Name: analysis of fatty acid content and composition in microalgae
Uploaded: 2013-10-01T09:00:00
Description: Watch this Scientific Journal Video about Analysis of Fatty Acid Content and Composition in Microalgae at JoVE.com

A part of this work was financially supported by the Institute for the Promotion of Innovation by Science and Technology&mdash;Strategic Basic Research (IWT-SBO) project Sunlight and Biosolar cells. Erik Bolder and BackKim Nguyen are acknowledged for their contribution to the optimization of the bead beating procedure.

1. Sample Preparation
There are two alternate protocols for sample preparation included as steps 1.1 and 1.2. Both methods are equally suitable and give similar results, but if a limited amount of algae culture volume is available, method 1.1 is recommended.
NOTE: For either protocol, prepare two additional bead beater tubes according to the entire protocol but without adding algae to them to be used as a blank. In this way, peaks in the GC chromatogram resulting from...

<ol>
	<li>Breuer, G., Lamers, P. P., Martens, D. E., Draaisma, R. B., Wijffels, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+nitrogen+starvation+on+the+dynamics+of+triacylglycerol+accumulation+in+nine+microalgae+strains.">The impact of nitrogen starvation on the dynamics of triacylglycerol accumulation in nine microalgae strains.</a> Bioresource Technology. 124, 217-226 (2012).</li><li>Hu, Q., Sommerfeld, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microalgal+triacylglycerols+as+feedstocks+for+biofuel+production:+perspectives+and+advances.">Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances.</a> The Plant Journal. 54 (4), 621-639 (2008).</li><li>Chisti, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodiesel+from+microalgae.">Biodiesel from microalgae.</a> Biotechnology Advances. 25 (3), 294-306 (2007).</li><li>Draaisma, R. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+Between+Extraction+of+Lipids+and+Fatty+Acids+from+microalgal+biomass.">Comparison Between Extraction of Lipids and Fatty Acids from microalgal biomass.</a> JAOCS. 71 (9), 955-959 (1994).</li><li>Lee, J. Y., Yoo, C., Jun, S. Y., Ahn, C. Y., Oh, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+several+methods+for+effective+lipid+extraction+from+microalgae.">Comparison of several methods for effective lipid extraction from microalgae.</a> Bioresour Technol. 101, 75-77 (2010).</li><li>Guckert, J. B., Cooksey, K. E., Jackson, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=lipid+solvent+systems+are+not+equivalent+for+analysis+of+lipid+classes+in+the+micro+eukaryotic+green+alga.">lipid solvent systems are not equivalent for analysis of lipid classes in the micro eukaryotic green alga.</a> Journal of Microbiological Methods. 8, 139-149 (1988).</li><li>Pruvost, J., Van Vooren, G., Cogne, G., Legrand, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+biomass+and+lipids+production+with+Neochloris+oleoabundans+in+photobioreactor.">Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor.</a> Bioresource Technology. 100 (23), 5988-5995 (2009).</li><li>Griffiths, M. J., Hille, R. P., Harrison, S. T. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+micro-method+for+the+estimation+of+oil+content+and+composition+in+seed+crops.">A micro-method for the estimation of oil content and composition in seed crops.</a> J. Sci. Food Agric. 28 (7), 635-638 (1002).</li><li>Chen, W., Zhang, C., Song, L., Sommerfeld, M., Hu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+Nile+red+method+for+quantitative+measurement+of+neutral+lipids+in+microalgae.">A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae.</a> Journal of Microbiological Methods. 77 (1), 41-47 (2009).</li><li>Cooper, M. S., Hardin, W. R., Petersen, T. W., Cattolico, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+"green+oil"+in+live+algal+cells.">Visualizing "green oil" in live algal cells.</a> Journal of Bioscience and Bioengineering. 109 (2), 198-201 (2010).</li><li>Lamers, P. P., van de Laak, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotenoid+and+fatty+acid+metabolism+in+light-stressed+Dunaliella+salina.">Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina.</a> Biotechnology and Bioengineering. 106 (4), 638-648 (2010).</li><li>Santos, A. M., Janssen, M., Lamers, P. P., Evers, W. A., Wijffels, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+oil+accumulating+microalga+Neochloris+oleoabundans+under+alkaline-saline+conditions.">Growth of oil accumulating microalga Neochloris oleoabundans under alkaline-saline conditions.</a> Bioresour Technol. 104, 593-599 (2012).</li><li>Mulders, K. J. M., Weesepoel, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+pigment+accumulation+in+nutrient-depleted+Isochrysis+aff.+galbana+T-ISO.">Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO.</a> J. Appl. Phycol. , (2012).</li><li>Kliphuis, A. M., Klok, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+modeling+of+Chlamydomonas+reinhardtii:+energy+requirements+for+photoautotrophic+growth+and+maintenance.">Metabolic modeling of Chlamydomonas reinhardtii: energy requirements for photoautotrophic growth and maintenance.</a> J. Appl. Phycol. 24 (2), 253-266 (2012).</li><li>Lamers, P. P., Janssen, M., De Vos, R. C. H., Bino, R. J., Wijffels, R. 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The described method can be used to determine the content as well as the composition of total fatty acids present in microalgal biomass. Fatty acids derived from all lipid classes, including storage (TAG) as well as membrane lipids (phospholipids and glycolipids), are detected. All fatty acid chain lengths and degrees of saturation that are present in the microalgae can be detected and distinguished. The method is based on mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to F...

Fatty acids are one of the major constituents of microalgal biomass and typically make up between 5-50% of the cell dry weight1-3. They are mainly present in the form of glycerolipids. These glycerolipids in turn mainly consist of phospholipids, glycolipids, and triacylglycerol (TAG). Especially the fatty acids present in TAG are of commercial interest, because they can be used as a resource for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities3-6. Microalgae can grow in sea water based cultivation media, can have a much higher areal productivity than terrestrial plants, and can be cultivated in photobioreactors at locations that are unsuitable for agriculture, possibly even offshore. For these reasons, microalgae are often considered a promising alternative to terrestrial plants for the production of biodiesel and other bulk products3-6. Potentially no agricultural land or fresh water (when cultivated in closed photobioreactors or when marine microalgae are used) is needed for their production. Therefore, biofuels derived from microalgae are considered 3rd-generation biofuels.
The total cellular content of fatty acids (% of dry weight), the lipid class composition, as well as the fatty acid length and degree of saturation are highly variable between microalgae species. Furthermore, these properties vary with cultivation conditions such as nutrient availability, temperature, pH, and light intensity1,2. For example, when exposed to nitrogen starvation, microalgae can accumulate large quantities of TAG. Under optimal growth conditions TAG typically constitutes less than 2% of dry weight, but when exposed to nitrogen starvation TAG content can increase to up to 40% of the microalgal dry weight1.
Microalgae mainly produce fatty acids with chain lengths of 16 and 18 carbon atoms, but some species can make fatty acids of up to 24 carbon atoms in length. Both saturated as well as highly unsaturated fatty acids are produced by microalgae. The latter include fatty acids with nutritional benefits (&omega;-3 fatty acids) like C20:5 (eicosapentaenoic acid; EPA) and C22:6 (docosahexaenoic acid; DHA) for which no vegetable alternatives exist1,2,4,7. The (distribution of) fatty acid chain length and degree of saturation also determines the properties and quality of algae-derived biofuels and edible oils4,8.
To develop commercial applications of microalgal derived fatty acids, reliable analytical methods for quantification of fatty acid content and composition are needed.
As also pointed out by Ryckebosch et al.9, analysis of fatty acids in microalgae distinguishes itself from other substrates (e.g. vegetable oil, food products, animal tissues etc.) because 1) microalgae are single cells surrounded by rigid cell walls, complicating lipid extraction; 2) microalgae contain a wide variety of lipid classes and the lipid class distribution is highly variable7. These different lipid classes have a wide variety in chemical structure and properties such as polarity. Also, lipid classes other than acyl lipids are produced; 3) microalgae contain a wide variety of fatty acids, ranging from 12-24 carbon atoms in length and containing both saturated as well as highly unsaturated fatty acids. Therefore, methods developed to analyze fatty acids in substrates other than microalgae, might not be suitable to analyze fatty acids in microalgae.
As reviewed by Ryckebosch et al.9, the main difference between commonly used lipid extraction procedures is in the solvent-systems that are used. Because of the large variety of lipid classes present in microalgae, each varying in polarity, the extracted lipid quantity will vary with solvents used10-12. This leads to the inconsistencies in the lipid content and composition presented in literature9,10. Depending on the solvent system used, methods based on solvent extraction without cell disruption through, for example, bead beating or sonication, might not extract all lipids because of the rigid structure of some microalgae species9,13. In the case of incomplete lipid extraction, the extraction efficiency of the different lipid classes can vary14. This can also have an effect on the measured fatty acid composition, because the fatty acid composition is variable among lipid classes7.
Our method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography in combination with a flame ionization detector (GC-FID). An internal standard in the form of a triacylglycerol (tripentadecanoin) is added prior to the analytical procedure. Possible losses during extraction and incomplete transesterification can then be corrected for. The method can be used to determine the content as well as the composition of the fatty acids present in microalgal biomass. All fatty acids present in the different acyl-lipid classes, including storage (TAG) as well as membrane lipids (glycolipids, phospholipids), are detected, identified and quantified accurately and reproducibly by this method using only small amounts of sample (5 mg). This method does not provide information about the relative abundance of different lipid classes. However, the method can be extended to separate lipid classes from each other1. The concentration and fatty acid composition of the different lipid classes can then be determined individually.
In literature several other methods are described to analyze lipids in microalgae. Some methods focus on total lipophilic components15, whereas other methods focus on total fatty acids9,16. These alternatives include gravimetric determination of total extracted lipids, direct trans-esterification of fatty acids combined with quantification using chromatography, and staining cells with lipophilic fluorescent dyes.
A commonly used alternative to quantification of fatty acids using chromatography is quantification of lipids using a gravimetric determination17,18. Advantages of a gravimetric determination are the lack of requirements for advanced and expensive equipment like a gas chromatograph; ease to set up the procedure, because of the availability of standardized analytical equipment (e.g. Soxhlet); and a gravimetric determination is less time-consuming than chromatography based methods. The major advantage of using chromatography based methods on the other hand is that in such a method only the fatty acids are measured. In a gravimetric determination the non-fatty acid containing lipids, like pigments or steroids, are also included in the determination. These non-fatty acid containing lipids can make up a large proportion (&gt;50%) of total lipids. If one is only interested in the fatty acid content (for example for biodiesel applications), it will be overestimated when a gravimetric determination is used. In addition, in a gravimetric determination the accuracy of the analytical balance used to weigh the extracted lipids determines the sample size that needs to be used. This quantity is typically much more than the amount needed when chromatography is used. Finally, another advantage of using chromatography over gravimetric determination is that chromatography provides information about the fatty acid composition.
Another alternative to our presented method is direct transesterification16,19,20. In this method lipid extraction and transesterification of fatty acids to FAMEs are combined in one step. This method is quicker than a separate extraction and transesterification step, but combining these steps limits the solvents that can be used for extraction. This might negatively influence extraction efficiency. Another advantage of a separate lipid extraction and transesterification step is that it allows for an additional lipid class separation between these steps1. This is not possible when direct transesterification is used.
Other commonly used methods to determine the lipid content in microalgae include staining the biomass with lipophilic fluorescent stains such as Nile red or BODIPY and measuring the fluorescence signal21,22. An advantage of these methods is that they are less laborious than alternative methods. A disadvantage of these methods is that the fluorescent response is, for various reasons, variable between species, cultivation conditions, lipid classes, and analytical procedures. As an example, several of these variations are caused by differences in uptake of the dye by the microalgae. Calibration using another quantitative method is therefore needed, preferably performed for all the different cultivation conditions and growth stages. Finally, this method does not provide information about the fatty acid composition and is less accurate and reproducible than chromatography based methods.
The presented method is based on the method described by Lamers et al.23 and Santos et al.24 and has also been applied by various other authors1,25-27. Also other methods are available that are based on the same principles and could provide similar results9,28.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent and equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>tripentadecanoin (C15:0 TAG)</td>
 <td>Sigma Aldrich</td>
 <td>T4257</td>
 <td>CAS Number 7370-46-9</td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Sigma Aldrich </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Lipidox</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>TAG or FAME standards of all fatty acids expected in sample</td>
 <td>Larodan</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Beadbeater</td>
 <td>Bertin Technologies</td>
 <td>Precellys 24</td>
 <td></td>
 </tr>
 <tr>
 <td>beadbeater tubes</td>
 <td>MP Biomedicals</td>
 <td>Lysing matrix E 
 116914500</td>
 <td></td>
 </tr>
 <tr>
 <td>GC-FID</td>
 <td>Hewlett-Packer</td>
 <td>HP6871</td>
 <td></td>
 </tr>
 <tr>
 <td>GC column</td>
 <td>Supelco</td>
 <td>Nukol 25357</td>
 <td></td>
 </tr>
 <tr>
 <td>Positive displacement pipette 100-1000&mu;l</td>
 <td>Mettler Toledo</td>
 <td>MR-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Positive displacement pipet tips C-1000</td>
 <td>Mettler Toledo</td>
 <td>C-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Pipetvuller, Pi-Pump 2 ml</td>
 <td>VWR</td>
 <td>612-1925</td>
 <td></td>
 </tr>
 <tr>
 <td>glass tubes</td>
 <td>VWR</td>
 <td>SCERE5100160011G1</td>
 <td></td>
 </tr>
 <tr>
 <td>TUBE 16 X 100 MM BOROSILICATE 5.1 1 * 1.000</td>
 <td>VWR</td>
 <td>SCERE5100160011G1</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon coated screw-caps</td>
 <td>VWR</td>
 <td>SCERKSSR15415BY10</td>
 <td></td>
 </tr>
 <tr>
 <td>STUART SCIENTIFIC SB2 test tube rotator</td>
 <td>VWR</td>
 <td>445-2101</td>
 <td></td>
 </tr>
 <tr>
 <td>Heated Evaporator/Concentrator</td>
 <td>Cole-Parmer</td>
 <td>YO-28690-25</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of fatty acid content and composition in microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A typical chromatogram that is obtained via this process is shown in Figure 1. FAMEs 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 (fewer double bonds) have shorter retention times. The used GC column and protocol do not intend to separate fatty acid isomers (same chain length and degree of saturation, but different positions of double bonds), but this could be achieved by using a different GC column and ...

Watch this Scientific Journal Video about Analysis of Fatty Acid Content and Composition in Microalgae at JoVE.com

Analysis of Fatty Acid Content and Composition in Microalgae

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

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The Calibration and Use of Capacitance Sensors to Monitor Stem Water Content in Trees

Water transport and storage through the soil-plant-atmosphere continuum is critical to the terrestrial water cycle, and has become a major research focus ...

Combining Eye-tracking Data with an Analysis of Video Content from Free-viewing a Video of a Walk in an Urban Park Environment

As individuals increasingly live in cities, methods to study their everyday movements and the data that can be collected becomes important and valuable. ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

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

There is significant interest in the study of microalgae for engineering applications such as the production of biofuels, high value products, and for the ...