05-commercial production of dha using heterotrophic microalgae .pdf

8/10/2019 05-Commercial Production of DHA using heterotrophic microalgae .pdf

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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


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1. Classification of unsaturated fatty acids

Fatty acids with 2 or more double bonds

(1) Polyunsaturated fatty acid (PUFA)

(2) Highly unsaturated fatty acid (HUFA)

Fatty acids with longer chain (20 C) and more (3) double bonds

E.g. EPA, DHA

(4) Essential fatty acids

C CC

C CC

C .. C

OH

O

C

3 6

(3) n-3n-6 series


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

DPA (n-6)DHA

EPA

DPA (n-3)

ARA


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2. Functions of HUFAs

(2) Energy storage

(1) Precursors of eicosanoids

- ARA EPA

- Inflammation

- Diseases (Self-immune, cancer, )

- DHA in brain & retina

- Triacylglycerol

(3) Membrane structure

- Cell membrane, organelles

(4) Nerve conduction


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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

(1) mitigating inflammation reaction

(2) increasing acetylcholine level in brain of patient with Alzheimers disease

(3) maintaining membrane fluidity that influencing neurotransmission

n-3DHA

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

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


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3. DHA is an essential and important fatty acid

- 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

(1) For human

(2) For marine fish

- 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


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Deformation of grouper larvae related to insufficient DHA intake


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(1) Deep sea fish oil

(3) Microalgae

- Minor source, expensive

- 10-50% DHAeasy to purify-No contaminants; suitable for pregnant

women and infants

- Edible for vegetarians

- Environmental friendly

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

- 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


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DHA products from animal sources


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- From Gold Circle Farms

- 150 mg/egg

- 90 mg/100 g milk powder

DHA products form plant sources (1)


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- From plant oil?Exaggerated and false advertisement

- From microalga

DHA products form plant sources (2)


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With no contaminants

Suitable for pregnant women,

infants, and vegetarians

With Contaminants (heavy metals,

PCBs, dioxin etc.)

Higher DHA content

Easy to purify

Lower DHA content

Difficult to purify

More expensive

Environmental friendly

Cheaper

Not environmental friendly

DHA from microalgaeDHA from fish oil

5. Marine microalgae can produce high-quality DHA


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(2) Heterotrophic microalgae

rich in DHADinophyta:Crypthecodinium

Labyrinthulomycota:Schizochytrium,

ThraustochytriumAuratiochytrium

(1) Autotrophic microalgaerich in DHA

Haptophyta:Isochrysis

Chromophyta:Pavlova

6. Heterotrophic microalga is a nice candidate for DHA production

Can be produced with lower cost!


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DHA products made by heterotrophic microalgae

Company

Martek (USA)

OmegaTech (USA)

Bio-Marine (USA)

Advanced BioNutrition (USA)

Nutrinova (Germany)

species

Crypthecodinium cohnii

Schizochytrium sp.

Schizochytrium sp.

Schizochytrium sp.

Ulkenia

product

DHA in Infant milk, feed

Health food, feed

Feed

Feed

Health food


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Martin Marietta

Martek 1985 DHA

Martek 525 90

DHA 99% DHA75

43 million

DHA--

Martek Biosciences: the most successful company producing algal DHA

7 St t i f d l i l l DHA i d t i T i


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1. Find new algal species rich in DHA which have not beenpatented.

7. Strategies for developing algal-DHA industry in Taiwan

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 P d


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(1) Isolation and identification of heterotrophic algal strainsfrom a range of marine habitats in Taiwan

(2) Biomass, total fatty acid content, and DHA yieldcomparison of these strains

(3) Optimization of DHA production

8. Procedures

(4) Toxicity assay

(5) Patent and tech transfer

9 Collection dates and sites of algal like microorganism strains established


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

BL13

BL3

76

10 Identification of heterotrophic algal strains based on 18SrDAN sequences


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Aurantiochytrium

Oblongichytrium

Aplanochytrium

BL5

Thraustochytrium sp. ATCC 26185

BL8

BL4

BL7

BL2

BL6

BL14

BL9

TN3

BL11

Aurantiochytrium mangrovei RCC893

BL10

BL1

Aurantiochytrium limacinum NIBH SR21

HK8a

KL2a

HK8

HK1

KL2

Thraustochytriidae sp. #32

Sicyoidochytrium minutum SEK 354

HK10

HK5

Thraustochytrium striatum ATCC 24473

Ulkenia profunda KMPB N3077a

Ulkenia sp. SEK 214

Parietichytrium sarkarianum SEK 351

B otryochytrium radiatum Raghukumar 16...

Thraustochytrium aureum ATCC 34304

KL1

Thraustochytrium k innei KMPB 1694d

Schizochytrium sp. SEK 210

Schizochytrium aggregatum ATCC 28209

Thraustochytrium aggregatum KMPB N-BA...

Thraustochytrium pachydermum KMPB N-B...

Labyrinthula sp. N8

Labyrinthula sp. L72

Aplanochytrium stocchinoi

Aplanochytrium minutum

S1a

Oblongichytrium multirudimentale KMPB.. .

Oblongichytrium minutum KMPB N-BA-77

HK 9

TN6

Oblongichytrium sp. S EK 347

Crypthecodinium cohnii ATCC 30336

99

99

99

89

99

96

91

19

98

99

99

93

99

88

88

44

14

99

93

72

72

99

80

97

18

9587

97

90

93

95

27

99

99

85

99

84

99

99

97

99

92

49

71

0.02

Thraustochytrium (sp 1)





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

11 Identification of heterotrophic algal strains based on morphologies


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11. Identification of heterotrophic algal strains based on morphologies

These characteristics support the identifications based on 18SrDNA.

Morphological characteristics of BL10


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Morphological characteristics of BL10

50 m

Vegetative cell

Ameboid cell

Zoospore

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


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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)

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

Fatty acid profiles can be a nice chemotaxonomical characteristic.

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


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0 50 100 150 200

ATCC30336

HK9

TN6

HK5

HK10

S1a

KL1

KL2

HK8a

HK8

KL2a

HK1

BL10

BL1

BL11

TN3

BL14

BL9

BL6

BL13

BL3

BL7

BL2

BL8BL4

BL5

ATCC26185

Biomass and total fatty acid (TFA) production (mgL-1

)

TFA

Biomass

0 10 20 30

EPA & DHA yield (mgL-1

)

EPA

DHA

C p , y , , y

14. Optimal conditions for DHA production by using BL10


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* 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

Time (min)

5 10 15 20 25 30

DHA

C16:0

C14:0 DPA

6%

46%

7%

37%

p p y g

0

10

20

30

40

50

60

0 1 2 3 4 5Time (day)

Biom

ass(gL-1)

BL10 is the best algal strain for DHA production


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Strain

BL10

G13/2S

N-2

S31

SR21ONC-T18

ATCC30772

DHA cont. (gL-1)

17.0

6.1

2.7

0.3

4.24.5

19.0

Biomass (gL-1)

60

63

13

6

3526

109

Species

Aurantiochytrium sp.

Schizochytrium sp.

S. sp.

S. sp.

S. limacinumThraustochytrium sp.

Cryptothecodinium cohnii

BL10 >15

Marine fish 0.03-2.90

Fresh water fish 0.04-0.75

Crab & shrimp 0.03-0.35

Egg


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y y p

Initial W (g) Final W (g) W (g) RelativeW (%)

F control 181.816.8 233.316.7 51.53.7 29%4%F exp 185.816.8 246.713.8 60.811.0 33%8%

M control 247.810.6 355.59.2 107.710.1 44%6%M exp 252.311.4 369.522.3 117.214.9 46%5%

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

- No weight loss- No diarrhea

15. Achievement about BL10: 08 EAS Best Poster Award


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5. c eve e t about 0: 08 S est oste wa d

15. Achievement about BL10: paper published in scientific journal


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p p p j

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


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Patent

Tech transfer

Yang H.-L., Chen Y.-M., Lu C.-K., 2009. Application of a

Algal Strain Rich in DHA (DHA).Submitted, accession number 98107877.

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 VedanBiotechnology Corporation with authorization fee of 25,100,000

NTD.

Competition

2009 Crazy Idea Competition, bronze medal.


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We thank NSC and NCKU for their financial supports.

Acknowledgements

Thanks for your attention.

05-commercial production of dha using heterotrophic microalgae .pdf

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