Analysis of Fatty Acid Content and Composition in Microalgae

Summary

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.

Abstract

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 (ω-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.

Introduction

Fatty acids are one of the major constituents of microalgal biomass and typically make up between 5-50% of the cell dry weight^1-3. 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 (ω-3 fatty acids), and food commodities^3-6. 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 products^3-6. 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 3^rd-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 intensity^1,2. 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 (ω-3 fatty acids) like C20:5 (eicosapentaenoic acid; EPA) and C22:6 (docosahexaenoic acid; DHA) for which no vegetable alternatives exist^1,2,4,7. The (distribution of) fatty acid chain length and degree of saturation also determines the properties and quality of algae-derived biofuels and edible oils^4,8.

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 used^10-12. This leads to the inconsistencies in the lipid content and composition presented in literature^9,10. 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 species^9,13. 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 acids^9,16. 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 determination^17,18. 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 (>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 transesterification^16,19,20. 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 signal^21,22. 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 authors^1,25-27. Also other methods are available that are based on the same principles and could provide similar results^9,28.

Protocol

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 extraction of components from materials used can be identified and quantified.

1.1. Sample Preparation Protocol Option 1: Recommended When a Limited Amount of Algae Culture is Available

1. Determine the algae dry weight concentration (g/L) in the culture broth, for example as described by Breuer et al.¹.
2. Transfer a volume of culture broth that contains 5-10 mg algae dry weight to a glass centrifuge tube. Calculate the exact amount of biomass transferred using the biomass concentration determined in step 1.1.1.
3. Centrifuge 5 min at 1,200 x g.
4. Discard part of the supernatant, leave approximately 0.25 ml in the tube.
5. Re-suspend the algae in the remaining supernatant by gentle pipetting the pellet up and down and transfer the complete cell pellet to a bead beater tube using a 200 μl pipette.
6. Rinse the centrifuge tube and glass pipette with ±0.15 ml milliQ water and transfer the liquid to the same bead beater tube.
7. Centrifuge bead beater tubes for one minute at maximum rpm to make sure no air bubbles remain in the bottom of the tubes. It is possible to store closed bead beater tubes at -80 °C.
8. Lyophilize the bead beater tubes containing the sample. It is possible to store the closed bead beater tubes at -80 °C.

1.2. Sample Preparation Protocol Option 2

1. Centrifuge an undetermined volume of algae culture broth and discard supernatant. It is not necessary to determine the biomass concentration or to measure the volume used.
2. Measure or calculate the osmolarity of the culture medium using the concentration of the main salts present in the culture medium.
3. Wash the cell pellet by resuspending the cell pellet in the same volume, as used in step 1.2.1, of an ammonium formate solution, which approximates the osmolarity of the culture medium. An equiosmolar ammonium formate solution prevents cell lyses during washing of the cells.

NOTE: Washing the cells is necessary to remove salts present in the culture medium. This is especially important for fatty acid analysis in marine microalgae because of the high salt concentrations in their culture medium. If salts would still be present, this would cause overestimation of the amount of cell dry weight which will be determined later in this protocol. Ammonium formate is used to wash cells because this solution will completely evaporate during lyophilization and not leave any residue.

4. Centrifuge algae suspension and discard supernatant.
5. Lyophilize the cell pellet.
6. Weigh 5-10 mg lyophilized microalgae powder into a bead beater tube. Record the exact weight.

2. Cell Disruption and Lipid Extraction

NOTE: This section describes an extensive cell disruption procedure. Possibly, some of the cell disruption steps are redundant and less extensive cell disruption might yield the same results for some microalgae species or for biomass derived from certain cultivation conditions. This would need validation for each specific situation however. Therefore the proposed extensive disruption protocol is recommended as a universal method suitable for all microalgal material.

1. Weigh an exact amount of tripentadecanoin (triacylglycerol containing three C15:0 fatty acids) and add it to an exactly known amount of 4:5 (v/v) chloroform:methanol. In this way the concentration of tripentadecanoin is exactly known. Aim for a concentration of 50 mg/L. Tripentadecanoin serves as the internal standard for fatty acid quantification.
2. Add 1 ml chloroform:methanol 4:5 (v/v) containing tripentadecanoin to each beating tube (specified in the reagents and equipment table). Check that there are no beads remaining inside the cap of the beadbeater tube, this will result in leaking of the tubes. Use a positive displacement pipette for accurate addition of solvents.
3. Bead beat the bead beater tubes 8x at 2,500 rpm for 60 sec, each time with a 120 sec internal between each beating.
4. Transfer the solution from the bead beater tubes to a clean heat-resistant 15 ml glass centrifuge tube with Teflon insert screw-caps. Be sure to transfer all the beads from the bead beater tube as well.
5. To wash the bead beater tube, add 1 ml chloroform:methanol 4:5 (v/v) containing tripentadecanoin into the bead beater tube, close cap and mix, and transfer this solution to the same glass tube from step 2.4. Wash the tube three times (in total 4 ml of chloroform:methanol 4:5 (v/v) containing tripentadecanoin is used per sample - 1 ml added in step 2.2 and 3 ml for 3 washing steps).
6. Vortex the glass tubes for 5 sec and sonicate in a sonication bath for 10 min.
7. Add 2.5 ml of MilliQ water, containing 50 mM 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) and 1 M NaCl, which is set to pH 7 using a HCl solution. This will cause a phase separation between chloroform and methanol:water. 1 M NaCl is used to enhance the equilibrium of lipids towards the chloroform phase.
8. Vortex for 5 sec and then sonicate for 10 min.
9. Centrifuge for 5 min at 1,200 x g.
10. Transfer the entire chloroform phase (bottom phase) to a clean glass tube using a glass Pasteur pipette. Make sure not to transfer any interphase or top phase.
11. Re-extract the sample by adding 1 ml chloroform to old tube (containing the methanol:water solution).
12. Vortex for 5 sec and then sonicate for 10 min.
13. Centrifuge for 5 min at 1,200 x g.
14. Collect the chloroform phase (bottom phase) using a glass Pasteur pipette and pool with the first chloroform fraction from step 2.10.
15. If difficulties are experienced in collecting the entire chloroform fraction in the previous step, repeat steps 2.11-2.14. Otherwise proceed with step 2.16.
16. Evaporate the chloroform from the tube in a nitrogen gas stream. After this step samples can be stored at -20 °C under a nitrogen gas atmosphere.

3. Transesterification to Fatty Acid Methyl Esters

1. Add 3 ml methanol containing 5% (v/v) sulfuric acid to the tube containing the dried extracted lipids (tube originally containing the chloroform fraction) and close the tube tightly.
2. Vortex for 5 sec.
3. Incubate samples for 3 hr at 70 °C in a block heater or water bath. Periodically (every ±30 min) ensure that the samples are not boiling (caused by an improperly closed lid) and vortex tubes. During this reaction fatty acids are methylated to their fatty acid methyl esters (FAMEs).
4. Cool samples to room temperature and add 3 ml MilliQ water and 3 ml n-hexane.
5. Vortex for 5 sec and mix for 15 min with a test tube rotator.
6. Centrifuge for 5 min at 1,200 x g.
7. Collect 2 ml of the hexane (top) phase and put in fresh glass tube using a glass Pasteur pipette.
8. Add 2 ml MilliQ water to the collected hexane phase to wash it.
9. Vortex for 5 sec and centrifuge for 5 min at 1,200 x g. After this step it is possible to store the samples at -20 °C under nitrogen gas atmosphere (phase separation not necessary).

4. Quantification of FAMEs Using Gas Chromatography

1. Fill GC vials with the hexane phase (upper phase) using a glass Pasteur pipette.
2. Place the caps on the GC vials. Make sure the caps are completely sealed, and cannot be turned, to prevent evaporation from the vial.
3. Refill the GC wash solvents (hexane), empty the waste vials, and put sample vials in auto-sampler. Before running the actual samples on the GC, first run 2 blanks containing only hexane on the GC.
4. Run the samples on a GC-FID with a Nukol column (30 m x 0.53 mm x 1.0 μm). Use inlet temperature of 250 °C and use helium as carrier gas, pressure of 81.7 kPa and split ratio of 0.1:1, total flow rate: 20 ml/min. FID detector temperature of 270 °C with H₂ flow rate of 40 ml/min, air flow rate of 400 ml/min and He flow rate of 9.3 ml/min. Set injection volume to 1 μl/sample. Pre and post wash injector with n-hexane. Initial oven temperature is set to 90 °C for 0.5 min and then raised with 20 °C/min until an oven temperature of 200 °C is reached. Oven temperature is then maintained at 200 °C for 39 min. Total run time is 45 min.

Results

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 ...

Discussion

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...

Materials

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