Extraction of lipids from microalgae: optimization of an analytical method
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1 Extraction of lipids from microalgae: optimization of an analytical method Eline Ryckebosch K.U.Leuven Campus Kortrijk Research Unit Food & Lipids Belgium
2 Outline Introduction Materials & Methods Results & Discussion Conclusion Future work Extraction of lipids from microalgae 2
3 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 Extraction of lipids from microalgae 3
4 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 Extraction of lipids from microalgae 4
5 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 Extraction of lipids from microalgae 5
6 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) Extraction of lipids from microalgae 6
7 Materials & Methods (3) Total lipid extractionbased on 1 Chl/MeOH (1:1) Washing with water? Crude lipids are weighed Neutral lipid extraction Petroleumether (bp C) Crude lipids are weighed 1 Wrolstad et al., Handbook of Food Analytical Chemistry, John Wiley & sons Inc Extraction of lipids from microalgae 7
8 Materials & Methods (4) Lipid classes: SPE (Silica) based on 1 Neutral lipids: chloroform Glycolipids: acetone Phospholipids: methanol FAME: GC-FID Methylation: acid catalyzed 1 EC Wax column C (10 C/min), C (2 C/min), 215 C (44min) 1 Christie W.W. (2003) Lipid Analysis. Third Edition. The Oily Press Extraction of lipids from microalgae 8
9 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 Extraction of lipids from microalgae 9
10 Results & Discussion (2) Total lipid extraction Chloroform/methanol (1:1) With or without water? a Chl/MeOH total b Co-extraction of polar components when washstep with water is omitted Overestimation of amount of lipids Washing step is necessary % lipids Chl/MeOH + water Treatment Chl/MeOH Extraction of lipids from microalgae 10
11 Results & Discussion (3) Cell disruption methods total lipids 50 a a ab b ab 40 c c c c d % Lipids Total Ex 1 Ex 2 Ex 3 Ex fresh lyo lyo-son lyo-n2 lyo-bb Cell disruption method Extraction of lipids from microalgae 11
12 Results & Discussion (4) Same result on other algae? Nannochloropsis salina Scenedesmus obliquus a b a c Total Ex 1 Ex a c b d Total Ex 1 Ex 2 % Lipids 20 % Lipids Lyo Lyo + N2 + BB + son 0 Lyo Lyo + N2 + BB + son Cell disruption method Cell disruption method Extraction of lipids from microalgae 12
13 Lipid classes extracted 100 Results & Discussion (4) 80 a a % Lipids (of total lipids) c c NL GL PL 20 b b 0 EX 1 EX 2 Extraction Extraction of lipids from microalgae 13
14 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, Extraction of lipids from microalgae 14
15 Results & Discussion (6) Fatty acid composition: neutral lipid class NL TV EX1 TV EX2 C14:0 5.8 ± ± 0.02 C16: ± ± 0.2 C16:1n ± ± 0.1 C18:1n ± ± 0.1 C20:5n-3 6 ± ± Extraction of lipids from microalgae 15
16 Results & Discussion (7) GL TV EX1 TV EX2 C14:0 1.1 ± ± 0.2 Unknown 1 / 16 ± 2* C16:0 7.7 ± ± 0.6* C16:1n-7 19 ± 1 15 ± 2 Unknown 2 7 ± ± 0.3 C17:0 / 19 ± 2* C17:1n-7 22 ± ± 0.3* Unknown ± ± 0.06 C18:0 / 6.5 ± 0.5* C18:1n ± ± 0.6 C18:3n ± ± 0.07 C20:5n-3 37 ± ± 0.2* Extraction of lipids from microalgae 16
17 Results & Discussion (8) PL TV EX1 TV EX2 C14:0 15 ± ± 0.6 C16:0 25 ± ± 0.5 Unknown 1 12 ± 2* / C16:1n ± ± 8 C17:0 / 2 ± 2* C17:1n ± 0.4* / C18:0 / 2 ± 1* C18:1n ± ± 0.3* Unknown ± ± 3 C18:2n ± 0.2* / C20:4n ± 0.5* / C20:5n-3 13 ± 8 18 ± 9 C24:0 7.0 ± ± 0.8 C22:6n-3 1 ± 1 3 ± Extraction of lipids from microalgae 17
18 Results & Discussion (9) Cell disruption methods neutral lipids d i 25 % Lipids a c g a h c g Total Ex 1 Ex 2 Ex 3 Ex 4 Ex 5 5 e b f Cell disruption method Extraction of lipids from microalgae 18
19 Lipid classes extracted Results & Discussion (10) 100 a a 80 % Lipids (of neutral lipids) NL GL PL 20 b c b c 0 lyo lyo-bb Cell disruption method Extraction of lipids from microalgae 19
20 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 Extraction of lipids from microalgae 20
21 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 Extraction of lipids from microalgae 21
22 Lab Aquatic Biology Algal Applications Lipid analysis Harvesting Large scale extraction Coordinates Eline Ryckebosch K.U.Leuven Campus Kortrijk Food & Lipids Extraction of lipids from microalgae 22
23 GL TV1 TV2 C14:0 1.1 ± ± 0.2 Unknown 1 / 16 ± 2* C16:0 7.7 ± ± 0.6* C16:1n-7 19 ± 1 15 ± 2 Unknown 2 7 ± ± 0.3 C17:0 / 19 ± 2* C17:1n-7 22 ± ± 0.3* Unknown ± ± 0.06 C18:0 / 6.5 ± 0.5* C18:1n ± ± 0.6 C18:3n ± ± 0.07 C20:5n-3 37 ± ± 0.2* Extraction of lipids from microalgae 23
24 PL TV1 TV2 C14:0 15 ± ± 0.6 C16:0 25 ± ± 0.5 Unknown 1 12 ± 2* / C16:1n ± ± 8 C17:0 / 2 ± 2* C17:1n ± 0.4* / C18:0 / 2 ± 1* C18:1n ± ± 0.3* Unknown ± ± 3 C18:2n ± 0.2* / C20:4n ± 0.5* / C20:5n-3 13 ± 8 18 ± 9 C24:0 7.0 ± ± 0.8 C22:6n-3 1 ± 1 3 ± Extraction of lipids from microalgae 24
25 NL TV1 TV2 C14:0 5.8 ± ± 0.02 C16: ± ± 0.2 C16:1n ± ± 0.1 C18:1n ± ± 0.1 C20:5n-3 6 ± ± Extraction of lipids from microalgae 25
26 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 Extraction of lipids from microalgae 26
27 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 Extraction of lipids from microalgae 27
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