Omega-3 Polyunsaturated fatty acid production by microalgae · LOGO Omega-3 Polyunsaturated fatty...

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Omega-3 Polyunsaturated fatty acidproduction by microalgae

Aurantiochytrium mangrovei Sk-02 as the effect of carbon sources

and growth environments.

Miss Nutnicha Chodchoey

Ph.D Candidate

[email protected]

www.themegallery.com

Representative examples of naturallyoccurring PUFAs

Omega-6

� Linoleate (LA),

� γ-Linolenate (GLA),

� Dihomo-ω-linolenate (DHGLA)

� Arachidonate (AA)

Omega-3

� α- Linolenate (ALA),

� Eicosapentaenoate (EPA),

� Docosapentaenoate (DPA)

� Docosahexanenoate (DHA)

(Gill and Valivety, 1997)


Health benefits of DHA

� Prevent human cardiovascular diseases, cancer and Alzheimer’s disease.

� Plays an important role in infant brain and retinal development.

� Increases HDL level (good cholesterol).

� Lower triacylglycerol level.


Aurantiochytrium sp., a “natural high DHA” organism

� Heterotrophic marine eukaryote.

� Widely found in marine environments, especially mangrove forests.

� Cultivated commercially as an aquafeed.

� Exact pathway of PUFA-synthesis not established, due to extremely low levels of intermediates.


Objectives

� To study the effect of carbon sources on fatty

acid composition in Aurantiochytrium mangrovei

Sk-02

� To study the effect of salinity and temperature on

lipid class composition, especially phospholipid

class.


Materials and Methods

� Medium composition and cell cultivation.

151751040 g/l glucose




301151020 g/l safflower oil

301151020 g/l olive oil

Cultivation Temp.

(°°°°C)C)C)C)

MnCl2(mg/l)

Artificial sea salts

(g/l)

Yeast extract

(g/l)

Carbon source


Materials and Methods

� The samples were taken until carbon source was almost finished in the culture medium to analyze :

1. Residual glucose : Enzymatic kit.

2. Cell density : Measured by spectrophotometer

at 660 nm

3. Biomass (g/l) : The weight of freeze

dried cells.


Materials and methods

4. Fatty acid analysis

Lipids in the cells were methyl esterified by 4% H2SO4 in methanol. Fatty acids in the form of methyl ester were

analysed by GC.

The unsaturation index (UI)

= [(1.0 * %monoene) + (2.0 * % diene) + (3.0 * % triene) + (4.0 * % tetraene) + (5.0 * pentaene) + (6.0 * % hexaene) ]/100


5. Lipid class separation

• Aminopropyl was used (Adapted from Kaluzny et al., 1985).

• 20 mg of lipids from the cells was extracted by the Bligh and Dryer method(1959).

• The samples were evaporated under nitrogen gas.

•Fatty acid composition from each fraction was analyzed by GC.

16MethanolPhospho-

lipid (PL)

PC,PE

16Choroform:

Methanol:

3N HCl

(200 : 100 : 1)(modified method of Banni et

al. ,2001)

Acidic

phospho-

lipid

(aPL)

PI,PS

82% v/v Acetic acid in

diethylether

Free fatty

acid (FA)

16Chloroform-2-propanol

2:1 (v/v)

Neutral lipid

(NL)

Volume

(ml)

SolventLipid fraction


Results

� The effect of carbon sources on biomass and fatty acid production.

0

2

4

6

8

10

12

14

16

18

20

20 g/l Olive oil 20 g/l Safflower oil 40 g/l Glucose

Bio

mass a

nd

to

tal

fatt

y a

cid

(g

/l)

Biomass (g/l)

Total fatty acid (g/l)36.4%w/w 39.6%w/w 48.9%w/w


Results

� Main fatty acids found in the cells grown on various carbon sources.

0%

20%

40%

60%

80%

100%

20 g/l Olive oil

20 g/l Safflower oil

40 g/l Glucose

% w

/w M

ain

fatt

y a

cid

s

C22:6n3

C22:5n6

C20:5n3

C18:2n6

C18:1n9

C16:0


Results

• Fatty acid composition (% total fatty acid) of olive oil and safflower oil.

0.4C20:1n9

0.2C20:0

0.4C18:3n3

13.2C18:2n6c

68.7C18:1n9c

1.9C18:0

1.5C16:1

13.7C16:0

%(w/w)

TFA

Fatty acid

Olive oil

0.5C22:5n6

2.2C22:6n3

0.3C20:1n9

0.2C20:0

0.3C18:3n3

71.5C18:2n6c

12.2C18:1n9c

1.6C18:0

11.1C16:0

%(w/w)

TFA

Fatty acid

Safflower oil


Results

� The effect of salinity and cultivation temperatures on growth and fatty acid production.

0

5

10

15

20

25

30

0 50 100 150 200 250

Time (h)

OD

660

15 C, 15 g/l Sea salts




0

2

4

6

8

10

12

14

16

18

15 C, 15 g/l SS 15 C, 75 g/l SS 30 C, 15 g/l SS 30 C, 75 g/l SS

Bio

ma

ss

an

d t

ota

l fa

tty

ac

id (

g/l

)

Biomass (g/l)

Total fatty acid (g/l)


Results

� Fatty acid composition of A. mangrovei Sk-02 as the effect of salinities (15 g/l or 75 g/l sea salts) and cultivation temperatures (15 °°°°C or 30 C or 30 C or 30 C or 30 °°°°C)C)C)C).

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

15 C,15 g/l

SS

15 C, 75 g/l

SS

30 C,15 g/l

SS

30 C, 75 g/l

SS

Fatty a

cid

com

positi

on (

%w

/w)

C22:6n3

C22:5n6

C20:5n3

C20:3n6

C18:0

C16:0

C15:0

C14:0


Results

� Lipid class composition of A. mangrovei Sk-02 grown in various conditions.

0

10

20

30

40

50

60

70

80

90

100

15 C, 15 g/l SS15 C, 75 g/l SS30 C, 15 g/l SS30 C, 75 g/l SS

Lip

id c

las

s c

om

po

sit

ion

(%

w/w

)

Phospholipid class

Free fatty acid class

Neutral lipid class

0

10

20

30

40

50

60

70

80

90

100

20 g/l Olive oil

20 g/l Safflower oil

Lip

id c

las

s c

om

po

siti

on

(%

w/w

)


Results

� Fatty acid composition in neutral lipid class and phospholipid class of

A. mangrovei Sk-02 grown on oil substrates.

0%

20%

40%

60%

80%

100%

20 g/l Olive oil 20 g/l Safflower oil

Fa

tty

ac

id (

%w

/w)

in n

eu

tra

l li

pid

cla

ss

0%

20%

40%

60%

80%

100%

20 g/l Olive oil 20 g/l Safflower oil

Fa

tty

ac

id (

%w

/w)

in p

ho

sp

ho

lip

id c

las

s

C22:6n3

C22:5n6

C20:5n3

C18:2n6c

C18:1n9c

C18:0

C16:0

Neutral lipid class Phospholipid class

1.91.2UI 3.72.5UI


Results

� Fatty acid composition in neutral lipid class and phospholipid class of A.

mangrovei Sk-02 grown on various condition (40 g/l glucose as a c-source).

0%

20%

40%

60%

80%

100%


Fa

tty

ac

id (

%w

/w)

in n

eu

tra

l lip

id c

las

s

0%

20%

40%

60%

80%

100%


Fa

tty

ac

id (

%w

/w)

in p

ho

sp

ho

lipid

cla

ss

C22:6n3

C22:5n6

C20:5n3

C20:3n6

C18:0

C16:0

C15:0

C14:0

Neutral lipid class Phospholipid class

1.02.02.62.7UI 4.03.54.14.5UI


Conclusions

� As compare to oil substrates, glucose can support more PUFA production in the cells.

� From the results of lipid class composition, most lipids are stored in the cells mainly in the form of neutral lipid (>>>>60%w/w TAG) .

� The lower the temperature, the more PUFA-especially DHA-are produced in the cells.

� Most DHA produced in the cells are located mostly in phospholipid membrane.


What’s next ?

� Less omega-3 production will be from fish.

� Dried cells can be directly mixed to the feed.

� The membrane of this microalgae consists of

galactose polymer with sulfate group (immunostimulant).

� DHA can support the growth and survival rate of shrimps.

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