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 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 N2 gas flow. The residue is 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 (5 inches 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, scintillation vials and storage vials 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. 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.
 

 

SECTION: Prepare glassware
14. Latex bulbs are required for Pasteur pipets
SECTION: Prepare reagent
15. Folch solvent (CHCl3: MeOH=2:1 v/v)
SECTION: Prepare reagent
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.
 
  
  

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

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

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

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

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

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

 

 

SECTION: Extraction
17.1.  Add 100 µL MilliQ directly onto the sample.
SECTION: Extraction
17.2. Freeze at -80 °C   10 min 
SECTION: Extraction
17.3. Remove vials from freezer.
SECTION: Extraction
17.4. Purge the dispensette, fill the tubing with solvent before dispensing solvent into sample tube.
SECTION: Extraction
17.5. Dispense 2.0 mL Folch solvent into sample tube.
SECTION: Extraction
18. If lipids samples have been hydrolyzed for carbohydrate and solvent has already been added, go to the vortex step directly. 

SECTION: Extraction
19. Vortex  28 min by using a tube insert.
 

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

 

SECTION: Extraction
23. Place one pasteur pipet (#P1) into each tube

 

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

 

 

SECTION: Extraction
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).
SECTION: Extraction
26. Add 50 µL MilliQ and 1 mL Folch solvent to the residue.
SECTION: Extraction
27. Vortex the eight samples at the highest speed to loosen the filter.
SECTION: Extraction
28. Vortex the eight samples by using a tube insert while transferring supernatant from another eight samples to centrifuge tubes.
SECTION: Extraction
29. Sonicate  2 min 
SECTION: Extraction
30. Vortex the second eight samples by using a tube insert while transferring supernatant from the first eight samples to centrifuge tubes.

 

SECTION: Extraction
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).

SECTION: Extraction
32. Add 50 µL MilliQ and 1 mL Folch solvent to the residue.
SECTION: Extraction
33. Vortex while transferring supernatant from another set of samples to centrifuge tubes.
SECTION: Extraction
34. Sonicate  2 min 
SECTION: Extraction
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).

SECTION: Extraction
36. Add 50 µL MilliQ and 1 mL Folch solvent to the residue.
SECTION: Extraction
37. Vortex while transferring supernatant from another set of samples to centrifuge tubes.
SECTION: Extraction
38. Sonicate  2 min 
SECTION: Extraction
39. Use the pasteur pipet (#P1) to gently lift the filter upwards and transfer all liquid to the centrifuge tube (#T1).

SECTION: Extraction
40. Leave the pasteur pipet (#P1) in the tube with filter for a while, the tip gradually sucks liquid dripping from filter. 
SECTION: Separation
42. Centrifuge at 3200 rpm, 5 min 

 

SECTION: Separation
44. Use pasteur pipet (#P1) to remove supernatant as much as possible. Do not disturb the debris.
SECTION: Separation
45. Return pipet (#P1) back to the tube with filter.
SECTION: Separation
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.


 


 


 

 

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

 

 

SECTION: 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 5 mL 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. 
 


SECTION: Purification
54.  Freeze dried extract and excess extract (in chloroform) at  -80 °C.
SECTION: Separation
43. Filter and cell debris stay between the two layer.
SECTION: Separation
46. Add 500 ul KCl solution, vortex and then centrifuge at 3200 rpm, 5 min 
 

SECTION: Separation
47. Use #P1 to remove supernatant as much as possible. Do not disturb the debris.
SECTION: Separation
48. Turn on heat block to 37 °C , use a thermometer to monitor the actual temperature.
 


 

SECTION: Separation
49. Place tubes in the heater. 

 


SECTION: Extraction
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.
SECTION: Purification
53. Dry extract at 37 °C under a stream of N2 gas (<2 psi) for about 30 min  (Generally 2 mL/5 min).