Commercial Production of DHA using
Heterotrophic Microalgae
Yi-Min Chen (陳逸民), Huey-Lang Yang (楊惠郎)
Institute of Biotechnology, National Cheng Kung University
Chung-Kuang Lu (盧重光)
National Museum of Marine Biology & Aquarium
Su-Fen Chen (陳淑芬)
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-3、n-6 series：
C
C
C
C
3
C …..
C
C
OH
C
C
6
O
(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 Alzheimer’s disease
(3) maintaining membrane fluidity that influencing neurotransmission
吃素不見得健康！
多吃n-3不飽和脂肪酸，尤其是DHA有益身體健康！
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.
That’s 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% DHA；easy 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
•
淵自美國太空及國防工業公司Martin Marietta研究藻類於太空長程航行中
之應用，發現藻類是一含豐沛醫藥資源。
•
Martek於1985創立，專利一株富 DHA異營藻株並研發其應用。
•
Martek現有 525 員工，年營業額近 90億台幣。
•
DHA已是 99% 美國嬰兒食品及奶粉必要成份， 含DHA之奶粉已在75國銷
售，每年近 43 million 嬰兒使用。
‧ 高價水產餌料及畜產營養添加劑之應用。
‧ 國際DHA健康食品研發潮流--現正迅速由嬰兒保健食品進入成人，孕婦及
銀髮族預防醫藥之方向。
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
0
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
1
2
3
4
5
37%
Time (day)
DHA
5
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
6
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)
ΔW (g)
Relative ΔW (%)
F， control
F， exp
181.8±16.8
185.8±16.8
233.3±16.7
246.7±13.8
51.5±3.7
60.8±11.0
29%±4%
33%±8%
M， control
M， exp
247.8±10.6
252.3±11.4
355.5±9.2
369.5±22.3
107.7±10.1
117.2±14.9
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 (富含DHA之藻株及其應用).
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藻株生產技術)。
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: what’s 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
量程，安全性評估
一.吳文騰: 經由程序工程建立
生產DHA 之先導試量產產
程開發
二.蕭世裕: 食用安全性評估
八.楊惠郎:其他富含長鏈不飽
和脂肪酸微藻
+
新保健食品產品之開發
二.蕭世裕: 憂鬱症改善效能評估
三.楊燿榮: 對於非酒精性脂肪性肝炎的保護作用
四.黃朝慶:母體DHA 的補充對於緩和幼兒腦缺氧缺
血以及腦室周圍白質軟化症所造成腦傷之效益
五.林秋烽:多醣類成份免疫促進效果評估
六.吳天賞:活性寡糖與多醣體之分離與構造研究
七.李春芳:畜產食料增加羊乳與羊肉DHA成份之開
發
八.楊惠郎:水產養殖的應用
Acknowledgements
We thank NSC and NCKU for their financial supports.
Thanks for your attention.