To begin, prepare the inoculum to study the growth kinetics of Chlorella sorokiniana and Haematococcus pluvialis Prepare the required volume of sterile culture medium according to standard laboratory procedures. Configure the light source for both the photobioreactor and the flask to the desired color spectrum. Cover both systems to prevent external light interference.
After adjusting the photoperiod settings, connect the carbon dioxide duration system to the photobioreactor to provide carbon dioxide for 12 hours to chlorella. Next, inoculate the culture vessel to achieve a starting cell density of 300, 000 cells per milliliter or an optical density of 0.180 at 550 nanometers. Sample the culture at regular intervals of 12 hours for Chlorella sorokiniana and eight hours for Haematococcus pluvialis.
Place 40 milliliters of the sample into a plastic conical centrifugation tube. Centrifuge the tube at 3000 G for 20 minutes at 15 degrees Celsius. Decant the supernatant and add three milliliters of 90%pure acetone solution to resuspend the cells, then transfer the cells into a glass tube covered with aluminum foil to prevent oxidation and mix using a vortex.
Sonicate the suspended sample in an ice bath for two cycles of five minutes each, and let the sample rest at four degrees Celsius for 16 hours. After resting, sonicate the sample again for two cycles of five minutes each under the same conditions, and centrifuge the sample at 3000 G for 20 minutes at 15 degrees Celsius. Separate the pigment extract using a Pasteur pipette and transfer it to another clean tube protected from light.
Then place the pigment extract in a quartz cell and read it in a spectrophotometer at 600, 647, and 664 nanometers. Calculate chlorophyll concentrations using the equation. Chlorophyll content of Chlorella sorokiniana was highest in treatment four, under conditions of high carbon dioxide addition, purple light, and high light intensity.
Main effects plot showed that light intensity had a lower effect compared to carbon dioxide and purple light effects. In Chlorella sorokiniana, a consistent increase in chlorophylls A and B was seen throughout the experiment, with chlorophyll A surpassing chlorophyll B at 50 hours. A decrease in chlorophyll concentration was observed in Haematococcus pluvialis at 50 hours, with chlorophyll B decreasing significantly while chlorophyll A remained higher.
