The overall goal of this procedure is to measure the total fatty acid content and composition in micro algae. This is accomplished by first preparing a sample containing a known amount of lyophilized algae biomass. The second step is to disrupt the micro algal biomass.
Next, the lipophilic components are extracted and isolated. The final step is transesterification of the ACell lipids to fatty acid methyl esters. Ultimately, gas chromatography is used to quantify the fatty acids.
The main advantage of this technique over existing methods like tric determinations, is that this method is very accurate and specifically measures all the fatty acids and all other lipophilic components. Demonstrating the procedure will be NDI Afer, a technician from our laboratory Before starting this procedure, determine the algae dry weight concentration in grams per liter in the culture broth as detailed in the listed reference. Transfer a volume of culture broth that contains five to 10 milligrams of algae dry weight twig glass centrifuge tube.
Calculate the exact amount of biomass transferred using the biomass concentration determined previously centrifuge the sample for five minutes at 1200 Gs.Discard part of the supernatant, leaving approximately 0.25 milliliters in the tube. Next resus. Suspend the algae in the remaining supernatant by gently pipetting the pellet up and down, and transfer the cell mixture to a bead beater tube.
Using a 200 microliter pipette, rinse the centrifuge tube and pipette tip with plus or minus 0.15 milliliters of milli cube water and transfer the liquid to the bead beater tube. Centrifuge the bead beater tube for one minute at maximum RPM to make sure no air bubbles remain in the bottom of the tube. When finished, lyophilize the material in the tube following this.
Prepare a 50 milligram per liter solution of trip Penta Deano in a fortified volume ratio of chloroform to methanol. Using a positive displacement pipette, add one milliliter of the tripod, no solution to the beading tube. Check that there are no beads remaining inside the cap of the bead beater tube as this will result in leaking of the tubes bead.
Beat the beater tube eight times at 2, 500 RPM for 60 seconds each time with a 122nd interval between each beading. Next, transfer the solution in beads from the bead beater tube to a clean heat resistant 15 milliliter glass centrifuge tube with a Teflon insert screw cap. Wash the bead beater tube with one milliliter of the tripod, no solution, and transfer the wash to the glass centrifuge tube.
Repeat two more times. Vortex the sample for five seconds and then sonicate it for 10 minutes. Following sonication at 2.5 milliliters of a pH seven milli cube water solution containing 50 millimolar triss, and one molar sodium chloride to the centrifuge tube.
Once the sample has been vortexed and sonicate a second time, centrifuge it for five minutes at 1200. When finished, carefully transfer the bottom chloroform phase to a clean glass tube using a glass pasti pipette, repeat the extraction with one milliliter of chloroform. When finished, collect the chloroform phase using a glass posterior pipette and pool with the first chloroform fraction.
Evaporate the chloroform using a nitrogen gas stream to afford the dried extracted lipids. Next, add three milliliters of methanol containing 5%sulfuric acid to the tube containing the dried extracted lipids and close it tightly. Vortex the sample for five seconds.
Incubate the samples for three hours at 70 degrees Celsius in a block heater. Periodically ensure that the samples are not boiling and vortex the tube after each time the samples are checked. Once the sample is cooled to room temperature at three milliliters of milli cube water and three milliliters of n heane to the tube following vortex mixing.
Agitate the sample for 15 minutes with a test tube rotator. After removing the samples from the test tube rotator, centrifuge them for five minutes at 1200 Gs.Using a glass past pipette, collect two milliliters of the top hexane phase and transfer it to a fresh glass tube. To wash the collected hexane phase, add two milliliters of milli Q water to the glass tube.
Then vortex the sample for five seconds and centrifuge it for five minutes. At 1200 Gs, transfer the hexane phase from the glass tube to a GC vial using a glass pastier pipette. Then place a cap on the GC vial and tighten it to prevent solvent evaporation.
Place the sample vial in the auto sampler of the GC fit instrument. Finally, run the sample on the instrument with a new call column using a total flow rate of 20 milliliters per minute and an injection volume of one microliter. See the text protocol for column specifications and chromatographic conditions?
A typical chromatogram that is obtained via this process is shown here. Fatty acid methyl esters are separated by size and degree of saturation by the GC column, and protocol used shorter chain length fatty acids and more saturated fatty acids have shorter retention times. The used GC column and protocol do not intend to separate fatty acid isomers, but this could be achieved by using a different GC column and protocol.
The fatty acid content and composition of C Desus UEX 3 93 under both nitrogen sufficient and deprived conditions are displayed here. The total fatty acid contents were 8.6 plus or minus 0.5%and 44.7 plus or minus 1.7%under nitrogen replete and a nitrogen deprived cultivation conditions respectively. These values demonstrate that fatty acid composition and content are highly affected by nitrogen starvation.
In ESBL palmitic acid and oleic acid are the two most abundant fatty acids. See the text protocol for details on calculating the fatty acid content and composition? The fatty acid content and composition of Odum Trico UEX 6 4 0 under both nitrogen sufficient and deprived conditions are shown here.
Similar to oblique UUs, the fatty acid content and composition are highly affected by nitrogen starvation. Under nitrogen sufficient and deprived conditions, the total fatty acid contents were 11.7 plus or minus 0.9%and 30.5 plus or minus 1.1%respectively. P trium also produces substantial amounts of highly unsaturated fatty acids such as I csap, Penta IC acid.
In addition, very long chain fatty acids such as ligner acid can also be detected by this method. This procedure can be extended by including a lipid clot separation step between the lipid extraction and a transesterification step to obtain additional insight into the lipid class composition of micro LG as described in the references in the protocol text.
