Rapid extraction of total lipids from microalgae

Ying-Yu Hu, Zoe V. Finkel

Published: 2022-06-08 DOI: 10.17504/protocols.io.dm6gpr9jdvzp/v4

Abstract

In this protocol, total lipids from miroalgae is extracted with Folch solvent (2:1 chloroform-methanol v/v) and the addition of 5% water. 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. The extract is then mixed with 0.88% potassium chloride solution to form a biphasic system, where in between the two phases is the thin distinct cell debris layer. The lower phase with extracted lipids is collected and dried under N₂gas flow. The residue is stored under -80 ºC for further measurement.

Citation

FOLCH J, LEES M, SLOANE STANLEY GH A simple method for the isolation and purification of total lipides from animal tissues J Biol Chem, 1957, 226, 497-509

Citation

Axelsson M, Gentili F 2014 A single-step method for rapid extraction of total lipids from green microalgae. PloS one https://doi.org/10.1371/journal.pone.0089643

Before start

Steps

Collect microalgae samples

Precombust GFF filter at 450°C for 4h 0m 0s

Rinse forceps with 95% ethanol, air-dry.

Equipment

Value	Label
Filter forceps	NAME
blunt end, stainless steel	TYPE
Millipore	BRAND
XX6200006P	SKU
http://www.emdmillipore.com/	LINK

Note

Wipe-dry forceps can cause carbon contamination of samples.

Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (5 inches 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

Value	Label
FreeZone® 2.5 L Benchtop Freeze Dryers	NAME
Labconco®	BRAND
700202000	SKU

Follow protocol to hydrolyze the sample.

Hydrolysation treatment can improve the extraction efficiency:

Prepare glassware

10.

Precombust the centrifuge tubes, scintillation vials and storage vials at 500°C for 6h 0m 0s

11.

Precombust pasteur pipets at 500°C for 2h 0m 0s

Equipment

Value	Label
Disposable Soda-Lime Glass Pasteur Pipets	NAME
5 3/4"	TYPE
Fisherbrand	BRAND
13-678-6A	SKU
https://www.fishersci.com/us/en/home.html	LINK

12.

Rinse caps with 95% ethanol and air-dry prior to use

13.

Rinse serological pipets and the reagent bottle for dichromate reagent with 95% ethanol until there is not stain and with chloroform for the final rinse. Air-dry.

Equipment

Value	Label
VWR® Volumetric Pipets, Reusable, Color Coded, Class A	NAME
0.5 mL and 5 mL	TYPE
VWR	BRAND
10546-004 and 10546-014	SKU

Equipment

Value	Label
PYREX® Media Bottles	NAME
Corning®	BRAND
1395-100	SKU

14.

Latex bulbs are required for Pasteur pipets

Prepare reagent

15.

Folch solvent (CHCl₃: MeOH=2:1 v/v)

15.1.

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)

15.2.

Attach dispensette to the bottle, mix well.

Equipment

Value	Label
Bottle-top dispenser	NAME
BrandTech Dispensette® S	BRAND
4731330	SKU

15.3.

Label bottle with MSDS label.

16.

KCl solution (0.88% )

16.1.

Weigh the pyrex media bottle and tare.

Equipment

Value	Label
PYREX® Media Bottles	NAME
Corning®	BRAND
1395-100	SKU

16.2.

Directly weigh 0.44g KCl in the bottle.

16.3.

Top bottle with MilliQ water to 50g

A	B
KCl (g)
Final (g)

Extraction

17.

If lipids samples are not processed for carbohydrate, transfer freeze dried samples and blanks into muffled centrifuge tubes

Note

It takes about 7 to 8 hours to process 16 samples.

Equipment

Value	Label
Disposable Glass Screw-Cap Centrifuge Tubes	NAME
10 mL	TYPE
Corning®	BRAND
99502-10	SKU

Equipment

Value	Label
Polypropylene Screw Caps	NAME
Linerless, 15-415	TYPE
Kimble Chase	BRAND
73805-15415	SKU

17.1.

Add 100µL MilliQ directly onto the sample.

17.2.

Freeze at -80°C 0h 10m 0s

17.3.

Remove vials from freezer.

17.4.

Purge the dispensette, fill the tubing with solvent before dispensing solvent into sample tube.

17.5.

Dispense 2.0mL Folch solvent into sample tube.

18.

If lipids samples have been hydrolyzed for carbohydrate and solvent has already been added, go to the vortex step directly.

19.

Vortex 0h 28m 0s by using a tube insert.

Equipment

Value	Label
VWR ANALOG VORTEX MIXER	NAME
VWR	BRAND
10153-838	SKU
With tube insert	SPECIFICATIONS

20.

Sonicate 0h 2m 0s

21.

Vortex 0h 30m 0s by using a tube insert.

22.

Prepare one set of precombusted tubes (#T1), label the tubes, cap is not required.

Equipment

Value	Label
Disposable Glass Screw-Cap Centrifuge Tubes	NAME
10 mL	TYPE
Corning®	BRAND
99502-10	SKU

23.

Place one pasteur pipet (#P1) into each tube

Equipment

Value	Label
Disposable Pasteur Pipet	NAME
9 inch	TYPE
VWR	BRAND
14672-380	SKU

24.

Prepare another set of precombusted tubes (#T2) for supernatant. Cap the tube to avoid contamination.

Equipment

Value	Label
Disposable Glass Screw-Cap Centrifuge Tubes	NAME
10 mL	TYPE
Corning®	BRAND
99502-10	SKU

Equipment

Value	Label
Polypropylene Screw Caps	NAME
Linerless, 15-415	TYPE
Kimble Chase	BRAND
73805-15415	SKU

25.

Work on eight samples first. Use the pasteur pipet (#P1) to gently lift the filter upwards and transfer liquid (as much as possible) to the centrifuge tube. Keep the pasteur pipet (#P1) in its corresponding empty tube (#T1).

26.

Add 50µL MilliQ and 1mL Folch solvent to the residue.

27.

Vortex the eight samples at the highest speed to loosen the filter.

28.

Vortex the eight samples by using a tube insert while transferring supernatant from another eight samples to centrifuge tubes.

29.

Sonicate 0h 2m 0s

30.

Vortex the second eight samples by using a tube insert while transferring supernatant from the first eight samples to centrifuge tubes.

Note

Same alternate routine for the following steps.

31.

Use the pasteur pipet (#P1) to gently lift the filter upwards and transfer all liquid to the centrifuge tube (#T1). Keep the pasteur pipet (#P1) in its corresponding empty tube (#T1).

32.

Add 50µL MilliQ and 1mL Folch solvent to the residue.

33.

Vortex while transferring supernatant from another set of samples to centrifuge tubes.

34.

Sonicate 0h 2m 0s

35.

Use the pasteur pipet (#P1) to gently lift the filter upwards and transfer all liquid to the centrifuge tube (#T1). Keep the pasteur pipet (#P1) in its corresponding empty tube (#T1).

36.

Add 50µL MilliQ and 1mL Folch solvent to the residue.

37.

Vortex while transferring supernatant from another set of samples to centrifuge tubes.

38.

Sonicate 0h 2m 0s

39.

Use the pasteur pipet (#P1) to gently lift the filter upwards and transfer all liquid to the centrifuge tube (#T1).

40.

Leave the pasteur pipet (#P1) in the tube with filter for a while, the tip gradually sucks liquid dripping from filter.

41.

Pipette the pasteur pipet in water layer up and down to rinse off organic extract. Put the pipet back in the tube with filter.

Separation

42.

Centrifuge at 3200rpm

Equipment

Value	Label
General-purpose benchtop centrifuge	NAME
IEC CENTRA CL2	TYPE
Thermo	BRAND
00427 0F	SKU

43.

Filter and cell debris stay between the two layer.

44.

Use pasteur pipet (#P1) to remove supernatant as much as possible. Do not disturb the debris.

45.

Return pipet (#P1) back to the tube with filter.

46.

Add 500 ul KCl solution, vortex and then centrifuge at 3200rpm

Note

Volume of Folch solvent to KCl is about 4 to 1.

47.

Use #P1 to remove supernatant as much as possible. Do not disturb the debris.

48.

Turn on heat block to 37°C , use a thermometer to monitor the actual temperature.

Equipment

Value	Label
LSE digital dry bath heater	NAME
Corning	BRAND
6885-DB	SKU

Equipment

Value	Label
Blocks for Corning® LSE Digital Dry Bath Heaters	NAME
Corning	BRAND
480124	SKU

49.

Place tubes in the heater.

Note

Organic layer turns foggy when temperature is lower than 37°C

50.

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

If it has already been the end of the day, keep samples at -80°C

Note

Clear vial helps to check if there is water drops or impurities in lipids extract after dried.

Equipment

Value	Label
Glass Vials PTFE/SILiCone SEPTA Clear	NAME
16 mL	TYPE
Thermo Scientific	BRAND
B7990-4	SKU
https://www.fishersci.com/us/en/home.html	LINK

Equipment

Value	Label
Screw Vial Convenience Kit, 12mL solid top PTFE cap	NAME
Thermo Scientific	BRAND
B7800-12A	SKU

51.

Dry organic phase extract at 37°C under a stream of N₂ gas (<2 psi) for about 0h 30m 0s .

A	B	C
	Time	Gas cylinder pressure
Start
End

Equipment

Value	Label
Reacti-Vap Evaporator	NAME
Thermo Scientific	BRAND
TS-18825	SKU

Purification

52.

The lipids extract might still have water residue (which can't be dried by nitrogen gas) or water soluble impurities.

Redissolve it with 5mL chloroform by using glass serological pipet, transfer certain amount of chloroform dissolved extract for lipids measurement (based on the estimation, <100 ug) into a new vial. Log the actual volume transferred.

Equipment

Value	Label
Safetypette	NAME
Jencons	BRAND
75856-442	SKU

53.

Dry extract at 37°C under a stream of N₂ gas (<2 psi) for about 0h 30m 0s (Generally 2 mL/5 min).

A	B	C
	Time	Gas cylinder pressure
Start
End

54.

Freeze dried extract and excess extract (in chloroform) at -80°C.

Rapid extraction of total lipids from microalgae

Abstract

Before start

Steps

Collect microalgae samples

Prepare glassware

Prepare reagent

Extraction

Separation

Purification

推荐阅读