Evaluating Methods for Optimal Phycocyanin Extraction

 Introduction

    For the past few weeks while I have waited to become part of the program, I have been working with spirulina platensis, which is a multifilamentous prokaryotic cyanobacterium. The predominant phycobiliprotein in spirulin is C-phycocyanin, otherwise known as C-PC, or may be referred to going forward as simply phycocyanin. Phycocyanin presents as a blue color when extracted from spirulina, and is used in the industry as a dye in food or other various products, a marker in biomedical research, among other things. It also has standard qualifications for specific uses, measured by calculating absorbance levels, with 0.7 and above being food grade, 3.9 considered reactive grade, and any value above 4.0 to be analytical grade (Patil et al., 2006). So far I have been utilizing various methods to attempt the highest level of purity and concentration possible, while also trying to make the process as efficient and simple as can be accomplished. This is predominantly done by a sonication method to lyse the spirulin cells, and then undergoing 2 centrifuge cycles. Prior to the past week, I had been able to acquire a purity above food grade on a small scale, and we were looking to upgrade this process to a larger scale, with the goal of 1.0 mg of total phycocyanin to be extracted in mind. I had decided to go forward with a 1 part spirulin to 5 parts distilled water/phosphate buffer, in 50 ml tubes, with no glass beads. The glass beads had been used in previous trials, but I was also looking to expedite the process by using the sonicating machine which could potentially sonicate the solutions in 2 to 4 minutes, rather than multiple hours in the ultrasonic bath. Hence, no glass beads were used and I went forward with a total of 4 50 ml tubes, each containing 6 grams of spirulin. 2 of the 4 tubes were diluted with 30 ml of deionized water, while the second pair was diluted with 30 ml of 0.1 M phosphate buffer, previously filtered and had a pH of 7.0. I also adjusted the times that each tube would be sonicated for, with 1 deionized water and 1 phosphate buffer being sonicated for 120 seconds, and 1 of each sonicated for 240 seconds, each trial going 30 seconds on and 3 seconds off, in order to evaluate any obvious differences in doubling the sonication time. During sonication it was obvious that a large percentage of spirulin was being lost to the machine, and the solution became a viscous sludge. I decided to modify the methods starting the next week. 

Methods

Considering the previous week was unsuccessful during the sonication process, I attempted to save the material by individually emptying each 50 ml tube into a blender, and diluting the solution until deemed necessary. Tube 1 (deionized water) was diluted with approximately 100 ml of additional deionized water, and blended for a total of 10 seconds, with 10 pulses and 1 second intervals. After blending, the solution was distributed to three 50 ml to prepare for centrifugation. Tube 2 (deionized water) also received an approximate 100 ml to dilute the solution, and blended in an identically to Tube 1. After blending, Tube 2 was distributed to three 50 ml tubes to prepare for centrifugation. The consistency of both solutions after blending were low in viscosity. Tube 3 (phosphate buffer) received  15 ml of additional phosphate buffer for the dilution process, and blended for a total of 10 seconds, with 10 pulses and 1 second intervals. It was distributed back into one 50 ml tube. Tube 4 (phosphate buffer) received 15 ml of additional phosphate buffer to achieve dilution and blended identically to all the previous tubes. Tube 4 was distributed into a 50 ml tube after blending. Tubes 3 and 4 were less viscous than prior to blending and dilution, but not as low in viscosity as Tubes 1 and 2. All of the blended solutions presented with a layer of foam, which was disposed of once the liquid was extracted from the blending apparatus with a 10 ml pipette. All resulting 50 ml tubes of blended solutions were placed in the ultrasonic bath for 60 minutes to finish sonication process. Tubes 1 through 4 were then centrifuged at 9500 RPM for 30 minutes at 4 degree Celsius. This step was repeated after extracting the supernatant from the original set into new tubes, for another 30 minutes at 9500 RPM at 4 degrees Celsius. The resulting solutions were individually  tested using the nanodrop, for the absorbance values at A620, for C-PC, A652, for APC, and A280, for the total protein content. The concentration of the solutions were calculated with the equation C-PC (mg/ml)=[A620 — 0.474(A652)]/5.34 (Patel et al, 2004). In order to calculate the purity level the equation A620/A280 was used (Patil et al, 2006). 

Results

Tube 1 had the values of 20.35, 14.44, and 13.84 for A620, A652, and A280 respectively. The purity was calculated to be approximately 1.47 and the concentration 2.5 mg/ml. There was a total of 132 ml of solution, resulting in an approximate 324.56 mg totally yield. Tube 2 had the values 22.19, 15.16, and 14.57 for A620, A652, and A280. The purity level was found to be 1.52 and the concentration was 2.74 mg/ml, with a final volume of 140ml, giving a total yield of approximately 383.6 mg. Tube 3 had the values 23.65, 14.74, and 14.08 for A620, A652, and A280. The purity level was found to be 1.68, and the concentration 3.1 mg/ml with a volume of 73 ml for a total yield of 228.75 mg. Tube 4 had 24.24, 18.83, and 9.36 for A620, A652, and A280. The purity level was determined to be 2.59, and the concentration 2.8 mg/ml. The volume of the solution was 68 ml and the total yield was found to be 190.4 mg. 

Conclusion

Tubes 1 and 2, which were diluted with deionized water, had slightly lower purity values than Tubes 3 and 4, which had phosphate buffer rather than water. The concentration of Tubes 2 and 3 were almost identical, while Tube 1 was only slightly lower, and Tube 4 slightly greater by a noticeable amount. Tubes 1, 2 and 3 were all relatively close in purity levels, with Tube 4 being considerably greater; which indicates that the sample may have not been an accurate representation of the the entirety of the solution. Further tests should be considered to ensure that human error did not affect any values, and I will likely take multiple nanodrop readings in the future and average the values for a more accurate result. A greater number of trials would also be able to reveal if there is any consistency among these trends that phosphate buffer is a more effective solvent for phycocyanin extraction, as well as go forward testing the blending and ultrasonic bath method in comparison to previously used techniques, such as the introduction of glass beads. Although objectively successful results in terms of potential yield to be extracted from these four solutions, the exact method for greatest results is still unclear.  

References

Patel, A., Mishra, S., Pawar, R., & Ghosh, P. K. (2004). Purification and characterization of C-phycocyanin from cyanobacterial species of marine and freshwater habitat. Protein Expression and Purification, 40(2), 248–255. https://doi.org/10.1016/j.pep.2004.10.028 

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