Extraction of lipids from microalgae:

optimization of an analytical method

Eline Ryckebosch

K.U.Leuven Campus Kortrijk

Research Unit Food & Lipids
Belgium
 Outline

• Introduction
• Materials & Methods
• Results & Discussion
• Conclusion
• Future work

1-6-2010 Extraction of lipids from microalgae 2

 Introduction

• Microalgae
− unicellular aquatic photosynthetic organisms
− exist individually, in chains or in groups
− high growth rate, high biomass production
and low land use
− often contain high amounts of lipids
− contain valuable, often unusual fatty acids

1-6-2010 Extraction of lipids from microalgae 3

 Introduction

• Aim
Optimal protocol for an analytical method for lipid
analysis from microalgae

• Key processes for lipid analysis of microalgae:

− Lipid extraction
− Separation of lipid classes
− FAME-determination

1-6-2010 Extraction of lipids from microalgae 4

 Materials & Methods (1)

• Microalgae
− Phaeodactylum tricornutum
− Scenedesmus obliquus
− Nannochloropsis salina

• Cultivation & Harvest

− WC medium (+ 30 g/L salt),
continuously aerated, 12h:12h light/dark
− Harvest: centrifugation

1-6-2010 Extraction of lipids from microalgae 5

 Materials & Methods (2)

• Pretreatment
− Lyophilization (lipolysis?)
− Isopropanol/boiling water
(deactivation of endogenic lipases)
− Antioxidants (prevention of oxidation)

• Cell disruption
− Sonication (sonication bath, 15 min)
− Nitrogen (liquid nitrogen, 3 x freezing/thawing)
− Bead-beating (5 mm beads; 30 Hz; 2 x 1.5 min)
1-6-2010 Extraction of lipids from microalgae 6
 Materials & Methods (3)

• Total lipid extractionbased on 1

− Chl/MeOH (1:1)
− Washing with water?
− Crude lipids are weighed

• Neutral lipid extraction

− Petroleumether (bp 30-50°C)
− Crude lipids are weighed

1 Wrolstad et al., 2005. Handbook of Food Analytical Chemistry, John Wiley & sons Inc.

1-6-2010 Extraction of lipids from microalgae 7

 Materials & Methods (4)

• Lipid classes: SPE (Silica)based on 1

− Neutral lipids: chloroform
− Glycolipids: acetone
− Phospholipids: methanol

• FAME: GC-FID
− Methylation: acid catalyzed1
− EC Wax column
− 100-180°C (10°C/min), 180-215°C (2°C/min),
215°C (44min) 1
Christie W.W. (2003) Lipid Analysis. Third Edition. The Oily Press
1-6-2010 Extraction of lipids from microalgae 8
 Results & Discussion (1)

• Influence of pretreatment in the method was tested:

− Lyophilization
− Isopropanol/boiling water (endogenic lipases)
− Antioxidants
No influence on total lipid content, lipid class
composition and fatty acid profile was observed
• Thus:
− Lyophilization does not cause lypolysis
− Endogenic lipases do not cause lipolysis
− No significant oxidation takes place during extraction
1-6-2010 Extraction of lipids from microalgae 9
 Results & Discussion (2)
Chl/MeOH total

• Total lipid extraction 60

b

− Chloroform/methanol (1:1) 50

− With or without water? 40

% lipids
30

Co-extraction of polar
20

10

components when wash- 0

Chl/MeOH + water Chl/MeOH

step with water is omitted Treatment

Overestimation of amount of lipids

Washing step is necessary

1-6-2010 Extraction of lipids from microalgae 10

 Results & Discussion (3)

• Cell disruption methods – total lipids

50
a a ab b ab

c c c c d
40

Total
30
Ex 1
% Lipids

Ex 2
Ex 3
Ex 4
20

10

0
fresh lyo lyo-son lyo-N2 lyo-BB

Cell disruption method

1-6-2010 Extraction of lipids from microalgae 11
 Results & Discussion (4)

• Same result on other algae?

Nannochloropsis salina Scenedesmus obliquus
40 35
a a Total a b Total
c Ex 1 Ex 1
Ex 2 30 d Ex 2
b
30 c
25

% Lipids
% Lipids

20
20
15

10
10

0 0
Lyo Lyo + N2 + BB + son Lyo Lyo + N2 + BB + son

Cell disruption method Cell disruption method

1-6-2010 Extraction of lipids from microalgae 12
 Results & Discussion (4)

• Lipid classes extracted

100

a a
80
% Lipids (of total lipids)

NL
60 GL
PL

40

c c

20 b b

0
EX 1 EX 2

Extraction
1-6-2010 Extraction of lipids from microalgae 13
 Results & Discussion (5)

• Fatty acid composition: total extract

TV EX 1 TV EX 2
C14:0 6,63 5,92
C16:0 28,84 29,88
C16:1n-7 41,95 44,49
C17:0 1,78 1,09
C18:1n-9 8,15 8,12
C20:5n-3 8,51 7,61

1-6-2010 Extraction of lipids from microalgae 14

 Results & Discussion (6)

• Fatty acid composition: neutral lipid class

NL TV EX1 TV EX2
C14:0 5.8 ± 0.4 5.90 ± 0.02
C16:0 32.0 ± 0.7 32.3 ± 0.2
C16:1n-7 47.6 ± 0.4 46.9 ± 0.1
C18:1n-9 8.1 ± 0.3 8.4 ± 0.1
C20:5n-3 6±1 6.474 ± 0.002

1-6-2010 Extraction of lipids from microalgae 15

 Results & Discussion (7)
GL TV EX1 TV EX2
C14:0 1.1 ± 0.4 2.2 ± 0.2
Unknown 1 / 16 ± 2*
C16:0 7.7 ± 0.4 1.6 ± 0.6*
C16:1n-7 19 ± 1 15 ± 2
Unknown 2 7±4 3.3 ± 0.3
C17:0 / 19 ± 2*
C17:1n-7 22 ± 3 1.6 ± 0.3*
Unknown 3 2.6 ± 0.5 2.14 ± 0.06
C18:0 / 6.5 ± 0.5*
C18:1n-9 1.5 ± 0.2 2.5 ± 0.6
C18:3n-3 1.2 ± 0.2 1.22 ± 0.07
C20:5n-3 37 ± 1 28.2 ± 0.2*
1-6-2010 Extraction of lipids from microalgae 16
 Results & Discussion (8)
PL TV EX1 TV EX2
C14:0 15 ± 4 9.1 ± 0.6
C16:0 25 ± 7 20.6 ± 0.5
Unknown 1 12 ± 2* /
C16:1n-7 11.7 ± 0.5 26 ± 8
C17:0 / 2 ± 2*
C17:1n-7 0.8 ± 0.4* /
C18:0 / 2 ± 1*
C18:1n-9 11.1 ± 0.2 8.3 ± 0.3*
Unknown 2 1.0 ± 0.4 3± 3
C18:2n-6 1.6 ± 0.2* /
C20:4n-6 0.9 ± 0.5* /
C20:5n-3 13 ± 8 18 ± 9
C24:0 7.0 ± 0.6 8.1 ± 0.8
C22:6n-3 1± 1 3± 2
1-6-2010 Extraction of lipids from microalgae 17
 Results & Discussion (9)

• Cell disruption methods – neutral lipids

35

d
30
i

25

Total
a Ex 1
% Lipids

20 a
Ex 2
h Ex 3
15 c Ex 4
Ex 5
c
10 g g

e b
5
f
0
1 2 3 4 5 6

Cell disruption method

1-6-2010 Extraction of lipids from microalgae 18

 Results & Discussion (10)
• Lipid classes extracted
100 a a

80
% Lipids (of neutral lipids)

60 NL
GL
PL

40

20
b c b c

0
lyo lyo-BB

Cell disruption method

1-6-2010 Extraction of lipids from microalgae 19
 Conclusions

• Optimal protocol for lipid analysis

− Lyophilized algae
− No extra pretreatment necessary
− Total lipid extraction (Chl/MeOH 1:1 + water)
− No cell disruption necessary
− One extraction is sufficient for most applications
− Fatty acid composition GL or PL  second extraction
− Neutral lipids through separation of total lipids on SPE

1-6-2010 Extraction of lipids from microalgae 20

 Future work

• Rapid method for lipid determination of microalgae

(see Poster)
• Screening algae for nutritionally interesting fatty
acids
• Cultivate microalgae to maximize the amount of the
interesting fatty acid in the lipid fraction
• Properties of this nutritionally valuable algal oil

1-6-2010 Extraction of lipids from microalgae 21

 Lab Aquatic Biology
Algal Applications
Lipid analysis Harvesting Large scale extraction

Coordinates
Eline Ryckebosch
K.U.Leuven Campus Kortrijk
Food & Lipids
+32 56 24 64 01
Eline.Ryckebosch@kuleuven-kortrijk.be
1-6-2010 Extraction of lipids from microalgae 22
 GL TV1 TV2
C14:0 1.1 ± 0.4 2.2 ± 0.2
Unknown 1 / 16 ± 2*
C16:0 7.7 ± 0.4 1.6 ± 0.6*
C16:1n-7 19 ± 1 15 ± 2
Unknown 2 7±4 3.3 ± 0.3
C17:0 / 19 ± 2*
C17:1n-7 22 ± 3 1.6 ± 0.3*
Unknown 3 2.6 ± 0.5 2.14 ± 0.06
C18:0 / 6.5 ± 0.5*
C18:1n-9 1.5 ± 0.2 2.5 ± 0.6
C18:3n-3 1.2 ± 0.2 1.22 ± 0.07
C20:5n-3 37 ± 1 28.2 ± 0.2*
1-6-2010 Extraction of lipids from microalgae 23
 PL TV1 TV2
C14:0 15 ± 4 9.1 ± 0.6
C16:0 25 ± 7 20.6 ± 0.5
Unknown 1 12 ± 2* /
C16:1n-7 11.7 ± 0.5 26 ± 8
C17:0 / 2 ± 2*
C17:1n-7 0.8 ± 0.4* /
C18:0 / 2 ± 1*
C18:1n-9 11.1 ± 0.2 8.3 ± 0.3*
Unknown 2 1.0 ± 0.4 3±3
C18:2n-6 1.6 ± 0.2* /
C20:4n-6 0.9 ± 0.5* /
C20:5n-3 13 ± 8 18 ± 9
C24:0 7.0 ± 0.6 8.1 ± 0.8
C22:6n-3 1±1 3±2
1-6-2010 Extraction of lipids from microalgae 24
 NL TV1 TV2
C14:0 5.8 ± 0.4 5.90 ± 0.02
C16:0 32.0 ± 0.7 32.3 ± 0.2
C16:1n-7 47.6 ± 0.4 46.9 ± 0.1
C18:1n-9 8.1 ± 0.3 8.4 ± 0.1
C20:5n-3 6±1 6.474 ± 0.002

1-6-2010 Extraction of lipids from microalgae 25

 Results & Discussion (9)

• Cell disruption causes no clear differences in total

extracted lipids (4 extractions)
• Beat beating gives a higher amount of total lipids
during 1st extraction
• 1st extraction extracts > 90% of total lipids
• First and second extraction gives the same lipid class
composition
• Fatty acid composition first and second total lipid
extraction is similar
• Small differences in fatty acid composition of GL & PL
1-6-2010 Extraction of lipids from microalgae 26
 Results & Discussion (12)

• Pretreatment causes large differences in neutral

extracted lipids
• Using bead beating the highest amount of neutral
lipids are extracted
• The first extraction with bead beater extract almost
all neutral lipids
• Cell disruption with bead beater doesn’t extract
more of the polar lipid classes

1-6-2010 Extraction of lipids from microalgae 27