloring properties. This pigment is commercially produced from Spirulina platensis microalgae, in the form of photoautotrophic cultures and in open environments in large ponds or pools in tropical or subtropical areas at the edges of oceans. Different techniques are used in order to extract phycocyanin from spirulina microalgae.. Each technique has its own advantages and disadvantages besides different efficiency. These methods include freezing-defrosting, enzymatic, ultrasound, high hydrostatic pressure, ultracentrifuge, ultra homogenization, extraction using water and various solvents. Of course recently, the production of recombinant phycocyanin has been considered as a suitable option for the production of heterotrophic phycocyanin. The purpose of the current research was to cultivate Spirulina platensis, evaluation of the microalgae growth process, and comparison of the efficiency of different methods in the extraction of phycocyanin pigment.

 The pure sample of Spirulina platensis microalgae was prepared from Algaeology Laboratory, Biology Department of Tarbiat Modares University. For the cultivation of spirulina, Zarrouk culture medium with different compositions was used, and after cultivation in smaller scales (100 and 500 ml), the final cultivation was carried out in volumes of 5 and 50 liters. After cultivating the microalgae and exposing them to fluorescent light with appropriate light lux intensity (3500 to 8000) and a period of 12 hours of darkness and 12 hours of light, the samples were placed at 29 °C for 16 days. In order to evaluate the growth process of the algal mass, the absorbance of the solution containing the algal cells was read at a wavelength of 540 nm. After preparing the dry mass of spirulina microalgae, four methods of ultrasound, freezing-defrosting, enzymatic and mineral solvent technique were used to extract phycocyanin. In the next steps, the efficiency of each method was evaluated by measuring the concentration and purity of phycocyanin. In addition, the effect of applying the purification process by ammonium sulfate on the concentration and purity of the extracted pigment was also evaluated. This research was conducted in a completely randomized design and SPSS and EXCEL softwares were used for statistical analysis and drawing of diagram, respectively. Data were analyzed using one-way analysis of variance and the difference between the means was evaluated by Duncan's test at 95% confidence level.

 The results showed that microalgae growth from day 0 to 14 had an upward trend and the resulting changes were significant at all times, except days 14 and 16 (p<0.05). Also, after passing the short resting phase (2 days), the microalgae entered the logarithmic growth phase and continued to grow until the 14th day, but between the 14th and 16th days, the growth was almost constant. In the following, it was found that the mass produced after 16 days is 1120 mg/l. The concentration of phycocyanin extracted in enzymatic and ultrasound methods (1.815 and 1.786 mg/ml, respectively) had no significant difference (p>0.05) and was at a higher level than the other two methods (p<0.05); In addition, the pigment concentration was higher in the freezing-defrosting technique (1.535 mg/ml) than in the mineral solvent method (1.121 mg/ml). After purification of the pigment using ammonium sulfate, the pigment concentration and purity increased significantly in each method (p<0.05). The results of this research showed that by choosing the optimal method and applying the purification process using ammonium sulfate, the extraction efficiency of phycocyanin from Spirulina microalgae (Spirulina platensis) could be increased.

 Based on the results of this research, the growth trend of Spirulina platensis in Zarrouk culture medium was  ascending first and then constant (during 16 days). Ultrasound technique and enzymatic method (lysozyme enzyme) to extract phycocyanin pigment from Spirulina platensis microalgae have more efficiency than freezing-defrosting and inorganic solvent (hydrochloric acid) methods. Also, purification of the extracted pigment using 40% ammonium sulfate increases the concentration and purity of phycocyanin in each method.