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.
Hydrolyzation 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
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 one 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. Dispose the pasteur pipet (#P1), place clean pasteur pipet (#P2) into the centrifuge tube (#T1)
SECTION: Extraction
41. Prepare a set of clean centrifuge tubes (#T3) for the next section.
SECTION: Remove filter debris
42. Centrifuge at 3200 rpm, 5 min 

 

SECTION: Remove filter debris
43. Use clean pasteur pipet (#P2) to transfer as much as possible supernatant to #T3. Do not disturb the debris.
SECTION: Remove filter debris
44. Return pipet (#P2) back to #T1 tube.
SECTION: Remove filter debris
45. Add  1 mL Folch solvent and 50 µL MilliQ into the centrifuge tube with debris (#T2).
SECTION: Remove filter debris
46. Vortex the mixture.
SECTION: Remove filter debris
47. Centrifuge at 3200 rpm, 5 min 

SECTION: Remove filter debris
48. Use pasteur pipet (#P2) to transfer as much as possible supernatant to #T3. Do not disturb the debris.
SECTION: Remove filter debris
49. Add  1 mL Folch solvent and 50 µL MilliQ into the centrifuge tube.
SECTION: Remove filter debris
50. Vortex the mixture.
SECTION: Remove filter debris
51. Centrifuge at 3200 rpm, 5 min 
SECTION: Remove filter debris
52. Use pasteur pipet (#P2) to transfer as much as possible supernatant to #T3. Do not disturb the debris.
SECTION: Remove filter debris
53. Add  1 mL Folch solvent and 50 µL MilliQ into the centrifuge tube.
SECTION: Remove filter debris
54. Vortex the mixture.
SECTION: Remove filter debris
55. Centrifuge at 3200 rpm, 5 min 
SECTION: Remove filter debris
56. Use pasteur pipet (#P2) to transfer as much as possible supernatant to #T1, do not disturb the debris. Keep the pipet (#P2). 
SECTION: Remove filter debris
57. Shake the tube (#T1), and then use pasteur pipet (#P2) to transfer all fluid to #T3. 
SECTION: Separation
58. Generally, it yields a final volume of 7.5 mL for samples collected on 25 mm GFF filter; final volume is 7 mL for sample collected on 47 mm GFF filter.
SECTION: Separation
59. In order to obtain a biphasic system so that the extract can be separated from the water, the final composition of CHCl3:MeOH:H2O is 8:4:3 (v/v), i.e. 2.67:1.33:1.

 

SECTION: Separation
60. Depending on the size of filter, add corresponding volume of 0.88% KCl to #T3.
25 mm GFF: 1.5 mL KCl
47 mm GFF: 1.4 mL KCl
SECTION: Separation
61. Vortex  1 min by using a tube insert.
SECTION: Separation
62. Vortex each tube for about 10 s  individually.


SECTION: Separation
63. Centrifuge at 3200 rpm, 5 min or until biphasic layers separate completely.

SECTION: Separation
64. Remove most of upper aqueous phase with the pasteur pipet (#P2).
SECTION: Separation
65. Use #P2 to transfer the lower organic phase to a 12 mL storage vial.
Do not disturb the cell debris in between the two phases.
 

 



 

 

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

 

 

SECTION: Separation
67. 
 

SECTION: Separation
68. The lipids extract might still have water residue (which can't be dried by nitrogen gas). 

(1) If the extract is partially used for lipids measurement:

Redissolve it with chloroform by using glass serological pipet, transfer certain amount of chloroform dissolved solution for lipids measurement based on the estimation. 

 

 

(2) If the extract is totally used for lipids measurement:

Freeze dry to remove water.
SECTION: Separation
69.  Freeze at  -80 °C.
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.