Rapid extraction of total lipids from microalgae

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

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

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:

Precombust the centrifuge tubes at 500°C for 6h 0m 0s

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

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

Latex bulbs are required for Pasteur pipets

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)

Attach dispensette to the bottle, mix well.

Equipment

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

Label bottle with MSDS label.

KCl solution (0.88% )

Weigh the pyrex media bottle and tare.

Equipment

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

Directly weigh 0.44g KCl in the bottle.

Top bottle with MilliQ water to 50g

A	B
KCl (g)
Final (g)

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

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

Add 100µL MilliQ directly onto the sample.

Freeze at -80°C 0h 10m 0s

Remove vials from freezer.

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

Dispense 2.0mL 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.

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

Sonicate 0h 2m 0s

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

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

Place one pasteur pipet into each tube (#T1)

Equipment

Value	Label
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

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

Centrifuge at 3200rpm

Equipment

Value	Label
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 1mL Folch solvent to the residue.

Sonicate 0h 2m 0s

Vortex by using a tube insert for about 0h 10m 0s

Centrifuge at 3200rpm

Equipment

Value	Label
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 1mL Folch solvent to the residue.

Sonicate 0h 2m 0s

Vortex by using a tube insert for about 0h 10m 0s

Centrifuge at 3200rpm

Equipment

Value	Label
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 1mL Folch solvent to the residue.

Sonicate 0h 2m 0s

Vortex by using a tube insert for about 0h 10m 0s

Centrifuge at 3200rpm

Use the pasteur pipet to transfer organic layer to #T2.

Add 1.5mL KCl solution to T2, vortex and then centrifuge at 3200rpm

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

Place tubes (#T2) in the heater.

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.

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

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

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, 20 to 100 ug) into a new vial. Log the actual volume transferred.

Equipment

Value	Label
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.