Evaluating Methods for Optimal Phycocyanin Extraction and Purification

 Introduction: 

    From the previous experimental DOE conducted last week, it was determined that this week would be focused on beginning a similar two factor DOE using chitosan and charcoal for purification, but in alternative methods. Research suggests that a charcoal column and the use of chitosan solution, introduced prior to charcoal adsorption, in crude extract can approximately triple purity values (Patil et al., 2006), so it is hypothesized that this combination will be the most successful in terms of purity levels. Scaling up the volume of Spirulina plantesis for large scale production of phycocyanin has been the goal of all previous experimentation, and will be evaluated now that sonication methods have to achieve desirable crude extract has been roughly determined. The initial pellet produced after the first centrifuge will also be tested for any remaining phycocyanin from a tube in the DOE, to establish a protocol for resuspension and secondary extraction for optimal product yield from any given amount of spirulina. 

Methods:

    The two factor DOE was begun with four runs and two trials for each run, ordered identically to previous experiment. Tubes 1A through 4B each received one gram of spirulina, 0.25 grams of glass beads, and ten milliliters of deionized water total. Half the amount of total water was introduced initially, then the gram of spirulina, then glass beads, and the remaining water last and vortexed and shaken vigorously for thirty seconds immediately to prevent clumping. Each tube was placed in the ultrasonic bath for 1.5 hours and removed for first centrifuge for fifteen minutes at 6500 RPM. The supernatant was removed with sterile pipettes and centrifuged again under identical conditions. Testing for purity and concentration values were conducted using a nanodrop. All tubes were stored in the freezer to preserve phycocyanin stability for the following week.

    The large scale trial initially continued the 1gram:10ml ratio of spirulina to deionized water, with a total of fifteen grams of spirulina and 150 milliliters of water. Blending was conducted for two minutes, in ten second intervals. An additional fifty ml of water was used to decrease spirulina lost to blade and cap of blender. The solution was shaken thoroughly and allowed to sediment in the existing container for twenty minutes prior to extraction. The liquid was removed from the foam byproduct and dispensed into tubes for centrifugation for twenty minutes at 4500 RPM at four degrees Celsius. The supernatant was extracted and centrifuged again under identical conditions. The final extract was tested for purity and concentration values with a nanodrop. 

    The initial pellet formed by tubes 3A and 4B were identical in volume and utilized for secondary experimentation. Pellet 4B was resuspended in ten ml of deionized water, vortexed for thirty seconds, and sonicated in ultrasonic bath for additional thirty minutes. Pellet 3A was resuspended identically, and immediately centrifuged. Each tube was centrifuged for fifteen minutes at 6500 RPM, and the supernatant extracted. The initial pellet from the large scale experiment was resuspended in 200 ml of deionized water, equal to total volume used previously. This tube was centrifuged for fifteen minutes at 4500 RPM at four degrees Celsius. The supernatant was extracted and tested for purity and concentration using a nanodrop. 

Results: 

     The results of each of the DOE tubes in Figure 1 show purity values that are not considered consistent, but are useable for further testing of purification methods. In Figure 2, the results of the first trial of large scale phycocyanin production had a purity of 1.516 and concentration of 2.391 mg/ml with a total of approximately 160 ml produced. The total yield is estimated at 382.56 mg before further purification. The values of pellets 3A, 4B, and 1 are shown in Figure 3. The additional sonication time did not appear to significantly affect purity levels of extract, 3A being 0.454 and 4B 0.465, but the concentration was slightly greater from pellet 4B which was 1.698 mg/ml compared to 1.479 mg/ml from pellet 3A. The pellet from trial one comparatively had a lower purity value at 0.323 and concentration value of 0.735 mg/ml. 

Tube	A620	A280	A652	Purity (A620/A280)	Conc. ((A620 - .474A652)/5.34)mg/ml
1A	2.637	1.210	2.473	2.179	2.743
1B	2.310	1.360	2.771	1.699	1.866
2A	2.354	0.787	2.426	2.991	2.255
2B	2.829	1.342	2.499	2.108	3.079
3A	2.671	1.726	2.162	1.548	3.083
3B	2.750	1.529	2.060	1.799	3.321
4A	2.353	1.062	2.175	2.216	2.476
4B	2.438	0.833	2.390	2.927	2.444

Figure 1. Values initially achieved for each crude extract. 

Trial	A620	A280	A652	Purity	Concentration
1	2.572	1.697	2.733	1.516	2.391

Figure 2. Values of large scale trial one. 

Pellet	A620	A280	A652	Purity	Concentration
3A	1.096	2.413	0.645	0.454	1.479
4B	1.343	2.889	0.920	0.465	1.698
1	0.545	1.688	0.322	0.323	0.735

Figure 3. Values of initial pellets after resuspension. 

 

Conclusion:

    The values for purity and concentration found in the pellets of 3A and 4B indicate that further testing should be done to validate that secondary sonication may not be necessary given the amount of time that must be provided for sonication, and how little it seemed to make an impact on results. The pellet of trial one had lower values, potentially indicating that further sonication could be more useful in larger pellets as opposed to smaller ones, given the sheer amount of mass involved in scaled up extraction. It is likely that secondary extraction practices will be recommended for optimal phycocyanin extraction, due to the simplicity and efficiency of the process. The purity and concentration of trial one for large scale experimentation can be improved upon, and further trials should initially expect a fifty ml addition to the solution to control spirulina loss, in order to maintain a 1:10 ratio. More time may also be needed after sonication for the solution to completely settle and cut down on any losses to the foam that was formed during blending. Tubes 1A through 4B for the DOE do not have extremely consistent values despite identical methodology, but can be utilized for further testing.    

References:

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