Evaluating Methods for Optimal Phycocyanin Purification

 Introduction: 

    Previous experiments with Spirulina plantesis, a cyanobacteria which has potential antioxidant properties frequently used for health benefits, has indicated that extracting phycocyanin, a phycobiliprotein with numerous industrial uses, needs more clarification in terms of precise methodology in order to attain accurate and consistent results. Prior tests have encountered issues with testing accuracy, and the inability to compare results to a positive control of some type. To combat the latter problem, a product called "blue spirulina" will be investigated as a potential positive control so that typical spirulina extraction may be further advanced. The product claims to be one hundred percent pure blue spirulina with no additives. Typical spirulina extractions will also be investigated prior to the purification process, with a primary focus on large scale production methods. The project will also be including the work of Artemio Chavez from this point forward. 

Methods: 

    An initial solution was produced for preliminary testing by Art, using 40.2 grams of dried spirulina and four hundred milliliters of  deionized water. The solution was sonicated using the blender method, in ten second intervals for a total of thirty seconds. The homogenized liquid was centrifuged for twenty minutes at 4500 rpm four degrees Celsius with the 4250 rotor. The supernatant was removed and dispensed into a 400 ml media bottle, and placed in a -20 degree Celsius freezer as well as the four pellets produced from centrifugation. 

    I then removed five ml of thawed spirulina supernatant and centrifuged the sample for fifteen minutes at 6500 RPM. Supernatant was removed and spun identically a second time. One gram of blue spirulina was added to five ml of water and hand-shaken until homogenized. This tube was centrifuged for fifteen minutes at 6500 RPM, and supernatant removed and centrifuged identically a second time. Each tube was tested for purity levels using the nanodrop by measuring absorbances at 620 nm, 280 nm, and 652 nm. Due to initially abnormal results the blue spirulina sample was diluted by removing 0.8 ml of the blue spirulina supernatant and diluting with ten ml of deionized water in order to attain an accurate and consistent reading from the nanodrop. After testing, each tube was left at room temperature and wrapped with aluminum foil to prevent light degradation for 48 hours to observe any changes in purity. 

    After 48 hours the tubes were centrifuged for fifteen minutes at 6500 RPM. The pellets produced were relatively small and both odd in coloring, predominantly white with green streaking. Both supernatants were slightly more opaque so each was diluted by removing one milliliter of supernatant and combining with five milliliters of deionized water, shaking each to ensure homogeneity prior to testing. The large stock solution originally made by Art was also tested to observe crude purity levels. 

Results:

    The first tests performed (Figure 1) resulted in an average purity value of 0.938 (A620/A280) for the standard spirulina extraction, which exhibited fairly consistent nanodrop values for each absorbance tested. The blue spirulina sample started very inconsistent and unreliable with negative values for readings two , three, and four. Readings five, six, and seven were post dilution and exhibited high consistency. The purity average of 2.041 for the blue spirulina. 

    Figure 2 was data acquired 48 hours after the data set from Figure 1. The average purity value of the standard spirulina extraction was 0.28. This is a difference of -0.658 in comparison to the prior reading from Figure 1. The average purity value of blue spirulina solution was 2.307. This was an increase of 0.266 in purity from Figure 1.The initial crude extract, referred to as stock solution, had an average purity of 0.516. 

	A620	A280	A652
Standard Spirulina
1	2.027	2.074	1.217
2	1.951	2.060	1.178
3	2.016	2.258	1.180
Blue Spirulina
1	0.541	1.140	0.772
2	-1.667	-0.848	-1.894
3	-2.884	-2.774	-3.002
4	-3.298	-2.833	-2.745
5	2.018	1.008	1.029
6	2.138	1.018	1.045
7	2.025	1.002	1.040

Figure 1

Standard Supernatant	A620	A280	A652
1	0.318	1.128	0.154
2	0.316	1.135	0.156
Blue Supernatant
1	0.341	0.145	0.138
2	0.346	0.153	0.145
Stock Solution
1	1.355	2.543	0.795
2	1.293	2.587	0.764

Figure 2

Discussion:

    It is concluded from this data that the crude extract originally produced needs to be evaluated for adjustments in methodology that can improve initial purity values. From prior experiments it has shown to be possible to achieve values higher than food grade after this initial process, and various times for sonication with the blender will likely be tested to ensure thirty seconds is not too short for proper lysing of the cells. The difference between the standard solution and the blue spirulina in terms of results after 48 hours exposed to identical conditions at room temperature is also concerning. It suggests that the standard spirulina may not behave identically than the blue spirulina in regards to which conditions are most ideal for purification. Further testing with more replicates will be carried out once the methodology for optimal crude extract is achieved.