Evaluating Methods for Optimal C-PC Extraction

 Introduction

In previous labs, we have successfully extracted enough phycocyanin from Spirulina plantesis within a liquid solution to meet our goal of one milligram of total product, but are still unsure as to which method is the most effective to attain the highest purity and concentration levels of C-PC possible. Prior methods included both deionized water and phosphate buffer, glass beads, no glass beads, and the ultrasonic bath as well as the blender.  To determine which factors are the most effective, we conducted a three factor DOE experiment, consisting of two trials, in which the factors would be phosphate buffer versus deionized water, the addition of glass beads versus none, and a combination of the blender and ultrasonic bath as opposed to the ultrasonic bath alone. Our hypothesis was that phosphate buffer, glass beads, and the blender/ultrasonic bath would significantly improve C-PC purity and concentration values. 

Methods

Fifty ml tubes were used for three grams of dried spirulin and thirty ml of solvent, and one gram of glass beads if needed. To prevent pellet formation at the bottom of each tube, fifteen ml buffer/water was added to each tube first, then three grams of spirulin, with the remaining fifteen ml on top. Pellet formation or clumping is suspected to hinder C-PC extraction by not allowing all the cells in the clump to lyse. Each tube was vigorously shaken and vortexed on the ten setting for thirty seconds each for even distribution.  The eight tubes to only be exposed to the ultrasonic bath were sonicated for a total of four hours, and the remaining eight where blended for a total of 10 seconds, with one second pulses and intervals. After blending, the solutions were distributed to their original tubes and put in the ultrasonic bath for one hour. All sixteen tubes were centrifuged at 9500 RPM at four degrees Celsius for thirty minutes. Their supernatants were then acquired and redistributed into new fifteen ml tubes to undergo a second round of centrifugation the following week. Below is a detailed chart of each tube tested. 


 

 

Tube

Factor A (Beads)

Factor B (Blender/bath)

Factor C (Phosphate Buffer)

AB

AC

BC

ABC

1

(+)

(+)

(-)

(+)

(-)

(-)

(-)

2

(-)

(+)

(-)

(-)

(+)

(-)

(+)

3

(+)

(-)

(-)

(-)

(-)

(+)

(+)

4

(-)

(-)

(-)

(+)

(+)

(+)

(-)

5

(+)

(+)

(+)

(+)

(+)

(+)

(+)

6

(-)

(+)

(+)

(-)

(-)

(+)

(-)

7

(+)

(-)

(+)

(-)

(+)

(-)

(-)

8

(-)

(-)

(+)

(+)

(-)

(-)

(+)

 


Results

No quantitative results were recorded this week, since all tubes need to be centrifuged twice before testing. Qualitatively, all of the supernatants presented very dark blue with a slight green tint. There was also a very large pellet produced in comparison to prior experiments. Approximately twenty five to thirty ml of supernatant was acquired from each fifty ml tube. 

Discussion 

The ongoing three factor DOE experiment, although not completed, will hopefully give clarity to which of the three factors are most effective and the best overall combination to get the highest purity and concentration. From the qualitative data observed, it is suspected that the values will be very similar due to the preliminary coloring which has given a rough expectation for results in previous experiments. We still suspect that the addition of glass beads, phosphate buffer, and blender/ultrasonic bath combination will be shown as significantly improving values, even if by minimal amounts. 

Comments

  1. EricaNovember 14, 2021 at 1:47 PM

    Leilani,
    Nice work. We look forward to reviewing your results table!

    ReplyDelete

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