May 18, 2026

Lipid compositional profiling of Auxenochlorella protothecoides x symbiontica UTEX 250-A

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx42mwl8j/v1
  • 1California Institute for Quantitative Biosciences (QB3), University of California, Berkeley;
  • 2Environmental Genomics and Systems Biology, Lawrence Berkeley National Laboratory;
  • 3Department of Chemistry, University of California, Berkeley
  • Jon YT Lin: Author for correspondence;
  • Sabeeha S. Merchant: Author for correspondence;
Jon YT Lin
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx42mwl8j/v1
Protocol Citation: Jon YT Lin, Felicia Wong, Kenzo Khoo, Trent Northen, Jeffrey Moseley, Sabeeha S. Merchant 2026. Lipid compositional profiling of Auxenochlorella protothecoides x symbiontica UTEX 250-A. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx42mwl8j/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 08, 2025
Last Modified: May 18, 2026
Protocol  Integer ID: 229280
Keywords: triacylglycerol, thin-layer chromatography (tlc), gas chromatography, stereochemical analysis, pancreatic lipase, fatty acid methyl ester (FAME), green algae, lipid compositional profiling of auxenochlorella protothecoide, algal lipid, analysis of lipid, lipid compositional profiling, lipid characterization, green alga auxenochlorella protothecoide, lipid extraction, derivatization of fatty acid, reliable protocol for lipid characterization, auxenochlorella protothecoide, lipid, fatty acid, triacylglycerol, algal strain, porcine pancreas lipase treatment
Funders Acknowledgements:
US DOE Office of Science, Biological and Environmental Research
Grant ID: DE-SC0023027
US HHS NIH, Eunice Kennedy Shriver National Institute of Child Health and Human Development
Grant ID: F32HD114387
Abstract
This method describes the analysis of lipids extracted from the green alga Auxenochlorella protothecoides x symbiontica UTEX 250-A. The protocol includes lipid extraction, derivatization of fatty acids (FA) for GC-MS analysis, and composition analysis of the FA at the sn-2 position of triacylglycerol (TAG). This protocol is adapted from widely used methods in the field of plant and algal lipids (Bligh and Dyer, 1959;  Luddy et al., 1964; Takeuchi et al., 2020), but it has been modified for optimal outcome with UTEX 250-A. The optimized steps include cell pretreatment, dichloromethane-based extraction, and porcine pancreas lipase treatment. Together, we provide a thorough and reliable protocol for lipid characterization in this algal strain.


Scheme for lipid extraction and fatty acid analysis of Auxenochlorella


Materials
Cell growth conditions
Auxenochlorella sp. UTEX 250-A cells are inoculated into ApM1 media (Ref. 9) for three stages, each with different concentrations of glucose and NH4+.


ABCDEF
StageDaysInoculation (%)Glucose (%)NH4+ (mM)Purpose
pre-seed3-4colony or patch215cell density normalization
seed18215log phase growth
lipid-induction5-1022 (d0) + 2 (d2)3.75TAG production



Equipment and device
AB
NameSource
Tissue Lyser IIQiagen 85300
FreeZone 2.5 LyophilizerLabconco 7670520
EquaVAP 54-Well EvaporatorAnalytical Sales and Services 23054
N-EVAP 112 OA-SYS Nitrogen EvaporatorOrganomation Associates
Latch-lid™ TLC developing chambersSupelco Z266000
PTFE multi-plate racks, for 20*20cmSupelco Z266051
TLC plate holder 25 platesSupelco Z265284
TLC plates, Silica gel 60 F254, 20*20 cmSupelco 1.05715
TLC saturation pads, 20*20 cmSupelco Z265225
Azure 600® Imaging StationAzure AZI600-01
MicrocentrifugeEppendorf 5417R
Microcentrifuge rotorEppendorf F45-30-11
CentrifugeEppendorf 5810R
Centrifuge rotor with tube adaptersEppendorf A-4-62

Tubes and vials
AB
NameSource
Threaded 2 mL tube and capFisher Sci. 02-681-358 and 02-681-343
Safe-lock 2 mL tubeEppendorf 0030123620
2 mL Screw top clear vialAgilent 5182-0715
2 mL Screw top amber vialAgilent 5182-0716
0.3 mL Screw top amber vial with insertAgilent 5188-6592
Screw cap for vials, blue, PTEF/red silicone septaAgilent 5185-5820
16x150 mm disposable round bottom threaded culture tubesCorning 888447-16X
Reusable Phenolic GPI 15-415 Threaded Screw CapCorning 9998-15
PTFE septa for screw capThermo Fisher Sci. B7995-15
0.5 mm dia. glass beadsBioSpec 11079105
4.0 mm dia. glass beadsFisher Sci. 11-312B
2.0 mm dia. zirconia beadsBioSpec 11079105

Chemicals and standards
Name ShortSourceNote
Acetone, ≥99.9%Sigma-Aldrich 650501-1L
Acetic acid glacial, ≥99.5%Avantor JT9522-3
Dichloromethane anhydrousDCMSigma-Aldrich 270997
Ethyl acetate, ≥99.8%EtOAcSigma-Aldrich 270989-1L
Methanol, ≥99.9% (GC)MeOHSupelco MX0486
Sodium chlorideNaCl0.9% (w/v)
Potassium chlorideKCl0.88% (w/v)
Calcium chlorideCaCl222% (w/v)
Tridecanoic acid (13:0)13:0Sigma-Aldrich 91988-5G50 mg/mL in DCM
Hydrogen chloride solution, 3M in methanolHCl-MeOHSupelco 90964
n-Hexane, ≥99% (GC), ACS reagentSupelco 1.04367
Heptane, ≥99% (GC)Sigma-Aldrich 32287-2.5L
Tris(hydroxymethyl)aminomethane hydrochlorideTris-HCl 1M solution, pH 8.0
Sodium Taurocholatebile acidSigma-Aldrich T4009-1G
Lipase from porcine pancreasSigma-Aldrich L3126-100G
Lipid Standard, Mono-, Di-, & Triglyceride Mix-40MGT/D/MSigma-Aldrich 1787-1AMPMake a 10 mg/mL solution in DCM
PremulineSigma-Aldrich 206865-1GMake a 5% (w/v) stock in water
Milli-Q Water (Ultrapure/Type 1) was used to prepare the solutions of KCl, NaCl, and CaCl2, which were then sterilized by filtration through a bottle-top vacuum filter (500 mL, 0.22 µm pore size,PES membrane).




Protocol materials
Nitrogen gas, UHP
Liquid Nitrogen
Safety warnings
The organic solvents used here are highly volatile and toxic. Every step involving the use of organic solvents should be operated in a fume hood. Wear gloves and a mask to prevent direct contact and inhalation of organic solvents.
Sample collection
55m
(Optional) Pre-weigh empty 2 mL threaded tubes with an accuracy of at least 0.1 mg. Perform this step if dry-weight biomass is required.
15m
Collect 2 mL of the UTEX 250-A cell culture in the tube.
30m
4000 x g, Room temperature, 00:05:00 . Discard the supernatant carefully. The cell pellet can be stored at -80 °C

10m
Lipid extraction[1-3]
16h 5m
(Optional) Lyophilize the cell pellet for Overnight . Measure the tube again to obtain the dry biomass (mg).
Note
Lyophilization of the cell pellet is optional. For quick fatty acid analysis without lipid/biomass quantification, fresh and frozen pellets can be processed directly as Step 5. However, lipid yield will decrease in non-lyophilized samples.

16h
Add one of the two types of beads into each tube:
  • Option 1: two 4 mm glass beads and 200 mg of 0.5 mm glass beads.
  • Option 2: 250 mg of 2 mm zirconia beads (about 12 beads).
Note
Distribute beads and cell pellet by vortexing and/or tapping the tube. This will prevent the cell pellet from getting stuck at the bottom during bead-beating and lowering the extraction efficiency.

Grind the cells using a TissueLyser II (or equivalent homogenizer) at 30 Hz for 00:01:00 twice. If using Liquid Nitrogen , flash-freeze pellets before each grinding interval. Visually inspect for sufficient homogenization. If cell chucks are observed, repeat this step.
Note
The degree of homogenization is critical for the extraction efficiency. For non-lyophilized frozen cell pellets, it is necessary to keep pellets flash frozen using Liquid Nitrogen , before placing the tubes in the cold TissueLyser II adapter pre-chilled at -20 °C (DO NOT freeze at -80 °C ). Grind the cells with TissueLyser II at 30 Hz for 1 minute, and then chill the tubes with Liquid Nitrogen . Repeat the grinding step (total grinding time: 2 minutes). This will ensure the pellet is ground evenly.


Expected result


Figure 1. Yields of lipids (% dry-weight biomass) extracted from samples ground with glass beads or zirconia beads at low temperature (liquid nitrogen; LN2) or room temperature (RT). No significant difference was observed. Cells were grown in the lipid-inducing medium (refer to materials) for 10 days.




Prepare lipid extraction solution by mixing dichloromethane (DCM) and methanol in a 2:1 (v/v) ratio.
ABC
x 1x 12
Dichloromethane (mL)112
Methanol (mL)0.56
Example of preparing lipid extraction solution for 1 and 12 reactions

Note
Always make a fresh solution before extraction. Keep the solution in a temporary amber glass bottle with a PTFE-lined cap.

Add 1.5 mL of the lipid extraction solution to the tube. Cap it and vortex for 00:00:03 .
4000 x g, 4°C, 00:05:00 , transfer the supernatant (crude extract) to a new 2 mL safe-lock tube.
Note
The cell debris should turn pale. Avoid transferring the cell debris and beads to the new tube. If using 0.5mm glass beads, pipetting can easily lead to transfer of these beads and skew final lipid yields.

Expected result

Figure 2. Cell debris (white arrow) and beads after centrifugation. Only transfer the supernatant for the downstream process. The pale color is expected after solvent extraction.


Immediately mix the crude extract above with 325 µL of 0.88% potassium chloride in water (w/v).

3000 x g, 4°C, 00:05:00 . Using a pipette or glass pasteur pipette, carefully transfer the bottom lipid phase to a 2 mL clear screw top vial. Visually inspect vial and remove any transferred beads or upper layer blobs. Store at -20 °C in the dark.
Note
After removing the vial from -20 °C storage, allow it to equilibrate to Room temperature before opening to avoid condensation forming inside.


Expected result


Figure 3. Separation of the lipid-containing organic layer after centrifugation.



5m
To obtain total lipid weight, pre-weigh the empty 2 mL screw-top clear vial (Agilent 5182-0715) and dry the lipid solution with Nitrogen gas, UHP completely, then weigh the vial to get the lipid weight (net).
Expected result
Typically, 2 mL of UTEX 250-A cell culture grown in the lipid-inducing medium (refer to materials) for 5 days to an OD750 between 15-16 produces 11-12 mg of lipids (primarily triacylglycerols), corresponding to 50-55% of the dry-weight biomass.

(optional) The extracted pellet in step 9 can be re-extracted (2-step) to maximize lipid yield as follows:
1. Add 1 mL of milliQ water, then vortex to resuspend the pellet.
2. Spin at 3000 x g, 4°C, 00:05:00 , discard the supernatant
3. Repeat steps 8 - 11. Combine the organic layer with the one from the previous step 11.
Note

Figure 4. If cell pellet is visibly yellow (eg. two right tubes), likely more lipid can be extracted. This additional wash step is recommended to increase lipid recovery.


Expected result


Figure 5. Yields of lipids (mg per mL of cell culture) extracted from lyophilized (Lyo) or fresh samples with the 1-step or 2-step extraction method. Lyophilization significantly increased the extraction efficiency, while the 2-step extraction improved it more with the fresh pellet than with the lyophilized one. Cells were grown in the lipid-inducing medium (refer to materials) for 10 days.



Dissolve the dried lipids with 1 mL of dichloromethane (methylene chloride), or adjust to a final concentration of 20 mg/mL based on the lipid weight.
Acid-catalyzed methanol transesterification[4]
Transfer 100 µL - 1 mL of lipids to a 16x150 mm threaded glass tube, mix with 5 µL of C13:0 fatty acid (5 mg/mL in DCM ) as the internal standard, then add 1 mL of 1 N Methanolic hydrogen chloride (HCl-MeOH). Close the tube with a new PTFE septum inside the cap.
ABC
x 3x 12
3N HCl-MeOH (mL)14
Methanol (mL)28
Preparation of 1N Methanolic hydrogen chloride (HCl-MeOH) for 3 and 12 reactions

Note
The PTFE septum prevents solvent from evaporating in the next step and dissolving unwanted chemicals from the cap. Make sure the tube is sealed properly.

Incubate in a waterbath at 80 °C for 00:30:00 .
Note
Check every 10 minutes for leaking. If there are leaks, take out the leaky tube, wait until it returns to room temperature, and then open it and add more 1N HCl-MeOH. Replace the cap and septum as needed to ensure a proper seal.


Wait until the tube has returned to room temperature before opening it. Add 1 mL of 0.9% (w/v) Sodium Chloride and 1 mL of n-hexane. Close the tube with the same cap and vortex it briefly.

3000 x g, 4°C, 00:05:00 . Transfer the top layer to a 2 mL amber screw top vial.
Note
Avoid getting the bottom layer.

Using an evaporator, dry the fatty acid methyl ester (FAME) solution with nitrogen. Resuspend it with the desired amount (500 µL -1 mL ) of n-hexane or heptane and store it at -20 °C .

Transfer 50 µL of FAME to a 300 μL amber vial with insert for GC-MS or GC-FID analysis.

Note
Can be diluted with hexane to adjust the FAME concentration for optimal GC-MS ion signals.

sn-2 fatty acids analysis[5-8]
Heat up the water bath to 40 °C

Transfer 4-5 mg total lipids in DCM (for example, 250 µL of 20 mg/mL lipid extract) to a 16x150 mm threaded glass tube. Dry it with Nitrogen gas, UHP .
Note
Total lipids rather than isolated triacylglycerols (TAGs) are used here, as the lipids of UTEX 250-A cells grown under the lipid-inducing conditions are highly enriched in TAGs (estimated to be >98%) (Fig. 6). Adding an extra step of TAG isolation from total lipids is therefore determined to be unnecessary, as the process is time-consuming and increases the risk of lipid loss and subsequent signal loss in GC-MS.
Figure 6. Comparison of fatty acid composition between total lipids and triacylglycerols (TAGs). TAGs are separated from total lipids through thin-layer chromatography (steps 26-30). The similar profiles reflect the dominance of TAGs in total lipids.




While drying the lipid solution, prepare the lipase solution as below:
Recipe for 0.1% bile acid (taurocholic acid) solution:
ABC
x 1x 12
Bile acid (mg)13
1M Tris-HCl, pH 8.0 (mL)13

Recipe for lipase solution, after preparing the bile acid solution:
ABC
x 1x 12
1M Tris-HCl, pH 8.0 (mL)112
22% CaCl2 (mL)0.11.2
0.1% Bile acid (mL)0.253
Lipase from porcine pancreas (mg)448
Total (mL)1.3516.25


After the solvent has evaporated, add 20 µL of DCM to the tube to dissolve the lipid, then add 1.35 mL of lipase solution, cap the tube, and vortex for 1-2s.
Inbuate the mixture at 40 °C for 00:01:00 .

Take the mixture out and vortex at full speed for 3s, put it back to the water bath for 10-20s, repeat this step for 00:02:00 .
Note
When processing multiple tubes, it is critical to ensure each mixture receives an equal amount of vortexing time. It is recommended to process no more than 12 tubes per batch and vortex 3-4 tubes at a time.
Expected result

Figure 7. Impact of vortex during the digestion of UTEX 250-A total lipids with lipase from porcine pancreas. The digested lipids were separated and visualized on a TLC plate (see steps 25-29). TAG: triacylglycerol, 1, 3 DAG: sn-1, 3 diacylglycerol, 1, 2 DAG: sn-1, 2 diacylglycerol, MAG: monoacylglycerol.



Incubate the mixture at 40 °C for another 00:10:00 .
Add 1 mL of DCM, vortex for 2-5s, and then centrifuge at3000 x g, 4°C, 00:05:00 . Carefully transfer the bottom layer to a 2 mL clear screw top vial. The digested lipids can be stored at -20 °C .
Note
Contamination with the solid material at the interface between the organic and aqueous layers will hinder the loading and separation of lipids on the TLC plate in the next step.

Prepare 20x20cm Thin Layer Chromatography Silica (TLC) plates and TLC developing tank as follows:
  • Bake TLC plates in an oven at 85 °C -120 °C for 00:30:00 .
  • Make a TLC developing solvent mix for neutral lipid separation:
AB
n-hexane (mL)50
EtOAc (mL)50
Acetic acid (mL)1
  • Pour the developing solvent into the TLC developing tank, insert a TLC saturation pad, and close the lid.
  • Let the tank sit for 00:30:00
Using a pencil, lightly draw a horizontal line 2 cm from the bottom edge of the baked TLC plate. Mark 9 equally spaced points along the line at 2 cm intervals to indicate the positions for loading 9 samples.
Condense the digested lipid solution with Nitrogen gas, UHP to 20 µL -40 µL

Load all of the lipid samples onto the marked points of the TLC plate.
(Optional) Load 5 µL of the TAG/DAG/MAG mix (10 mg/mL in DCM) on the first dot as a control.
Put the TLC plate on the TLC rack, then place the rack in the TLC chamber. Close the lid immediately.
Allow the separation of lipids to proceed for 00:40:00 .

Remove the TLC plate from the chamber, let it sit for 00:05:00 or until the solvent is fully evaporated.

1 g Spray the plate with 0.5 mg/mL primuline evenly with a spray bottle connected to an air outlet.

Preparation of 0.05% (w/v) primuline in 80% acetone:
AB
5% (w/v) primuline in water (mL)2
Acetone (mL)160
Water (mL)38
Total (mL)200

Note
Prepare the 5% (w/v) primuline stock solution by dissolving 1 g of primuline in 20 mL of water. Store the final 0.05% (w/v) primuline solution in the dark or wrap the bottle with foil.”


Wait until the plate is fully dry, transfer it to a dark chamber, then visualize the lipid with a UV lamp (340-365 nm). Use a pencil to circle 2-monoacylglycerol (2-MAG), then turn off the UV lamp.
Safety information
Wear appropriate PPE when working with UV light.

Expected result
Figure 8. Visualization of neutral lipids stained with primuline under exposure to UV 365 nm. 5 mg of lipids extracted from engineered UTEX 250-A are digested with sn-1,3-specific lipase and separated on a TLC plate. The 2-MAG fraction is boxed.


The TLC plate can be imaged using an Azure imaging system equipped with a trans UV365 filter.
Use a razor blade to score the outline of the circled 2-MAG spot, then scrape the spot from the TLC plate with the rounded end of a metal micro spatula. Collect the silica powder containing 2-MAGs with weighing paper and transfer into a 16x150 mm threaded glass tube. Clean tools between each use with methanol to avoid cross-contamination.


Add 1 mL of 1 N HCl-MeOH and 5 µL of C13:0 fatty acid (5 mg/mL in DCM). Proceed with the acid-catalyzed methanolysis step as described above for GC-MS / GC-FID analysis of sn-2 fatty acid composition.
Expected result

Figure 9. (A) GC chromatograms of FAME derived from UTEX 250-A lipids before (top) and after (bottom) the sn-1,3 lipase treatment. The red arrow indicates the 16:0 peak which was predominantly excluded from the sn-2 position of triacylglycerol in UTEX 250-A. (B) Fatty acid composition (% of total fatty acids) of UTEX 250-A lipids before and after the sn-1,3 lipase treatment. The remaining, undigested TAG and the digested 2-MAG samples were collected from the TLC plate. As indicated by the similarity in FA profiles between the total lipids and undigested TAG, the sn-1,3 lipase exhibited no significant fatty acyl substrate preferences.



Protocol references
1. Bligh, E. G. & Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol.37, 911–917 (1959).
2. Cequier-Sánchez, E., Rodríguez, C., Ravelo, Á. G. & Zárate, R. Dichloromethane as a Solvent for Lipid Extraction and Assessment of Lipid Classes and Fatty Acids from Samples of Different Natures. J. Agric. Food Chem. 56, 4297–4303 (2008).
3. Ren, X. et al. Current lipid extraction methods are significantly enhanced adding a water treatment step in Chlorella protothecoides. Microb Cell Fact 16, 26 (2017).
4. Takeuchi, T. et al. Chlamydomonas CHT7 is required for an effective quiescent state by regulating nutrient-responsive cell cycle gene expression. Plant Cell 32, 1240–1269 (2020).
5. Hsieh, F.-L., Chang, T.-H., Ko, T.-P. & Wang, A. H.-J. Structure and Mechanism of an Arabidopsis Medium/Long-Chain-Length Prenyl Pyrophosphate Synthase. Plant Physiology 155, 1079–1090 (2011).
6. Esperón-Rojas, A. A. et al. A Specific Thin Layer Chromatography Method for the Identification and Separation of Medium Chain Acylglycerols. J. Oleo Sci.67, 1397–1403 (2018).
7. Luddy, F. E., Barford, R. A., Herb, S. F., Magidman, P. & Riemenschneider, R. W. Pancreatic lipase hydrolysis of triglycerides by a semimicro technique. J Americ Oil Chem Soc 41, 693–696 (1964).
8. van Erp, H., Bates, P. D., Burgal, J., Shockey, J. & Browse, J. Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant Physiology 155, 683–693 (2011).
9. Moseley, J. Auxenochlorella ApM1/ApRM1 media recipes and stocks v2. 2025. https://doi.org/10.17504/protocols.io.q26g71pq8gwz/v2