Experimental Protocol for Biodiesel Production with Isolation of Alkenones as Coproducts from Commercial Isochrysis Algal Biomass

The overall goal of this procedure is to produce biodiesel from commercially available Isochyrsis Algal while allowing the co-isolation of long chain alkenones as valuable co-product. This method can help answer key questions in the renewable energy field. Such as, our algal biofuels, viable alternatives to petroleum based fuels.

The main advantage of this technique is that the algal we use is already grown industrially and can be purchased in multi-kilogram quantities from several commercial suppliers. Plus, these particular algal bio synthesize a unique sweet of lipids known as long chain alkenones that can be isolated as a co-product. Demonstrating the procedure will be John Williams, a graduate student in my lab.

To prepare the commercially available wet isochrysis paste, cut a small hole in a one kilogram package and squeeze approximately 300 grams on to a 150 by 75 millimeter crystallizing dish. The paste is approximately 80%water, dark green, nearly black and has a pungent odor. Air dry the paste to create a layer that is about 20 millimeters thick.

Scrape the dry biomass into cellulose extraction thimble of known mass. Then record the weight of the isochrysis biomass. Transfer about 50 to 60 grams of dry isochrysis into a soxhlet extraction apparatus.

Then, fill the soxhlet flask with 400 milliliters of hexanes. Turn on the condenser water and heat source and allow the soxhlet to cycle for one to two days. When the color of the solvent has gone from dark green to a feint yellow, the extraction is complete.

Cool it to room temperature and use a rotary evaporator to remove the hexanes. Finally, weigh the hexane algal oil. Begin by dissolving the hexane algal oil in its flask with 10 volumes of two to one methanol dichloromethane.

Next add a stir up bar and two and two thirds parts of the water compared to the oil volume and add a mass of potassium hydroxide that is half the oils weight. Now, attach the flask to a 500 millimeter long reflux condenser. Heat the mixture with stirring to 60 degrees celsius for three hours in a fume hood.

After cooling the reaction, remove the organic solvents on a rotary evaporator. Then, poor the remaining aqueous mixture into a one liter separatory funnel and add an equal volume of hexanes. Rinse the flask with the hexanes to ensure all the material transfers to the funnel.

Shake the funnel and allow the layers to separate. Next, drain the lower aqueous layer into a flask and poor off the top organic face into a second flask. Keep both layers.

Repeat the hexane extraction until the organic layer is a feint yellow green. Then combine the collected organic extracts and condense them on a rotary evaporator to isolate the neutral lipids. These lipids appear as a greenish solid and have a melting point between 60 and 70 degrees celsius.

Next, acidify the aqueous phase to a pH of two using six molar hydrochloric acid. Then, extract the free fatty acids from the acidified aqueous phase using hexanes and a separatory funnel as before. Finally remove the hexanes from the combined organic extracts on a rotary evaporator to obtain purified free fatty acids.

A dark green near black oily residue that will melt just above 30 degrees celsius. Dissolve the fatty acids using six volumes of methanol cholorform and then pour the solution into a thick welled high pressure reaction flask with a stir up bar rinsing to ensure all the fatty acids transfer to the reaction flask. Next, under a fume hood, add conenctrated sulfuric acid.

Seal the flask, and heat the mixture to 90 degrees celsius while stirring for an hour. After cooling the reaction to room temperature, pour it into a separatory funnel and add two volumes of water. Now, shake the separatory funnel, allow the faces to separate and drain the bottom layer into a pre-weighed round bottom flask.

Then, concentrate the solution on a rotary evaporator and record the mass of the resulting bio diesel the substance can now be analyzed using gas chromatagraphs. Neutral lipids can be scraped from the round bottom, weighed and transferred to a flask for purification. Begin with dissolving the neutral lipids in a minimal amount of dichloromethane.

Then, pipe head the solution on to a chromotagraphy column containing 100 grams of silica gel. Next, use light pressure to dilute the solution through the silica with about 150 milliliters of dichloromethane. A small layer of sand is added to the top of the column to prevent the silica from being disturbed when filling with solvent.

Collect the yelia wint in a 250 milliliter round bottom flask. Then remove the dichloromethane with a rotary evaporator. The result should be an orange colored solid.

Re-cryalize the solids in boiling hexanes. First, add an initial 100 milliliters and then continue adding boiling hexanes until the solution is homogeneous. Now, slowly cool the solution to room temperature to promote crystallization and collect the crystallized alkenones using a filtration apparatus.

Use a small volume of ice cold hexanes to rinse the flask. Prior to processing, the isochrysis paste was first dried to obtain a black, green, flaky material with a seaweed-like smell. By contrast, the powdered isochrysis was a yellow-brown finely milled dry powder that was used without additional processing.

Extraction from either material by soxhlet with hexanes gave algal oils with yields that were substantially higher from the paste than the powder. Acylglycerols in the algal oil were converted to water soluble carboxylate from which neutral lipids and free fatty acids were extracted at a near quantitative volume from either starting material. However, the ratio of the products was different.

Esterification produced fatty acid methyl esters. Also known as bio diesel, a dark green near black oily liquid. The yield exceeded 90%The product was decolorized as described in the text protocol.

FAME analysis of the purified bio diesel fuels showed similar but not identical profiles from the two starting materials. Filtering the dissolved neutral lipids through silica using DCM, give a reddish orange solid that was re-crystallized with hexanes and to analytically pure alkenones. After watching this video, you should have a good understanding of how to process commercially available isochyrsis into bio diesel with isolation of pure alkenones as a co-product.

Once mastered, this technique can consistently generate significant quantities of algal biodiesel and alkenones for further study. With its development, this technique has paved the way for us to critically examine the fuel properties of algal biodiesel by providing sufficient quantities with which to perform the various fuel tests as opposed to relying on predictions or simulations. Don't forget that working with strong with acids or bases and heating the contents within a sealed vessel can be extremely hazardous.

Be sure to wear proper personal protective gear and work in a fume hood while performing these steps.