Evaluating Methods for Optimal C-PC Extraction

Introduction: 

    Spirulina plantesis has been so far evaluated for methods of extraction of phycocyanin in terms of solvent used, lysing protocol, and materials involved. From previous DOE experiments, it was determined that water was comparable to phosphate buffer, beads optimal for minimal volumes, and blending and ultrasonic bath very similar to just utilizing an ultrasonic bath alone. Now that a basic protocol of crude extraction has been determined, the next step is purification of this crude extract for maximum purity, concentration, and total yield. Charcoal has been found to be beneficial for purification, in various methods, with even higher purity levels achieved with the addition of chitosan as well. It has been shown that a crude extract of 1.18 purity can achieve a level of 2.56 after purification with charcoal, and 3.96 with the use of chitosan and charcoal. Charcoal is used in amounts of 70-80 g/L (Patil et al., 2006). Chitosan is a water insoluble carbohydrate bipolymer which is often used for its adsorption properties to remove excess oil from wastewater. A two percent acetic acid solution can be utilized to evenly distribute a two percent chitosan solution (Ahmad et al., 2004). This week a two factor DOE was started in order to test charcoal and chitosan for purification purposes of crude extract. Factor A will be charcoal, and Factor B will be chitosan, in an experiment which will have two trials for accuracy of results.

Methods:

    One gram of spirulina was used in each fifteen ml tube, with ten ml of water added to each. The first five ml of solvent should be added first, then the spirulina, and finished with the last five ml of solvent in order to prevent clumping, which hinders the ability to lyse all of the cells possible. Half of a gram of glass beads were also added to each tube. The tubes were vortexed for thirty seconds and vertically shaken after the deionized water, spirulina, and beads were added. Tubes 1A and 1B were the control tubes and did not receive any additional supplements. Tubes 2A and 2B received 0.8 grams of charcoal and were vortexed again for thirty more seconds. Tubes 3A and 3B had one ml of  two percent chitosan solution added, the solution made by adding four ml of two percent acetic acid to 0.8 grams of dried chitosan and vortexed for thirty seconds before allowing to dilute evenly for one hour. The tubes were vortexed for thirty additional seconds to distribute the chitosan evenly within the tubes. Tubes 4A and 4B received one ml of chitosan solution and 0.8 grams of charcoal, and were vortexed for thirty seconds. All tubes were sonicated with the ultrasonic bath for two hours, and shook every half hour to ensure a lack of clumping. The tubes were then centrifuged for thirty minutes at 6500 RPM, and their supernatant removed and deposited into new tubes to undergo a second round of centrifugation at 6500 RPM for another thirty minutes. Results were acquired with a nanodrop device. 

Results: 

    Tube 1 and it’s two trials recorded the best results for purity on average, with tube 1A, a control tube, having the highest purity value of 2.522. The lowest value was from tube 4A, with a purity level of 0.996. As seen from the spreadsheet showing statistical significance of factors A and B in regards to purity levels acquired, chitosan is strongly negative in terms of significance. Factor A, charcoal, was not found to be statistically significant in comparison to the controls.    

Tube	A620	A280	A652	Purity	Conc.
1A	2.540	1.007	2.80	2.522	2.27
1B	2.732	1.258	2.617	2.172	2.79
2A	2.206	0.91	2.6	2.42	1.98
2B	2.803	1.453	2.618	1.93	2.93
3A	2.497	1.940	2.080	1.287	2.83
3B	2.498	1.992	2.105	1.254	2.81
4A	2.669	2.678	1.994	0.996	3.23
4B	2.834	2.167	2.491	1.308	3.09

Conclusion: 

                Although trying to achieve a higher purity value in this DOE, the opposite was produced. The charcoal, and particularly chitosan, were found to have negative impacts on purity values rather than beneficial ones. It has been determined that another DOE needs to take place, using the same factors but in different stages of the process. It is thought that acquiring a crude extract first, after two rounds of centrifugation, and then applying charcoal and/or chitosan in a traditional column setup as opposed to adding them in the beginning of the experiment may be much more beneficial. The chitosan may also need to be introduced as a different volume or amount, because its strong negative impact is concerning for future experiments. Only after applying the charcoal and chitosan to the crude supernatants will it be possible to determine how useful they really are for the purification process of phycocyanin from spirulin. 

References: 

Ahmad, A. L., Sumathi, S., & Hameed, B. H. (2004). Chitosan: A natural biopolymer for the adsorption of residue oil from oily wastewater. Adsorption Science & Technology, 22(1), 75–88. https://doi.org/10.1260/026361704323151015 

Patil, G., Chethana, S., Sridevi, A. S., & Raghavarao, K. S. M. S. (2006). Method to obtain C-phycocyanin of high purity. Journal of Chromatography A, 1127(1-2), 76–81. https://doi.org/10.1016/j.chroma.2006.05.073