TITLE: Rapid extraction of total lipids from microalgae
AUTHORS: Yingyu  Hu, Zoe  V Finkel

[DESCRIPTION]
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.
 

 



[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.






[STEPS]
SECTION: Collect microalgae samples
1. Precombust GFF filter at 450 °C for  240 min 
SECTION: Collect microalgae samples
2. Rinse forceps with 95% ethanol, air-dry.

 

 

SECTION: Collect microalgae samples
3. Filter microalgae in liquid media onto precombusted GFF filters, using gentle vacuum pressure (130 mm Hg).
SECTION: Collect microalgae samples
4. Rinse sample with filtered seawater
SECTION: Collect microalgae samples
5. Place sample filters in cryogenic vials
SECTION: Collect microalgae samples
6. Filter blank media (without cells) through precombusted GFF filter as blank.
SECTION: Collect microalgae samples
7. Flash freeze filters and stored at -80 °C 

SECTION: Collect microalgae samples
8. Freeze dry before measurement.
 

SECTION: Collect microalgae samples
9. Follow <Total particulate carbohydrate from microalgae> protocol to hydrolyze the sample.
Hydrolysation treatment can improve the extraction efficiency:
  

SECTION: Prepare glassware
10. Precombust the centrifuge tubes at 500 °C for  360 min 
SECTION: Prepare glassware
11. Precombust pasteur pipets at 500 °C for  120 min 
 

SECTION: Prepare glassware
12. Rinse caps with 95% ethanol and air-dry prior to use
SECTION: Prepare glassware
13. Latex bulbs are required for Pasteur pipets
SECTION: Prepare reagent
14. Folch solvent (CHCl3: MeOH=2:1 v/v)
SECTION: Prepare reagent
14.1. 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.
 
  
  

SECTION: Prepare reagent
14.2. Attach dispensette to the bottle, mix well.
 

SECTION: Prepare reagent
14.3. Label bottle with MSDS label.
SECTION: Prepare reagent
15. KCl solution (0.88 % )
 


SECTION: Prepare reagent
15.1. Weigh the pyrex media bottle and tare.
 

SECTION: Prepare reagent
15.2. Directly weigh 0.44 g KCl in the bottle.
  

SECTION: Prepare reagent
15.3. Top bottle with MilliQ water to 50 g
 

 
 
SECTION: Prepare for extraction
16. If lipids samples are not processed for carbohydrate, transfer freeze dried samples and blanks into muffled centrifuge tubes
 

 

SECTION: Prepare for extraction
16.1.  Add 100 µL MilliQ directly onto the sample.
SECTION: Prepare for extraction
16.2. Freeze at -80 °C   10 min 
SECTION: Prepare for extraction
16.3. Remove vials from freezer.
SECTION: Prepare for extraction
16.4. Purge the dispensette, fill the tubing with solvent before dispensing solvent into sample tube.
SECTION: Prepare for extraction
16.5. Dispense 2.0 mL Folch solvent into sample tube.
SECTION: Prepare for extraction
17. If lipids samples have been hydrolyzed for carbohydrate and solvent has already been added, go to the vortex step directly. 

SECTION: Extract lipids
18. Vortex  28 min by using a tube insert.
 

SECTION: Extract lipids
19. Sonicate  2 min 
SECTION: Extract lipids
20. Vortex  30 min by using a tube insert.
SECTION: Extract lipids
21. Prepare one set of precombusted tubes (#T1), label the tubes, cap is not required.

 

SECTION: Extract lipids
22. Place one pasteur pipet into each tube (#T1)

 

SECTION: Extract lipids
23. Prepare another set of precombusted tubes (#T2) for extract. Cap the tube to avoid contamination.

 

 

SECTION: Extract lipids
24. Centrifuge at 3200 rpm, 5 min 

 

SECTION: Extract lipids
26. Add 1 mL Folch solvent to the residue.
SECTION: Extract lipids
28. Vortex by using a tube insert for about 10 min 

SECTION: Extract lipids
27. Sonicate  2 min 
SECTION: Collect lipids extract
39. Centrifuge at 3200 rpm, 5 min 



SECTION: Collect lipids extract
40. Use the pasteur pipet to transfer organic layer to #T2. 
SECTION: Collect lipids extract
41. Add 1.5 mL KCl solution to T2, vortex and then centrifuge at 3200 rpm, 5 min 
 


SECTION: Collect lipids extract
42. Turn on heat block to 37 °C , use a thermometer to monitor the actual temperature.
 


 

SECTION: Collect lipids extract
43. Place tubes (#T2) in the heater. 

 


SECTION: Collect lipids extract
44. 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.
 

 

 

SECTION: Collect lipids extract
45. Dry organic phase extract at 37 °C under a stream of N2 gas (<2 psi) for about 30 min .

 

 

SECTION: Purify lipids extract
46. 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. 
 


SECTION: Purify lipids extract
47. Dry extract at 37 °C under a stream of N2 gas (<2 psi)  (Generally 2 mL/5 min).
 
 

SECTION: Purify lipids extract
48.  Store dried extract and excess extract (in chloroform) at  -80 °C.
SECTION: Extract lipids
25. Use the pasteur pipet to transfer organic layer to #T2. 
 

SECTION: Extract lipids
29. Centrifuge at 3200 rpm, 5 min 

 

SECTION: Extract lipids
30. Use the pasteur pipet to transfer organic layer to #T2. 

SECTION: Extract lipids
31. Add 1 mL Folch solvent to the residue.
SECTION: Extract lipids
32. Sonicate  2 min 
SECTION: Extract lipids
33. Vortex by using a tube insert for about 10 min 

SECTION: Extract lipids
34. Centrifuge at 3200 rpm, 5 min 

 

SECTION: Extract lipids
35. Use the pasteur pipet to transfer organic layer to #T2. 

SECTION: Extract lipids
36. Add 1 mL Folch solvent to the residue.
SECTION: Extract lipids
37. Sonicate  2 min 
SECTION: Extract lipids
38. Vortex by using a tube insert for about 10 min