Evaluation of the Effect of Growth Factors on Chlorophylls a and b Production from Microalgae

Our project evaluates how growth factor control affects the production of biotechnologically relevant secondary metabolites such as chlorophyll from microalgae, such as Chlorella sorokiniana and Haematococcus pluvialis at different scales. We have focused on establishing an analytical method to quantify chlorophylls in microalgae. Some emerging technologies for advancement in this historic field are the modeling and optimization of processes using computational technique.

However, in order to carry them out, it is necessary to obtain experimental information on the processes. The growth factors evaluated in Chlorella sorokiniana that have the greatest effect on chlorophyll production are the light color and the carbon dioxide supply flow. The changing light intensity does not significantly influence the production of this metabolite.

The findings in this field of study can be advanced by implementing growth factors that induce microalgae to increase the production of secondary metabolites in conjunction with rapid, simple, and reliable analysis techniques. Our work provide us with guidelines to investigate and understand the biochemical and the energetic processes carried out by microalgae with different substrates, evaluating the effect of environmental variables to optimize the production of your mass and cellular compounds for various applications. To begin, inoculate a flask containing one liter of growth medium with Chlorella sorokiniana at an approximate concentration of three times 10 to the power of six cells per milliliter.

Incubate the inoculated flask under a red light source with an intensity of 3, 600 to 4, 200 lux, maintaining a temperature between 22 to 26 degrees Celsius. Manually agitate the flask every 24 hours during the seven day incubation. In a sterile environment, transfer 10 milliliters of culture into a 15 milliliter conical tube and centrifuge it at 3000 G for 20 minutes at 25 to 30 degrees Celsius.

Then, remove the supernatant from the centrifuge tube and pull the biomass into a sterile 50 milliliter conical centrifugation tube. Measure the inoculum's optical density at 550 nanometers to determine the microalgal cell concentration before storing it at three to four degrees Celsius for future use. 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 photo bioreactor and the flask to the desired color spectrum. Cover both systems to prevent external light interference.

After adjusting the photo period settings, connect the carbon dioxide aeration system to the photo bioreactor 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 630, 647, and 664 nanometers. Calculate chlorophyll concentrations using the equation.

Chlorophyll content of Chlorella sorokiniana was highest in treatment for 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.