Commercial Production of DHA using

Heterotrophic Microalgae

Yi-Min Chen (), Huey-Lang Yang ()

Institute of Biotechnology, National Cheng Kung University
Chung-Kuang Lu ()
National Museum of Marine Biology & Aquarium
Su-Fen Chen ()
Department of Health and Nutrition,
Chia Nan University of Pharmacy and Science

1. Classification of unsaturated fatty acids

(1) Polyunsaturated fatty acid (PUFA)
Fatty acids with 2 or more double bonds

(2) Highly unsaturated fatty acid (HUFA)

Fatty acids with longer chain (20 C) and more (3) double bonds
E.g. EPA, DHA

(3) n-3n-6 series

C
C

C
C
3

C ..

C
C

OH

(4) Essential fatty acids

Biosynthetic pathway related to n-3/n-6 PUFA

EPA

ARA

DPA (n-3)

DHA

DPA (n-6)

2. Functions of HUFAs
(1) Precursors of eicosanoids
- ARA EPA
- Inflammation
- Diseases (Self-immune, cancer, )

(2) Energy storage

- Triacylglycerol

(3) Membrane structure

- Cell membrane, organelles

(4) Nerve conduction

- DHA in brain & retina

The imbalance of n-6/n-3 fatty acid uptake results in hyper-inflammation

1:1~4:1

4:1 ~ 10:1

10:1 ~ 30:1

Cardiovascular disease, self-immune disease, cancer, dementia, depression

The uptake of n-3 HUFA (EPA, DHA) is good for:

(1) mitigating inflammation reaction
(2) increasing acetylcholine level in brain of patient with Alzheimers disease
(3) maintaining membrane fluidity that influencing neurotransmission

n-3DHA

3. DHA is an essential and important fatty acid

(1) For human
- The predominant structural fatty acid in cerebral cortex of brain and
photoreceptors of retina.
Thats why sea food is highly brain-friendly!

- Reduction of chronic diseases risk

Cardiovascular diseases, arthritis, CNS diseases, fatty liver disease

- Dietary recommendation for n-3 HUFA (EPA+DHA):

0.5-1.0g/d/p

(2) For marine fish

- Also important for development of brain and retina
- Recommended content of n-3 HUFA in feed:
0.5%-2%

- Cause deformation in many fish larvae when insufficient

Deformation of grouper larvae related to insufficient DHA intake

4. DHA is a n-3 HUFA derived from marine

(1) Deep sea fish oil
- Main source; cheaper
- 5-15% DHA; variable with different sources
- Complex fatty acid composition; hard to purify
- Possible contaminant: heavy metals, PCBs or dioxin

(2) Seal oil

- Minor source, expensive
- 10-15% DHA
- May be contaminated

(3) Microalgae
- Minor source, expensive
- 10-50% DHAeasy to purify
- No contaminants; suitable for pregnant
women and infants
- Edible for vegetarians
- Environmental friendly

DHA products from animal sources

DHA products form plant sources (1)

- From Gold Circle Farms

- 150 mg/egg

- 90 mg/100 g milk powder

DHA products form plant sources (2)

- From microalga

- From plant oil?

Exaggerated and false advertisement

5. Marine microalgae can produce high-quality DHA

DHA from fish oil

DHA from microalgae

Cheaper

More expensive

Not environmental friendly

Environmental friendly

Lower DHA content

Higher DHA content

Difficult to purify

Easy to purify

With Contaminants (heavy metals, With no contaminants

PCBs, dioxin etc.)
Suitable for pregnant women,
infants, and vegetarians

6. Heterotrophic microalga is a nice candidate for DHA production

(1) Autotrophic microalgae
rich in DHA
Haptophyta: Isochrysis
Chromophyta: Pavlova

(2) Heterotrophic microalgae

rich in DHA
Dinophyta: Crypthecodinium
Labyrinthulomycota: Schizochytrium,
Thraustochytrium
Auratiochytrium

Can be produced with lower cost!

DHA products made by heterotrophic microalgae

Company

species

product

Martek (USA)

Crypthecodinium cohnii

DHA in Infant milk, feed

OmegaTech (USA)

Schizochytrium sp.

Health food, feed

Bio-Marine (USA)

Schizochytrium sp.

Feed

Advanced BioNutrition (USA)

Schizochytrium sp.

Feed

Nutrinova (Germany)

Ulkenia

Health food

Martek Biosciences: the most successful company producing algal DHA

Martin Marietta

Martek1985 DHA

Martek 525 90

DHA 99% DHA75

43 million

DHA--

7. Strategies for developing algal-DHA industry in Taiwan

1. Find new algal species rich in DHA which have not been
patented.
2. Focus on heterotrophic microalgae
3. Explore the possible candidates from marine habitats around
Taiwan
4. Patent the candidate and transfer the related tech to local company

8. Procedures
(1) Isolation and identification of heterotrophic algal strains

from a range of marine habitats in Taiwan

(2) Biomass, total fatty acid content, and DHA yield

comparison of these strains

(3) Optimization of DHA production
(4) Toxicity assay
(5) Patent and tech transfer

9. Collection dates and sites of algal-like microorganism strains established

7 6 BL13

10. Identification of heterotrophic algal strains based on 18SrDAN sequences

BL3

BL5

49

7 1 Thraustochytrium sp. ATCC 26185

Thraustochytrium (sp 1)

BL8

99
92

BL4
BL7

99
97

BL2
BL6

99

Thraustochytrium (sp 2)

BL14

99
85

BL9
TN3

2 7 BL11
84

Aurantiochytrium

Aurantiochytrium mangrovei RCC893

99

BL10

99
95

BL1

93

Aurantiochytrium limacinum NIBH SR21

HK8a

90

KL2a

99
14
44

Thraustochytrium (sp 3)

HK8
HK1

88

KL2

8 8 Thraustochytriidae sp. #32

Sicyoidochytrium minutum SEK 354

97
9 9 HK10
80

Thraustochytrium (sp 4)

HK5
Thraustochytrium striatum ATCC 24473

18

Ulkenia profunda KMPB N3077a

99
87

Ulkenia sp. SEK 214

Parietichytrium sarkarianum SEK 351

72

95

72

Botryochytrium radiatum Raghukumar 16...

Thraustochytrium aureum ATCC 34304
99

99

KL1
Thraustochytrium kinnei KMPB 1694d

97
93

Thraustochytrium (sp 5)

Schizochytrium sp. SEK 210

99

Schizochytrium aggregatum ATCC 28209

Thraustochytrium aggregatum KMPB N-BA...

Thraustochytrium pachydermum KMPB N-B...
Labyrinthula sp. N8

99

Labyrinthula sp. L72

Aplanochytrium stocchinoi

89
99

93

Aplanochytrium

Aplanochytrium minutum
S1a

Oblongichytrium multirudimentale KMPB...

99

Oblongichytrium minutum KMPB N-BA-77

98

HK9

19
91
96

TN6
Oblongichytrium sp. SEK 347
Crypthecodinium cohnii ATCC 30336

0 .0 2

Oblongichytrium

11. Identification of heterotrophic algal strains based on morphologies

These characteristics support the identifications based on 18SrDNA.

Morphological characteristics of BL10

Zoospore

Ameboid cell

Vegetative cell

50 m

12. Identification of algal strains based on their fatty acid profiles

1. Total HUFAs content lower than 30% of total fatty acids 2

1. Total HUFAs content higher than 40% of total fatty acids 4
2. (n-3 DPA content) > (n-6 DPA content)...... Oblongichytrium sp. (Type I)
2. (n-3 DPA content) < (n-6 DPA content)...... 3
3. C20:3n-6 is not present.....

Thraustochytrium sp. 5 (Type II)

3. C20:3n-6 is present...

Schizochytrium aggregatum (Type III)

4. (n-3 DPA content) > (n-6 DPA content).... 5

4. (n-3 DPA content) < (n-6 DPA content) ... 6
5. C20:3n-6 is not present.......

Thraustochytrium sp. 2 (Type IV)

5. C20:3n-6 is present.....

Thraustochytrium sp. 4 (Type V)

6. C20:3n-6 and C20:4n-6 are not present....

Aplanochytrium sp. (Type VI)

6. C20:3n-6 and C20:4n-6 are present......

Thraustochytriun sp. 1 (Type VII)

Thraustochytrium sp. 3 (Type VII)
Aurantiochytrium sp. 1 (Type VII)
Aurantiochytrium sp. 2 (Type VII)

Fatty acid profiles can be a nice chemotaxonomical characteristic.

13. Comparison of biomass, total fatty acid content, EPA, and DHA yield
ATCC26185

TFA
Biomass

BL5

EPA
DHA

BL4
BL8
BL2
BL7
BL3
BL13
BL6
BL9
BL14
TN3
BL11
BL1
BL10
HK1
KL2a
HK8
HK8a
KL2
KL1
S1a
HK10
HK5
TN6
HK9
ATCC30336

50

100

150

200 0
-1

Biomass and total fatty acid (TFA) production (mgL )

10

20

30
-1

EPA & DHA yield (mgL )

14. Optimal conditions for DHA production by using BL10

* 4.5L 6L in 10 L fermenter
* Basal medium: Peptone/YE/Ammonium sulfate = 4/8/1gL-1
* Feeding: Glc, 120 gL-1 , twice a day
* Aeration: 1 VVM
60

-1

Biomass (gL )

50
40
30
20
46%

10

C16:0

0
0

37%

Time (day)

DHA

6%

7%

C14:0

DPA

10

15

20
Time (min)

25

30

BL10 is the best algal strain for DHA production

Biomass (gL-1)

DHA cont. (gL-1)

BL10

60

17.0

Schizochytrium sp.

G13/2S

63

6.1

S. sp.

N-2

13

2.7

S. sp.

S31

0.3

S. limacinum

SR21

35

4.2

Thraustochytrium sp.

ONC-T18

26

4.5

Cryptothecodinium cohnii

ATCC30772

109

19.0

Species

Strain

Aurantiochytrium sp.

Food
BL10

DHA content (%)

>15

Marine fish

0.03-2.90

Fresh water fish

0.04-0.75

Crab & shrimp

0.03-0.35

Egg

<0.07

Meat

<0.03

Rice

<0.01

Vegetable

<0.01

Milk

<0.01

14. Rat acute toxicity assay for oral consumption of BL10

Initial W (g)

Final W (g)

W (g)

Relative W (%)

F control
F exp

181.816.8
185.816.8

233.316.7
246.713.8

51.53.7
60.811.0

29%4%
33%8%

M control
M exp

247.810.6
252.311.4

355.59.2
369.522.3

107.710.1
117.214.9

44%6%
46%5%

- No fatality (LD50 >> 8.0g/kg)

- No weight loss
- No diarrhea

15. Achievement about BL10: 08 EAS Best Poster Award

15. Achievement about BL10: paper published in scientific journal

15. Achievement about BL10: patent, tech transfer and winning prize in competition

Patent
Yang H.-L., Chen Y.-M., Lu C.-K., 2009. Application of a
Algal Strain Rich in DHA (DHA).
Submitted, accession number 98107877.

Tech transfer
Yang H.-L., Chen Y.-M., Lu C.-K., 2009. Technologies related
to the production of BL10 algal strain (BL10)
Authorized from National Cheng Kung University to Vedan
Biotechnology Corporation with authorization fee of 25,100,000
NTD.

Competition
2009 Crazy Idea Competition, bronze medal.

16. Researches about BL10: whats the next?

1.

1. Use of BL10 as a model organism

- Genome structure
- Fatty acid and natural product
synthetic pathway
- Osmotic regulation
2. Development of new products
related to BL10

Strategies for developing new products based on BL10

.:
DHA

.:
.:

.:
.:
.:DHA

.:
.:
.:DHA

.:

Acknowledgements
We thank NSC and NCKU for their financial supports.

Thanks for your attention.