Cultivation of Green Microalgae in Bubble Column Photobioreactors and an Assay for Neutral Lipids

This protocol provides instructions for the construction and use of a Bubble Column Photobioreactor system to grow microalgae, and should be of interest to anyone studying algel biofuels, wastewater treatment, or algel biology. We have found the spire reactor system to produce highly reputable results. The reactor system is also customizable to a wide rang of algae and costs far less than many commercial offerings.

Demonstrating the procedure will be Qichen Wang, a graduate student from my laboratory. To begin setup of the Bubble Column Photobioreactors, construct a set of vented lids from the plastic lids of the one liter glass bottles and hybridization tubes as described in the text protocol. Slip a 1/4 inch O ring over the treads of a 1/8th inch panel mount lure fitting, and slide this into the 1/4 inch hole drilled in the lid.

Slip a second 1/4 inch over the threads so that the lid is sandwiched between the two O rings. Then slip a lock nut onto the threads and tighten it to fix the panel mount lure in place. Now snap to lock rings onto the exposed male lure, projecting from the lid.

Repeat this procedure for each hole in the lid. For lids that will be used on the Bubble Column and bottle reactors, attach 1/8th inch female lure to barb fittings to 1 1/2 inch pieces of 1/8th inch ID PVC tubing. Attach these to each of exposed male lure fittings on the lid.

Connect a check valve to the free end of one of the 1/8th inch pieces. Then connect a male lure to barb fitting to the second piece of 1/8th inch tubing projecting from the lid. Click the rotating lock ring into place and fasten a 0.2 micron air filter to this.

Compleat assembly of the air deliver system, fish tanks, stir plates, and lights as descried it the text protocol. Concentrate the settled microalgae stock by removing the supernatent by using a vacuum pump. Leave less than 100 milliliters of medium in each bottle, but avoid removing settled algae.

Suspend and transfer the algae slurry to sterile 50 milliliter centrifuge tubes. Centrifuge 1, 000 times G for five minutes to further concentrate the algae. In the biosafety cabinet, remove enough supernatent to achieve a total volume of approximately 80 milliliters of algae concentrates for 12 photobioreactors.

Avoid vacuuming out the pellet. Transfer the algae concentrate to a steril container. Now, add six milliliters of algae slurry into each photobioreactor with a steril 10 milliliter serological pipette.

Swirl the bioreactors to mix algae into the medium. Draw a two milliliter sample from each bioreactor using a serological pipette and transfer to a two milliliter tube. Collect a two milliliter sample every 24 hours to monitor culture progress.

Check the sample for ph using test strips and adjust the reactor as needed. Tighten the bioreactor lids and place all bioreactors into the fish tank water bath. Adjust the aeration, carbon dioxide, and lighting to the appropriate levels for the species.

Rotate the bioreactor position each day after sampling. Apply 200 microliters of each culture sample in triplicate to wells of a 96 well microplate. Then measure optical density at 550 nanometers and 680 nanometers.

Measure a fixed volume of algae culture from each bioreactor with a graduate cylinder and transfer in centrifuge bottles. Centrifuge the culture 4, 696 times G for five minutes. Discard the supernatent by carefully vacuuming it out.

Transfer the pellets to labeled 50 milliliter tubes. Rinse the centrifuge bottles with distilled water and transfer the contents to the 50 milliliter tubes. Ensure the total volume does not exceed 45 milliliters.

After washing the algae pellets as described in the text protocol, discard the supernatent. Then, add 7.5 milliliters of distilled water to each 50 milliliter tube. Now, vortex the 50 milliliter tubes and transfer the algae slurries into pre weighed 15 milliliter tubes.

Rinse the 50 milliliter tubes with additional distilled water, and transfer the liquid to the 15 milliliter tubes keeping the total volume in those tubes to less than 12 milliliters. After centrifuging the 15 milliliter tubes and discarding the supernatent as before, freeze the tubes with pellets at minus 80 degrees Celsius for at least 30 minutes in preparation for freeze drying. Add 1.5 milliliters of folch solvent to each two milliliter tube containing 20 milligrams of freeze dried algae.

Pour approximately 0.5 milliliters of zirconia silica beads into each tube until the liquid level reaches two milliliters. Homogenize the algae samples in a bead mill for 20 seconds at a speed of 6.5 meters per second. Transfer the tubes to ices for 30 seconds to chill the samples.

Then, repeat five more times to fully extract the lipids. Filter the homogenate through a five milliliter syringe containing a stainless steel wire mesh disc to strain out the beads, collecting filtrate in a 15 milliliter tube. Wash the beads with 1.5 milliliters of folch solvent, pushing liquid through with the syring as necessary.

Repeat this wash 2 more times and collect all filtrate in the 15 milliliter tube, yielding a final volume of approximately six milliliters. Add 1.2 milliliters of 0.9%sodium chloride solution to the folch extract in the 15 milliliter tube, and vortex to mix well. Centrifuge the 15 milliliter tubes at 6, 000 times G for five minutes.

Record the bottom chloroform phase volume to the nearest 0.1 milliliter using lines on the side of the 15 milliliter tube. Then transfer the bottom phase to a glass vile using a glass pasture pipette. To perform the neutral lipid assay, dilute the lipid extracts and vegetable oil standard three fold with methanol.

For each diluted, sample add 80 microliters to a 96 well polypropylene microplate in quadruplicate. For the solvent blank, apply 80 microliters of folch solvent in quadruplicate. For standards, add 10, 30, 60, 90, and 120 microliters of the diluted vegetable oil standard also in quadruplicate.

Place the microplate in the fume hood on a preheated dry block heater at 55 degrees Celsius for 20 to 30 minutes until all solvent has evaporated. Remove the microplate from the heating block and let it cool to room temperature. Then, add 30 microliters of isopropyl alcohol to each well and mix by pipetting up and down.

Ensure all pipette channels are mixing the solution and resuspending the lipids, yielding a homogenous green liquid. Now, add 200 microliters of one microgram per milliliter a nile red solution to each well, and pipe it up and down 10 times to mix. Following a five minute incubation at room temperature, add 20 microliters of 50%bleach solution to each well, and pipe it up and down five times to mix well.

Leave the plat to incubate for 30 minutes at room temperature. After 30 minutes, read the florescence in the samples every five to 10 minutes at 530 nanometers excitation, 575 nanometers emission, with auto cut offs set to 570 nanometers until the signal from the algae samples stabilizes. This procedure yields a time course of algel optical density data at OD 550 nanometers.

Control cultures were grown on fresh anate NH4 medium. Treatment one, is co-cultures auxenochlorella protothecoides and azospirillum brasiliense grown on fresh innate NH4 medium. Treatment two, is a azinic A.Protothecoides grown on innate NH4 medium supplemented with 50 milligrams per milliliter IAA, which completely inhibited algae growth.

Treatment three, is azinic A.Protothecoides, grown on spent medium from A.Brasilienes. The growth curves show cultures of auxenochlorella protothecoides enter late logarithmic growth at 120 hours. Shown here are corelation curves between the optical density at 550 nanometers and the final dry weight concentration, using a second order polynomial fit.

Finally, the coloration can be applied to the time course optical density data to obtain a dry weight growth curve. The same treatments are used here, except that chlorella sorokiniana is cultured instead of auxenochlorella protothecoides. Percent dry weight neutral lipid obtained in the neutral lipid assay correlates well with triose glycerol content on a corresponding thin layer chromatography plate.

Unlike A.Protothecoides, the IAA treatment did not inhibit C.Sorkiniana growth. Following lipid extraction of the algae, the remaining cell pallet can be analyzed for its starch and cell wall content, providing a more detailed picture of energy storage products within the cell. The methods described here have enabled new discoveries in the field of algel biofuels and wastewater treatment.

Specifically, this system has been used to better understand how bacteria influence algel growth.