Jul 21, 2022
Version 5
Rapid extraction of total lipids from microalgae V.5
- Ying-Yu Hu1,
- Zoe V. Finkel1
- 1Dalhousie University
Protocol Citation: Ying-Yu Hu, Zoe V. Finkel 2022. Rapid extraction of total lipids from microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpr9jdvzp/v5Version created by Ying-Yu Hu
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: July 06, 2022
Last Modified: July 21, 2022
Protocol Integer ID: 66101
Keywords: lipids, microalgae, Folch solvent,
Abstract
In this protocol, total lipids from miroalgae is extracted with a mixture of water and Folch solvent (2:1 chloroform-methanol v/v). Filter and cell debris is commonly removed by filtration, which is laborious and time consuming. It is also the main reason to either cause sample loss and therefore underestimation; or contamination from filtration system and therefore overestimation. We now use centrifugation to remove filter debris, which stays in between the organic phase and water phase. The extracted lipids is dried under N2 gas flow, stored under -80 ºC for further measurement.
CITATION
CITATION
Guidelines
Biomass requirement
Considering that
(1) lipids are approximately 10~30% of microalgal dry mass
(2) the linear range for colorimetric lipid analysis is 4.2 to 80 μg, the low limit of quantitation is 20 μg
The minimum requirement of sample volume for total lipids is calculated as following:
V_L=20/Chl-a/(17.3/1.1)
If both total lipids and phospholipids are expected to be measured, the minimum sample volume needs to be at least doubled.
Protocol materials
Chloroform (HPLC grade)Merck MilliporeSigma (Sigma-Aldrich)Catalog #439142-4L
Step 14.1
MethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #34860
Step 14.1
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P3911-500G
Step 15.2
Safety warnings
Safety information
Operate chloroform in fume hood.
Safety information
Follow the disposal guidelines regarding the halogenated organic waste.
Collect microalgae samples
Collect microalgae samples
Precombust GFF filter at 450 °C for 04:00:00
4h
Rinse forceps with 95% ethanol, air-dry.
Equipment
Filter forceps
NAME
blunt end, stainless steel
TYPE
Millipore
BRAND
XX6200006P
SKU
Note
Wipe-dry forceps can cause carbon contamination of samples.
Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (130 mm Hg).
Rinse sample with filtered seawater
Place sample filters in cryogenic vials
Filter blank media (without cells) through precombusted GFF filter as blank.
Flash freeze filters and stored at -80 °C
Freeze dry before measurement.
Equipment
FreeZone® 2.5 L Benchtop Freeze Dryers
NAME
Labconco®
BRAND
700202000
SKU
Follow <Total particulate carbohydrate from microalgae> protocol to hydrolyze the sample.
Hydrolysation treatment can improve the extraction efficiency:
Prepare glassware
Prepare glassware
Precombust the centrifuge tubes at 500 °C for 06:00:00
6h
Precombust pasteur pipets at 500 °C for 02:00:00
Equipment
Disposable Soda-Lime Glass Pasteur Pipets
NAME
5 3/4"
TYPE
Fisherbrand
BRAND
13-678-6A
SKU
2h
Rinse caps with 95% ethanol and air-dry prior to use
Latex bulbs are required for Pasteur pipets
Prepare reagent
Prepare reagent
Folch solvent (CHCl3: MeOH=2:1 v/v)
Mix two parts of chloroform and one part of methanol in a 1 L amber bottle. Log the volume of each solvent for double checking the ratio.
| A | B | |
| Chloroform (mL) | ||
| Methanol (mL) |
Chloroform (HPLC grade)VWR InternationalCatalog #439142-4L
MethanolVWR InternationalCatalog #34860
Attach dispensette to the bottle, mix well.
Equipment
Bottle-top dispenser
NAME
BrandTech Dispensette® S
BRAND
4731330
SKU
Label bottle with MSDS label.
KCl solution (0.88 % )
Note
1.5 mL per sample
Weigh the pyrex media bottle and tare.
Equipment
PYREX® Media Bottles
NAME
Corning®
BRAND
1395-100
SKU
Directly weigh 0.44 g KCl in the bottle.
Potassium chlorideVWR InternationalCatalog #P3911-500G
Top bottle with MilliQ water to 50 g
| A | B | |
| KCl (g) | ||
| Final (g) |
Prepare for extraction
Prepare for extraction
If lipids samples are not processed for carbohydrate, transfer freeze dried samples and blanks into muffled centrifuge tubes
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU
Equipment
Polypropylene Screw Caps
NAME
Linerless, 15-415
TYPE
Kimble Chase
BRAND
73805-15415
SKU
Add 100 µL MilliQ directly onto the sample.
Freeze at -80 °C 00:10:00
10m
Remove vials from freezer.
Purge the dispensette, fill the tubing with solvent before dispensing solvent into sample tube.
Dispense 2.0 mL Folch solvent into sample tube.
If lipids samples have been hydrolyzed for carbohydrate and solvent has already been added, go to the vortex step directly.
Extract lipids
Extract lipids
Vortex 00:28:00 by using a tube insert.
Equipment
VWR ANALOG VORTEX MIXER
NAME
VWR
BRAND
10153-838
SKU
With tube insert
SPECIFICATIONS
28m
Sonicate 00:02:00
Vortex 00:30:00 by using a tube insert.
30m
Prepare one set of precombusted tubes (#T1), label the tubes, cap is not required.
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU
Place one pasteur pipet into each tube (#T1)
Equipment
Disposable Pasteur Pipet
NAME
9 inch
TYPE
VWR
BRAND
14672-380
SKU
Prepare another set of precombusted tubes (#T2) for extract. Cap the tube to avoid contamination.
Equipment
Disposable Glass Screw-Cap Centrifuge Tubes
NAME
10 mL
TYPE
Corning®
BRAND
99502-10
SKU
Equipment
Polypropylene Screw Caps
NAME
Linerless, 15-415
TYPE
Kimble Chase
BRAND
73805-15415
SKU
Centrifuge at 3200 rpm, Room temperature, 00:05:00
Equipment
General-purpose benchtop centrifuge
NAME
IEC CENTRA CL2
TYPE
Thermo
BRAND
00427 0F
SKU
5m
Use the pasteur pipet to transfer organic layer to #T2.
Note
If some debris precipitate at the bottom, gentle blow air bubble through pasteur pipet to relocate it to water layer. Avoid transferring water layer and debris into #T2
Add 1 mL Folch solvent to the residue.
Sonicate 00:02:00
Vortex by using a tube insert for about 00:10:00
10m
Centrifuge at 3200 rpm, Room temperature, 00:05:00
Equipment
General-purpose benchtop centrifuge
NAME
IEC CENTRA CL2
TYPE
Thermo
BRAND
00427 0F
SKU
Use the pasteur pipet to transfer organic layer to #T2.
Add 1 mL Folch solvent to the residue.
Sonicate 00:02:00
Vortex by using a tube insert for about 00:10:00
Centrifuge at 3200 rpm, Room temperature, 00:05:00
Equipment
General-purpose benchtop centrifuge
NAME
IEC CENTRA CL2
TYPE
Thermo
BRAND
00427 0F
SKU
Use the pasteur pipet to transfer organic layer to #T2.
Add 1 mL Folch solvent to the residue.
Sonicate 00:02:00
Vortex by using a tube insert for about 00:10:00
Collect lipids extract
Collect lipids extract
1h 30m
1h 30m
Centrifuge at 3200 rpm, Room temperature, 00:05:00
5m
Use the pasteur pipet to transfer organic layer to #T2.
Add 1.5 mL KCl solution to T2, vortex and then centrifuge at 3200 rpm, Room temperature, 00:05:00
Note
Volume of Folch solvent to KCl is about 4 to 1.
5m
Turn on heat block to 37 °C , use a thermometer to monitor the actual temperature.
Equipment
LSE digital dry bath heater
NAME
Corning
BRAND
6885-DB
SKU
Equipment
Blocks for Corning® LSE Digital Dry Bath Heaters
NAME
Corning
BRAND
480124
SKU
Place tubes (#T2) in the heater.
Note
Organic layer turns foggy when temperature is lower than 37 °C
Use a new pasteur pipet to transfer the lower organic phase to a clear 12 mL storage vial.
Do not disturb the cell debris in between the two phases.
Note
Clear vial helps to check if there is water drops or impurities in lipids extract after dried.
Equipment
Glass Vials PTFE/SILiCone SEPTA Clear
NAME
16 mL
TYPE
Thermo Scientific
BRAND
B7990-4
SKU
Equipment
Screw Vial Convenience Kit, 12mL solid top PTFE cap
NAME
Thermo Scientific
BRAND
B7800-12A
SKU
Dry organic phase extract at 37 °C under a stream of N2 gas (<2 psi) for about 00:30:00 .
| A | B | C | |
| Time | Gas cylinder pressure | ||
| Start | |||
| End |
Equipment
Reacti-Vap Evaporator
NAME
Thermo Scientific
BRAND
TS-18825
SKU
30m
Purify lipids extract
Purify lipids extract
30m
30m
The lipids extract might still have water residue (which can't be dried by nitrogen gas) or water soluble impurities.
Redissolve it with 5 mL chloroform by using glass serological pipet, transfer certain amount of chloroform dissolved extract for lipids measurement (based on the estimation, 20 to 100 ug) into a new vial. Log the actual volume transferred.
Equipment
Safetypette
NAME
Jencons
BRAND
75856-442
SKU
Dry extract at 37 °C under a stream of N2 gas (<2 psi) (Generally 2 mL/5 min).
| A | B | C | |
| Time | Gas cylinder pressure | ||
| Start | |||
| End |
Store dried extract and excess extract (in chloroform) at -80 °C .
Citations
FOLCH J, LEES M, SLOANE STANLEY GH. A simple method for the isolation and purification of total lipides from animal tissues
Axelsson M, Gentili F. A single-step method for rapid extraction of total lipids from green microalgae.
https://doi.org/10.1371/journal.pone.0089643