공학석사학위 청구논문

광생물반응기를 이용한 미세조류

Haematococcus pluvialis 의 고농도 배양과 천연

astaxanthin 의 축적에 영향을 미치는 인자의

최적화

Optimization of Effective Factors for High-density Haematococcus pluvialis Cultures and Astaxanthin

Accumulation in Photobioreactors

2001 년 8 월

인하대학교 대학원

생물공학과

박 은 경

ii

Optimization of Effective Factors for High-density Haematococcus pluvialis Cultures and Astaxanthin

Accumulation in Photobioreactors

by

Eun-Kyung Park

A THESIS

Submitted to the faculty of

INHA UNIVERSITY

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Biological Engineering

August 2001

iii

이 논문을 박은경의 석사학위논문으로 인정함.

2001 년 8 월

주심 김 동 일 (인)

부심 이 철 균 (인)

위원 윤 현 식 (인)

iv

To my family

v

CONTENTS

DEDICATION ...........................................................................................i

CONTENTS ..........................................................................................v

LIST OF FIGURES................................................................................ viii

LIST OF TABLES.....................................................................................x

ABSTRACT ...........................................................................................xi

1. INTRODUCTION.............................................................................. 1

1.1. Objevtives ...................................................................................................... 2

2. LITERATURE REVIEWS................................................................. 4

2.1. Agal Biotechnology..................................................................................... 4

2.2. Astaxanthin .................................................................................................. 6

2.2 1. Astaxanthin Market......................................................................... 7

2.2.2. Astaxanthin Structure ..................................................................... 9

2.2.3. Astaxanthin Activity..................................................................... 12

2.3. Current Methods of Astaxanthin Production......................................... 16

2.3.1. Chemical Synthesis....................................................................... 16

2.3.2. Extraction ....................................................................................... 19

2.3.3. Production by Yeast...................................................................... 19

2.3.4. Production by Microalgae............................................................ 20

3. MATERIALS AND METHODS...................................................... 21

3.1. Cell Lines ..................................................................................................... 21

3.2. Culture Media and Culture Conditions................................................... 23

3.3. Cell Analysis ............................................................................................... 24

vi

3.4. Pigment Analysis ........................................................................................ 25

3.5. Measurements of Light Intensity............................................................. 27

3.6. Other Measurement.................................................................................... 28

3.7. Photobioreactor........................................................................................... 29

4. RESULTSAND DISCUSSION ....................................................... 30

4.1. Astaxanthin Production............................................................................. 30

4.1.1. Haematococcus pluvialis ............................................................. 30

4.1.2. High Density Algal Cultures ....................................................... 33

4.1.2.1. Temperature and pH ............................................................. 33

4.1.2.2. Light........................................................................................ 34

4.1.2.3. Various Media........................................................................ 40

4.1.3. Induction of Astaxanthin Accumulation ................................... 42

4.1.3.1. Heating.................................................................................... 42

4.1.3.2. Light Intensity....................................................................... 43

4.1.3.3. Light Quality.......................................................................... 47

4.1.3.4. Nutrients Deficiencies .......................................................... 53

4.1.4. Scale up........................................................................................... 54

4.1.5. Astaxanthin Stability .................................................................... 58

4.2. Astaxanthin Analysis ................................................................................. 60

4.2.1. Pigments Extraction...................................................................... 60

4.2.2. TLC-plate and TLC-FID.............................................................. 62

4.2.3. Spectrophotometer........................................................................ 65

4.2.4. HPLC............................................................................................... 67

5. RECOMMENDATION.................................................................... 58

5.1. Photobioreactor Design............................................................................. 58

5.2. Process Design for Astaxanthin Production........................................... 61

6. CONCLUSION................................................................................ 75

vii

6.1. Conclusion................................................................................................... 75

6.2. Future Works ............................................................................................... 77

REFERENCES........................................................................................78

viii

LIST OF FIGURES

Figure 2.1. Applications of algal biotechnology................................................................ 5

Figure 2.2. Astaxanthin(3,3'-dihydroxy -β,β'-carotene-4,4'-dione) structures............. 11

Figure 2.3. Structure and composition of a Carophyll Pink beadlet . ................. 18

Figure 4.1. Life cycle of Haematococcus pluvialis ....................................................... 31

Figure 4.2. Cell growth characeristic; a) cell number (cell/mL): b) cell size

distribution (fL/cell)........................................................................................ 31

Figure 4.3. Astaxanthin concentration in combination of temp and pH. ..................... 33

Figure 4.4. Effect of light intensity on cell growth.......................................................... 37

Figure 4.5. Pigments concentration profiles under various light intensities. .............. 38

Figure 4.6. Comparison of media in various initial concentration................................ 41

Figure 4.7. Heating effect on astaxanthin accumulation................................................. 42

Figure 4.8. Comparison of cell size distribution under various light intensities. ....... 46

Figure 4.9. Effect of different light sources on cell growth. .......................................... 50

Figure 4.10. Pigments concentration profiles under different light sources.................. 51

Figure 4.11. Comparison of per cell volume distribution under different light

sources at 228 hr of cultivation..................................................................... 52

Figure 4.12. Designed bubble column photobioeactor. .................................................. 55

ix

Figure 4.13. Cell growth in photobioreactor with various light intensities................ 56

Figure 4.14. Astaxanthin concentration in photobioreactor under various light

intensity .......................................................................................................... 57

Figure 4.15. Comparison instability to light intensities of synthetic astaxanthin

and natural astaxanthin. ............................................................................... 59

Figure 4.16. Pigment extraction in different solvent with various time ..................... 61

Figure 4.17. Analysis method of TLC-plate and TLC-FID. ......................................... 63

Figure 4.18. Standard curve by TLC-FID........................................................................ 64

Figure 4.19. Astaxanthin standard curve with specatrophotometer. ........................... 66

Figure 5.1. Recommended two stage photobioreactor................................................... 71

Figure 5.2. Proposed flow chart of astaxanthin production system . ............................ 74

x

LIST OF TABLES

Table 2.1. Estimated value of algal biomass based on product value and

cell content ............................................................................8

Table 2.2. Market comparison of synthetic astaxanthin and natural

astaxanthin ........................................................................17

Table 3.1. Cell classification ................................................................22

Table 4.1. Comparison of Haematococcus pluvialis.................................31

Table 4.2. Astaxanthin contents per cell number under different light

intensity ..............................................................................39

Table 4.3. Results of nutrient deficient media ..........................................54

Table 5.1. Comparison of characteristics of various algal culture systems.69

Table 5.2. Astaxanthin yields reported elsewhere ....................................70

Table 5.3. Advantage and disadvantage of different harvesting methods ..73

xi

ABSTRACT

The developments of algal biotechnology related techniques such as natural

product screening techniques, cultivation methods, separation and purification,

as well as the availability of high-density large-scale photobioreactors have

made the commercialization of natural algal products possible. Proteins,

carbohydrates, fatty acids, enzyme, antibiotics, pharmaceuticals, bioactive

compounds, vitamins, and biofuels are the some of the commercialized or

soon to be commercialized products. Microalgal diversity over 30,000 species

has extended the use of microalgae to the areas of agricultures and

environmental industries as bio-fertilizers and bioremediation of soils. The

production of astaxanthin, superior antioxidant from Haematococcus pluvialis

is commercially possible.

Factors for high-density Haematococcus pluvialis cultures, culture

temperature, initial pH of medium, medium components and various light

intensities, and conditions for astaxanthin accumulation, heating, different

light sources (LEDs), high light intensity and nutrient deficient media were

examined to maximize astaxanthin production.

High density cell growth and maximum astaxanthin accumulation of 1.6

mg/L was obtained at initial pH 7.5 and 20 ~ 25℃, a high cell density of over

xii

2.7 g/L was obtained with 60 µE/(m2· s) or higher intensities and much lower

cell concentration (< 1.0 g/L) was obtained with lower light intensities (15 ~

30 µE/(m2· s) ). Light intensities over 60 µE/(m2· s) stimulated cell growth and

astaxanthin formation, while low light intensity, 15 µE/(m2· s), kept

Haematococcus cells in vegetative stage.

Astaxanthin accumulation is stimulated by heating, when compared to that

in control cultivation, and the maximum astaxanthin concentration of 6.5

mg/L was obtained at the light intensity of 160 µE/(m2· s). However, only 1.3

and 0.7 mg/L were obtained at 30 and 15 µE/(m2· s), respectively.

The supplement of 470 nm photons had more dramatic effect on final

astaxanthin concentration and specific astaxanthin contents than the

supplement of 680 nm photons. Although cell concentration in nutrient

deficient media such as nitrogen, phosphate and proteose-peptone were lower

than that in control media , specific astaxanthin content in nutrient deficient

media were higher than in control medium.

xiii

국 문 요 약

Astaxanthin 의 생산 능력을 가진 것으로 알려진 Haematococcus

pluvialis 는 진핵세포이며 단세포 녹조류이며 두 개의 flagella 를 가

지고 빛에 반응하는 광주성 (phototaxis)을 가지고, 점액질층을 가지

고 있어 배양 후반 (palmella 단계 이후) 에는 세포끼리 뭉쳐서 자란

다. 4∼10 µm 의 vegetative 세포, 10∼20 µm 의 palmella 세포, 20∼60

µm 의 aggregated 세포, 20∼60 µm 의 aplanospore 세포의 4 단계로 이

루어진 생장 특성을 보였다

고부가가치의 천연 astaxanthin 의 생산을 위한 H. pluvialis 의 고농

도 배양과 astaxanthin 의 축적율을 높이기 위하여 세포 농도를 증가

시키는 방법과 세포 내 색소를 증가시키는 방법 두 가지에 초점을

맞춰 본 연구를 수행하였다. H. pluvialis 의 최적 배양 온도와 pH 를

선정하기 위하여 20℃, 25℃, 30℃ 온도와 pH 4.5, 6.0, 7.5 의 배양 조

건을 수립한 결과 pH 7.5, 20℃ 와 25℃에서는 세포 생장과 색소 축

적면에서 우수함을 관찰 할 수 있었고, proteose-petone medium 내의

질소원과 proteose-peptone 의 고갈 시 세포의 생장의 특성과

astaxanthin 생산에 미치는 영향은 control 배지에서 세포 수와 세포의

농도가 0.98×106 cells/mL, 1.7 g/L 로 가장 높게 나왔으나, 세포 당

astaxanthin 의 농도는 2.57 mg/L, 단위 세포 당 astaxanthin 의 농도는

xiv

1.53 mg/g cell 로 NaNO3 이 없는 배지에서는 세포 수와 세포의 농도

가 0.28×106 cells/mL, 0.2 g/L, astaxanthin 의 농도는 0.24 mg/L, 단위 세

포 당 astaxanthin 의 농도는 9.6 mg/g cell 로 나온 결과 질소원과

peptone 이 고갈되면 세포의 생장은 억제되나 astaxanthin 의 생산은

20.02 mg/L 로 183% astaxanthin 의 축적을 촉진하는데 효과적이었다.

미세조류 배양에 가장 큰 영향을 주는 인자인 빛의 영향을 알아보

기 위하여, 15 ~ 160 µE/(m2· s) 의 다양한 광도 하에서 배양을 한 결과,

세포 생장을 촉진하는 광도는 60 µE/(m2· s) 이고 높은 광도일수록

astaxanthin 의 축적에 효과적으로 나타났다. 배양에 적당한 20, 25℃

에서 배양하다가 30℃로 24 시간 옮겨 배양하여 heating 효과를 주면

astaxanthin 생산에 효과적임을 알 수 있었다. 배지 내에 질소원과 같

은 영양원이 고갈 된 경우 aplanospore 세포의 크기가 control 배지의

aplanospore 세포의 크기보다 크고 붉은 색의 농도도 진한 것을 쉽게

관찰할 수 있었고, 다양한 파장의 광원에 대한 세포의 변화를 관찰

한 결과, 단일 파장을 발산하는 LEDs 를 광원으로 붉은 빛 계열의

680 nm, 푸른빛의 470 nm 와 60 µE/(m2· s) 광도의 형광등 빛의 control

조건에서 각각 배양하여 광원이 세포의 형태학적 구조에 미치는 변

화과정을 살펴보았더니, 470 nm 광원에서는 세포가 높은 농도로 관

찰되었고, 20 µm 이하의 세포가 전체 농도 중의 60%를 차지하여 세

포 생장을 촉진하는 영향을 미친다는 것과 세포 크기가 크고 뭉쳐

xv

서 자라며 높은 농도의 붉은 색의 색소 또한 관찰되어 aplanospore

세포 형태를 촉진시킨다는 것을 알 수 있었다.

- - 1

1. INTRODUCTION

Microalgae have many fundamental attributes that can be converted into

technical and commercial advantages such as a genetically diverse group of

organisms, the lowest form of plants, photosynthetic properties using sun light

and CO2, rapid growth, superior volumetric productivity, resistance to

environmental stress, simpler nutritional requirements, and potentials of

numerous unexplored species and strains.

Commercialization of microalgal biotechnology has been accelerated due to

the vast potential of microalgae as a source of valuable metabolites such as

proteins, carbohydrates, fatty acids, enzyme, antibiotics, pharmaceuticals,

bioactive compounds, vitamins , and biofuels and the areas of agricultures and

environmental industries as bio-fertilizers and bioremediation of soils [1-16].

Successful commercialization stories include Chlorella and Spirulina for

functional food and Dunaliella for β-carotene production [1-3, 17].

Freshwater microalga Haematococcus pluvialis is the most powerful producer

of astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione), a red keto-carotenoid,

and is receiving commercial interests due to its use as a preferred pigment in the

feeds, vitamin sources, a colorant, natural preservatives, a antioxidant, a

antiaging reagent, a anticancer agent, and an immunomodulator [1-5, 7, 17-26].

- - 2

Despite the accumulation of high level of astaxanthin in H. pluvialis, low growth

rate, thick cell wall and low cell density keep the best producer of astaxanthin

from commercial uses but advanced algal biotechnology has made it possible.

In this study, factors for high-density Haematococcus pluvialis cultures, the

culture temperature, initial pH of medium, various media and various light

intensities, and induction conditions for astaxanthin accumulation such as

heating, different light sources (LEDs), high light intensity and nutrient deficient

media were examined to maximize astaxanthin production.

1.1. Objectives

In order to maximize astaxanthin productivity, effective factors for optimal

conditions for high density algal cultures such as temperature, pH, media

compositions, light intensity, and factors for astaxanthin accumulation such as

heating, spectral quality, high light intensity, and nutrient deficiencies should be

examined.

• to find proper method for astaxanthin production by microalgal cultures and

select an astaxanthin producing species after literature review and preliminary

experiments

- - 3

• to determine analyzing method to monitor cell growth and astaxanthin

accumulation quantitatively

• to maximize astaxanthin productivity, cell growth characteristics and

morphological changes

• to optimize factors for high density algal culture should be tested such as

culture condition of temperature and pH, illumination intensity and optimal

medium

• to stimulate of astaxanthin accumulation rate and quantity, by applying

various unfavorable culture conditions such as heating, using nutrient deficient

media and high light intensity respectively, and induction condition such as

LEDs illumination

• to accomplish final purpose of astaxanthin commercialization, astaxanthin

instability to heat, light and oxygen

- - 4

2. LITERATURE REVIEWS

2.1. Algal Biotechnology

Algae are a photosynthetically diverse group of organisms, ranging in size

from cyanobacteria (0.2 ~ 2 µm) to giant kelps (60 m) and from small, single

celled forms to complex multicellular forms. Algae are important as primary

producers of organic materials at the base of food chains and provide oxygen.

The history of the commercial use of algal cultures spans about 60 years with

various applications since 1940s, for examples biofuel production and lipid,

protein from Chlorella and Scenedesmus in 1940s, waste water treatment in

USA in 1951, commercialization of algal products as health additives in Japan

and Tiwan in 1965, biofuel production and research by National Renewable

Energy Laboratory (SERI;Solar Energy Research Institute in 1973) in USA in

1970s, algal biotechnology is concentrated in Japan (RITE, Research Institute of

Innovative Technology for the Earth) in 1990s.

The improvement of photobioreactors for natural production, waste water

treatment and bioremediation and algal biotechnology for marine resource

screening, eutrophication and CO2 removal [1, 2] are processing by a number of

- - 5

scientist and engineer [1-3,5].

Figure 2.1. Applications of algal biotechnology

A l g a l B i o m a s s

; food

; feed

; biofuels, etc

M e t a b o l i t e s

; pharmaceuticals

; carotenoids

; amino acids, etc

E n v i r o n m e n t

; wastewater treatment

; heavy metal adsorption

; eutrophication

O t h e r s

; agricultures

; bioremediation

A l g a l

B io techno logy

- - 6

2.2. Astaxanthin

Astaxanthin(3,3'-dihydroxy-β,β'-carotene-4,4'-dione) is a red keto-carotenoid

and is receiving commercial interests due to its use as a preferred pigment in the

feeds of farmed fish and other marine animals, vitamin sources for poultry

industry, a colorant, natural preservatives and food additives in food industry, a

superior antioxidant to α-tocopherol (vitamin E) and β-carotene in cosmetic

industry, an antiaging reagent as a precursor of vitamin A, an anticancer agent by

singlet oxygen quenching, and an immunomodulator in pharmaceutical industry.

These useful properties have also been found to be effective in mammals and are

very promising possibilities as nutraceuticals and pharmaceuticals for humans.

- - 7

2.2.1. Astaxanthin Market

Animal feed market was $ 185 million in 1999 and currently growing at 8%

per year.

• Poultry and Livestock Market

Current market price is $ 2,500 ~ 4000$ per 1kg astaxanthin and astaxanthin

market is $ 125 million getting growing at 15% per year. Synthetic astaxanthin

(8%(w/w) Carophyll R by Hoffmann-La Roche) price is $ 2000/kg, extracted

astaxanthin (10%(w/w)) price is $ 7000/kg and natural astaxanthin price is

getting lower by developments of algal biotechnology. Currently natural

astaxanthin market is valued at $35 billion per annum and is expected to grow to

$49 billion by 2010.

• Near-Term Target Market and Long-Term Target Market

Considerations of astaxanthin activities, astaxanthin commercializations in

various industries such as nutraceuticals, food additives (exceed $ 3 billion

annually) and cosmetics, pharmaceuticals and environmental remediation are

expected.

- - 8

Table 2.1. Estimated value of algal biomass based on product value and cell

content [2]

Alga Product Cell content (% dry wt)

Value of product ($/kg)

Value of alga ($/kg)

Chlorella Health food 100 25 25.00

Dunaliella salina

Glycerol 40 2 0.80

Dunaliella salina β-Carotene 10 600 60.00

Haematococcus pluvialis

Astaxanthin 1 3000 30.00

Spirulina platensis

Phycocyanin 2 500 10.00

Phaeodactylum tricornutum

EPA 0.1 1900 190.00

- - 9

2.2.2. Astaxanthin Structure

• Stereoisomers

Astaxanthin has two chiral, carbons (numbered 3 and 3' on the two rings in

the structure), and thus there are three stereoisomers: (3S,3'S), (3R, 3'S), or

7(3R,3'R). The (3S,3'S) and (3R,3'R) stereoisomers are mirror images of each

other enantiomer and (3R,3'S) form is a meso and is optically inactive (Fig 2.1.).

• Geometric Isomers

Carbon-carbon double bonds can have the atoms attached to them arranged in

different ways. Several geometric isomers are possible: all-(E), (9Z), (13Z),

(15Z), (9Z,13Z), (9Z,15Z), (13Z,15Z), and (9Z,13Z,15Z) but thermodynamically

most stable form of the molecule is all-(E) astaxanthin. This is because in the all-

(E) form, the branching methyl (CH3) groups on the linear portion of the

molecule do not compete for space.

• Free or Esterified

Astaxanthin has two hydroxyl (OH) groups, one on each terminal ring. These

can be "free" (unreacted) hydroxyls, or can react with an acid (such as a fatty

acid) to form an ester. If one hydroxyl reacts with a fatty acid, the result is

termed a mono-ester. If both hydroxyl groups are reacted with fatty acids, the

result is termed a di-ester. Adding a fatty acid to form an ester makes the

- - 10

esterified end of the molecule more hydrophobic. The hydrophobicity of

astaxanthin (difficulty in dissolving in water), is known as the order of di-esters

> mono-esters > free form astaxanthin (Fig 2.1).

Astaxanthin occurs in several different forms which can be classified

according to stereoisomers, geometric isomers, and free or esterified forms. For

example, the predominant stereoisomer of astaxanthin found in krill (Euphausia

superba, a shrimp-like marine animal) is (3R,3'R), usually in esterified form. In

wild salmon, the predominant stereoisomer is (3S,3'S); in salmon flesh the

astaxanthin occurs as the free xanthophyll. The basidiomycete yeast

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) accumulates astaxanthin

as its major carotenoid; in this yeast, astaxanthin occurs as the (3R,3'R)

stereoisomer and is predominantly esterified. In the green alga Haematococcus

pluvialis, astaxanthin occurs as the (3S,3'S) stereoisomer. Astaxanthin from H.

pluvialis occurs primarily as monoesters (~80%) and diesters (~15%); the

predominant fatty acids that make up the esters are C18:1 and C20:0.

- - 11

Figure 2.1. Astaxanthin (3,3'-dihydroxy-β,β'-carotene-4,4'-dione) structures

- - 12

2.2.2. Astaxanthin Activities

Astaxanthin is made by Haematococcus, a fresh water microalga and is a

powerful, bioactive antioxidant and has demonstrated its efficacy in animal or

human;

- Macular Degeneration: the leading cause of blindness in the U.S.

- Alzheimer's and Parkinson's Disease: major neurodegenerative diseases.

- Cholesterol Disease: ameliorates the effects of LDL, the "bad" cholesterol.

- Stroke: repairs damage caused by lack of oxygen.

- Cancer: protects against several major types of cancer

• Pigmentaion

Salmon and trout, like other animals, can not synthesize astaxanthin by

themselves and they must obtain astaxanthin from their diets. In the wild, from

zooplankton (which presumably feed on the microalgae that are the original

producers of the carotenoid), or, in the case of commercial fish feeds used for

pen-raised fish, from intentionally added astaxanthin. It has been shown that in

fish and crustaceans, astaxanthin is essential for growth and plays a vitamin-like

role, and, in fact, astaxanthin is absorbed and deposited in fish flesh more

efficiently than are other similar xanthophylls (oxygenated carotenoids) such as

canthaxanthin, lutein, or zeaxanthin.

- - 13

Astaxanthin is thus commonly used to supplement fish feeds, and is approved

in the United States as a safe additive to salmonid fish feed (at up to 80 mg/kg

feed) in order to obtain the desired pink to orange-red color (Code of Federal

Regulations 21 CFR 73.35). Apart from imparting an attractive color,

astaxanthin has been shown to prevent the oxidation of fats in rainbow trout

during frozen storage, thus preventing rancidity [8, 13, 15, 27-35]. Astaxanthin

levels in the flesh of Atlantic salmon range from about 4 to 10 mg/kg, whereas

levels in wild Pacific salmon can be much higher, with a recent FDA study

reporting an average of about 14 mg/kg in coho salmon and about 40 mg/kg in

sockeye salmon. Thus, a reasonable serving portion of 4 ounces (one-fourth of a

pound, 113.4 g) of farmed Atlantic salmon would contain from 0.5 to 1.1 mg of

astaxanthin, whereas the same amount of sockeye salmon would contain 4.5 mg

of astaxanthin.

• Antioxidant

Astaxanthin has been shown to be a powerful quencher of singlet oxygen

activity in in vitro studies [1, 27, 28, 35-48], and is a strong scavenger of oxygen

free radicals, at least ten times stronger than β-carotene. Experiments with red

blood cells and mitochondria from rats have shown that astaxanthin is from 550

to 1000 times more effective at inhibiting lipid peroxidation than is vitamin E.

The results of these in vitro studies were confirmed in vivo with rats given

- - 14

dietary supplements of astaxanthin and subjected to oxidizing agents. The

antioxidative properties of astaxanthin have been demonstrated in a number of

different biological membranes. Other tests have shown that astaxanthin is up to

1000 times more powerful than vitamin E [49].

• Metabolic Effect

Astaxanthin may be effective as a prophylactic and/or therapeutic treatment of

Helicobacter infections of the mammalian gastrointestinal tract, and an oral

preparation has been developed for this purpose. Astaxanthin has been shown in

both in vitro experiments and in a study with human subjects to be effective for

the prevention of the oxidation of low-density lipoprotein. This suggests that it

could be used as a preventative for arteriosclerosis, coronary artery disease, and

ischemic brain damage; a number of astaxanthin-containing health products are

under development based on these findings. Astaxanthin has also been shown to

enhance production of LDL and especially HDL cholesterol in the bloodstream

of rats. Astaxanthin diesters appear to exert a synergistic effect on anti-

inflammatory agents, increasing the effectiveness of aspirin when the two are

administered together.

• Support of Eye Health

A recent study with rats indicates that astaxanthin is effective at ameliorating

retinal injury, and that it is also effective at protecting photoreceptors from

- - 15

degeneration. The results of this study suggest that astaxanthin could be useful

for prevention and treatment of neuronal damage associated with age-related

macular degeneration, and that it may also be effective at treating ischemic

reperfusion injury, Alzheimer's disease, Parkinson's disease, spinal cord injuries,

and other types of central nervous system injuries. In this study, astaxanthin was

found to easily cross the blood-brain barrier (unlike β-carotene), and did not

form crystals in the eye (unlike canthaxanthin).

• Cancer Deterrence and Immune Support

Dietary administration of Astaxanthin proved to significantly inhibit

carcinogenesis in the mouse urinary bladder [47, 48], rat oral cavity, and rat

colon. In addition, astaxanthin has been shown to induce xenobiotic -

metabolizing enzymes in rat liver, a process which may help prevent

carcinogenesis [15, 47, 48].

Astaxanthin has been shown to significantly influence immune function in a

number of in vitro and in vivo assays using animal models. The majority of this

work has been carried out by Harumi Jyonouchi and colleagues at the University

of Minnesota. Astaxanthin enhances in vitro antibody production by mouse

spleen cells stimulated with sheep red blood cells, at least in part by exerting

actions on T-cells, especially T-helper cells. Astaxanthin can also partially

restore decreased humoral immune responses in old mice. These

- - 16

immunomodulating properties are not related to pro-vitamin A activity, because

astaxanthin, unlike β-carotene, does not have such activity. Studies on human

blood cells in vitro have demonstrated enhancement by astaxanthin of

immunoglobulin production in response to T-dependent stimuli. Other

supporting data on astaxanthin and immune function, including studies on the

mechanisms of action involved [50-58].

2.3. Current Methods of Astaxanthin Production

2.3.1. Chemical Synthesis

Chemically synthesized astaxanthin, have complex isomers, enantiomers and

low market occupancy refer (Table2.2). Because astaxanthin have two chiral

centers, synthetic astaxanthin is produced as racemic mixture and application is

also restricted as feed only.

- - 17

Table 2.2. Market comparison of synthetic astaxanthin and natural astaxanthin.

1992 1996 2000 price

Syn. Nat. Syn. Nat. Syn. Nat. Per Kg

β-carotene 95 25 170 80 250 190 373-1770

Astaxanthin Canthanxanthin

120 10 210 40 305 150 2000-4000 1150-2315

Melanin 0 2 0 6 0 10

Sum 215 37 380 126 555 350

- - 18

Astaxanthin emulsified in antioxidants

Matrix (gelatine and carbohydrate)

Maize starch

Figure 2.2. Structure and composition of a Carophyll Pink beadlet

Carophyll Pink is a formulated product that provides the feed manufacturer

with a stable identical astaxanthin. This product has been designed and

formulated for addition pre-extrusion.

- - 19

2.2.2. Extraction

Extraction method is usually using strong acid and base or organic solvent.

These materials and resistance materials are become environmental problem.

Extraction from a shell of crustacea is very difficult to commercialize because of

difficulty of row material supplement, need of high value enzyme and a number

of employee. Nowadays, to overcome these disadvantages, enzyme method is

developed.

2.2.3. Production by Yeast

Although red yeast, Phaffia rhodozyma is also reported to produce astaxanthin

and gowth rate is faster than H. pluvialis, reported productivity is respectively

low as 5-35 mg/L, astaxanthin content is low in cell and instability of produced

forms as enantiomer (3R,3’R). Nowadays, many researcher have been studied to

overcome these problem by genetic engineering.

- - 20

2.2.4. Production by Microalgae

The krill, crawfish and Phaffia sources contain low concentrations of

astaxanthin. Feeds may require so much addition of these products for effective

pigmentation that it adds unwanted bulk and ash, decreases palatability, and

alters the nutrient balance of the diet. The astaxanthin derived from

Haematococcus algae is the most concentrated source of natural astaxanthin that

is available and provide sufficient astaxanthin in feeds for very effective

pigmentation.

- - 21

3. MATERIALS AND METHODS

3.1. Cell Lines

Haematococcus pluvialis was colleted by F. Mainx in 1931, isolated by

E.G. Pringsheim as axenic and renamed by George in 1976. Haematococcus

pluvialis is green unicellular algal has motility with two flagellates and sheath.

The green microalga, Haematococcus pluvialis, Flotow, Volvocales,

Chlorophyceae, (H. lacustris, UTEX 16, 32, 294) were obtained from the

University of Texas Culture Collection (Austin, TX, USA) to compare ability

of astaxanthin production. Three species of H. pluvialis were cultivated in

proteose-peptone medium. The experiments were carried out in 250 mL

Erlenmeyer flasks containing 120 mL of culture medium, after adjusting the

initial pH to 6.4. Seed culture was prepared at 20℃, 175 rpm under white

fluorescent lamp at the light intensity of 60 µE/(m2· s).

- - 22

Table 3.1. Cell classification.

Phylum Chlorophyta

Class Chlorophyceae

Order Volvocales

Family Haematococcaceae

Genus Haematococcus

species pluvialis

- - 23

3.2. Culture Media and Culture Conditions

Haematococcus pluvialis obtained from the University of Texas Culture

Collection were streaked on Proteose-peptone medium agar plates and kept at

20~25℃with cool-white fluorescent lights until visible single colonies were

formed and then these agar plates were kept at 4℃ in a refrigerator. Seed

cultures were usually prepared by suspending a single colony from a master

plate in a 250 mL Erlenmyer flask containing 120 mL of proteose-peptone

medium, whose composition consisted of NaNO3 250 mg/L, CaCl2·2H2O 25

mg/L, MgSO4·7H 2O 150 mg/L, K2HPO4 75 mg/L, KH2PO4 175 mg/L, NaCl

25 mg/L, and proteose-peptone 1 g/L in distilled water, after adjusting the

initial pH to 6.4. The seed culture flask was cultured in an illuminated shaking

incubator (model HB-201S, HanBaek Scientific, Puchon, Korea) at a constant

light intensity of 60 µE/(m2· s) and 175 rpm.

- - 24

3.3. Cell Analysis • Cell Number, Average Cell Size and Size Distribution

Cell concentration and cell size distribution were measured by computer-

controlled Coulter Counter (model Z2, Coulter Electronics, Inc., Miami, FL,

USA). The principle of sizing and counting particles of Coulter Counter is

based on measurable changes in electrical resistance produced by

nonconductive particles suspended in an electrolyte. A small opening

(aperture) between electrodes is the sensing zone through which suspended

particles pass. In the sensing zone each particle displaces its own volume of

electrolyte. Volume displaced is measured as a voltage pulse; the height of

each pulse being proportional to the volume of the particle. The quantity of

suspension drawn through the aperture is precisely controlled to allow the

system to count and size particles for an exact reproducible volume. After

culture samples were diluted with electrolyte solution, Isoton®Ⅱ (Coulter

Electronics, Ltd., Hong Kong, China) to about 104 cell/mL. Data from Coulter

Counter were converted by AccuComp® software and exported to Excel to

calculate cell numbers and cell size distributions and cell concentrations were

calculated from total cell number and the average cell size from the Coulter

Counter after multiplying proper calibration factor to convert the total cell

- - 25

volume (= cell number × average cell size) to dry cell weight. Information on

the cell cycle stages, cell growth kinetics, and morphological characteristics

was obtained by these data.

• Morphological Change

Cell pictures by digital camera (Nikon, CoolPix 900) were analyzed with

Imagescope 2.3 software (Imageline Inc., Korea) and cell division consist of 4

stage, cell cycle, morphological change such as loss of flagellates resulting

decrease of mobility, formation of red color carotenoids, and dramatic

morphological transformation results in carotenoid formation were observed

optically with microscope (×400) and haemacytometer.

3.4. Pigment Analysis

• Pigments Extraction

One mililiter of culture sample was centrifuged at 10,000 rpm for 10 min

and supernatant was removed. Collected algal cells were resuspended with 1

mL acetone and cell walls were broken by a specially-designed tissue

homogenizer for 2 min with 12 strokes. Two mililiters of acetone was added

and the sample was stored at 4℃ refrigerator for 20 min to extract pigments.

- - 26

These steps were repeated until the color of cell debris became white or

colorless. Supernatant that contained pigments was harvested after removing

cell debris by centrifugation at 10,000 rpm for 10 min.

• Pigments Analysis

Chlorophyll concentration and astaxanthin concentration were analyzed by

a spectrophotometer (model HP8453B, Hewlett Packard, Waldbronn,

Germany). Chlorophyll concentration was calculated by previously reported

equation [1] : chlorophyll a = (12.7 ×A663) - (2.69 ×A645), chlorophyll b =

(22.9 ×A645) - (4.64 ×A663). Astaxanthin concentration was calculated by a

calibration curve using synthetic astaxanthin (product number A9335, Sigma

Chemical Co., St Louis, MO, USA) as a standard. For astaxanthin

concentration less than 10 mg/L, the following calibration was used:

astaxanthin concentration (mg/L) = 0.0045 ×OD475.

More quantitative and qualitative measurements of astaxanthin were

analyzed by a HPLC (Younglin Instrument Co., Anyang, Korea) with two

M930D pumps and a M730D photodiode array (PDA) detector. A reversed-

phase C18 column (300×3.9 mm; µm, Waters Co., Milford, MA, USA) was

chosen for separation and analysis of extracted pigments.

- - 27

3.5. Light Measurement

Colored lights were acquired by 470 nm LEDs (DigiKey, Thief River Falls,

MN, USA) and 680 nm LEDs (Quantum Devices Inc., Barneveld, WI, USA)

have narrow spectral outputs, whose central wavelength is approximately 470

nm and 680 nm. These LEDs were powered by DC power supplies (Model

GP-233, LG Precision, Seoul, Korea) at the constant voltages and the

supplemental light intensities were controlled by adjusting the supplied

voltage. Light intensities of fluorescent lights were changed by number of

cool white fluorescent lamps, distance from samples, and addition of reflector.

The light intensities of LED units and cool white fluorescent lamps

were measured with a silicon photo cell (model 0560.0500, Testoterm GmbH

& Co., Germany) and with a quantum sensor (model LI-190SA, LI-COR,

Lincoln, NE, USA).

- - 28

3.6. Other Measurements

The pH of the culture broth and medium was measured by using the pH

meter, Istek model 720p (Istek, Seoul, Korea), which was calibrated with

buffer solutions (pH 4, 7, and 10).

To measure astaxanthin productivity, after each run of experiment, cell

weight was measured. First empty crucibles were weighed after drying in

oven for overnight. Fifty milliliters of culture broth was centrifuged at 1,500

rpm for 20 min. After discarding supernatant, the pellet was resuspended to 10

mL of distilled water. The resuspended cells were centrifuged again at the

same condition to remove media components. The washed cell then

transferred to and transferred to dried for over night at 90℃ under vacuum

condition. Before measuring the DCW, the crucibles were placed in

desiccator to cool down to room temperature.

- - 29

3.7. Photobioreactor

Column photobioreactor was made of Pyrex glass tubes (650 mm of height,

35 mm of internal diameter). Fluorescent light source (FL20D, OSRAM,

Korea) were used as light sources for photosynthesis and carotenoid synthesis.

Two fluorescent lamps were placed at a distance of 12 cm from

photobioreactor columns. Light intensity was measured with Quantum Sensor

(model LI-190SA, LI-COR, Lincoln, NE, U.S.A.) with Datalogger (model LI-

1400, LI-COR).

Air was introduced into the bottom of the column at 0.2 v/v flow rate.

- - 30

4. RESULTS AND DISCUSSION

4.1. Astaxanthin Production

4.1.1. Haematococcus pluvialis

Despite the accumulation of high level of astaxanthin in Haematococcus

pluvialis, low growth rate, thick cell wall and low cell density keep the best

producer of astaxanthin from commercial uses. Although a number of algal

researcher groups have been studied about H. pluvialis recently, ambiguities

of some characteristics and lack of downstream processing make

commercialization difficult. In this study, various experiments were tested to

clear characteristics of H. pluvialis growth and change of morphology. As H.

pluvialis grows, several changes were observed under microscope: loss of

flagellates resulting decrease of mobility, formation of red color carotenoids,

dramatic morphological transformation consist of 4 stages ((a) vegetative cell;

(b) encystment; (c) maturation;(d) germination) and aggregation of cells after

a week. As shown in Figure 4.1, 4.2 cell growth and morphological change

were significant and important to clear cell characteristics and to accumulate

astaxanthin in cells.

- - 31

Table 4.1. Comparison of Haematococcus pluvialis

UTEX 16 UTEX 32 UTEX 294

Cell conc.

Astaxanthin Conc.

a) b)

d) c)

Figure 4.1. Life cycle of Haematococcus pluvialis

- - 32

a)

b)

Figure 4.2. Cell growth characteristics

a) cell number (cell/mL)

b) cell size distribution (fL/cell)

; (a) vegetative stage (b) encystment (c) maturation (d) germination

0

50000

100000

150000

200000

250000

300000

0 20000 40000 60000 80000 100000Vol.(fL)

Dif

f. C

ell V

ol.(

fL/c

ell)

(a)

(b)

(c)

(d)

- - 33

4.1.2. High Density Algal Cultures

4.1.2.1. Temperature and pH

H. pluvialis showed mixotrophic growth on proteose-peptone media

with various combinations of temperature and pH conditions under

continuous illumination. The pH of culture broth eventually reached about pH

8.5 regardless of initial pH values. Higher cell density was obtained at

relatively low temperature. When grown under 20℃ and initial pH of 7.5,

about 1.6 mg astaxanthin/L culture was obtained.

0.0

0.1

0.2

0.3

0.4

1618

2022

2426

2830

34

56

78A

stax

anth

in

con

cen

trat

ion

(m

g/L

)

Tempe

ratu

re (

0 C)

pH

Figure 4.3. Astaxanthin concentration in combination of temperature and pH

- - 34

4.1.2.2. Light

There are many known factors that effect the astaxanthin accumulation in H.

pluvialis: nutrient limitation or supplement (especially iron, manganese,

nitrogen, and potassium), salt stress, other environmental factors such as

excess oxygen stress , high light intensity , blue light, dark condition, and high

temperature. Among these factors, light intensity found to effect most on H.

pluvialis culture. Cell division, cell cycle and thus morphological change of

cells, carotenoid formation and concentration, and chlorophyll concentration

of H. pluvialis were effected by light intensity.

Figure 4.4 shows the cell growth curves under various light intensities. All

the data points represented in Figure 4.4 were the average of triplicates and

the standard deviation of each average represented by error bars in Figure 4. 4.

Cell concentrations were calculated from total cell number and the average

cell size from the Coulter Counter after multiplying proper calibration factor

to convert the total cell volume (= cell number x average cell size) to dry cell

weight. The culture which kept under dark did not grow as expected

(represented by � in Figure 4.4). However, there found to be an optimal light

intensity for cell growth. Light intensities between 60 ~ 90 µE/(m2·s)

- - 35

(represented by s, ¢, and £) supported the cells the best, while lower light

intensity of 15 ~ 30 µE/(m2· s) (� and q) or higher light intensity of 160

µE/(m2· s) (¿) could not match the growth under optimal light intensity. The

highest cell concentrations under different light intensities were 1.1, 1.9, 2.2,

and 2.7 g/L under 15, 30, 60, and 75 µE/(m2· s), respectively. The highest cell

concentration obtained here 2.7 g/L was one of the highest among reported

values.

The cell size distribution was also affected by light intensities. Four sets of

Coulter Counter data were compared in Figures 4.4 and 4.5. The cell size of H.

pluvialis increases dramatically as cells accumulate astaxanthin. Figure 4.8 a)

is the histogram of the cells under 0, 15, 60, and 160 µE/(m2· s) at earlier phase

of the culture (at 96 hr). It clearly showed that the main peak was shifted to

bigger size as the light intensity increased. For example, the cells under 15

µE/(m2· s) (···· in Figures 4.4 and 4.5 had a peak near 60 µm3/cell. The peaks

under 60 and 160 µE/m2/s (– – and – ··, respectively, in Figures 4.5 a) and 4.5

b) were moved to 80 and 102 µm3/cell, indicating the increase in average cell

size. Again, higher light intensity stimulated astaxanthin accumulation and

thus increased astaxanthin productivity. As H. pluvialis cultures entered into

stationary phase, all the cultures started to accumulate astaxanthin. The

difference in histogram peaks decreased in stationary phase (Figure 4.8 b) at

- - 36

336 hr). However, the same trends that higher intensity made more cells of

larger volumes could be observed at later phase of the culture. Conclusively,

higher intensity stimulates astaxanthin production, while there seemed to be

optimal light intensity for growth. This data suggested that two-stage cultures

would be beneficial for high yield astaxanthin production.

- - 37

Figure 4.4. Effect of light intensity on cell growth. Growth curves under dark

condition (-�-); under 15 µE/(m2·s) (-�-); 30 µE/(m2·s) (-q-); 60 µE/(m2·s) (-s-);

75 µE/(m2· s) (-¢-); 90 µE/(m2· s) (-£-); and under 160 µE/(m2· s) (-¿-).

Time (hr)

0 100 200 300 400

Cel

l Con

cent

rati

on (

g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

- - 38

a)

b)

Figure 4.5. Pigments concentration profiles under various light intensities. a).

astaxanthin concentration (mg/L); b). total chlorophyll concentration (mg/L); C.

chlorophyll contents per cell (pg/cell) under dark condition (-� -); under 15 µE/(m2·s)

(-�-); 30 µE/(m2·s) (-q-); 60 µE/(m2· s) (-s-); 75 µE/(m2·s) (-¢-); 90 µE/(m2·s) (-

£-); and under 160 µE/(m2· s) (-¿-).

Time (hr)

0 100 200 300 400

Tot

al C

hlor

ophy

ll C

once

ntra

tion

(mg/

L)

0

1

2

3

4

5

6

Time (hr)

0 100 200 300 400

Ast

axan

thin

Con

cent

rati

on (m

g/L

)

0

1

2

3

4

5

6

7A

- - 39

Table 4.2. Astaxanthin contents per cell number under different light

intensity

Light Intensity

µE/(m2s)

Astaxanthin Contents

per cell (mg/g cell)

Relative Contents

0 0.19 1.00

15 0.53 2.77

30 0.76 3.93

60 2.00 10.40

75 1.46 7.60

90 1.93 10.07

160 3.47 18.06

- - 40

4.1.2.3. Various Media

To determine superior media for Haematococcus pluvialis cultures,

compared to proteose-peptone medium and MBBM (Modified Basal Bristol

Medium) with 4 different inoculatio n concentrations. Respectively proteose

peptone medium was superior to MBBM but to determine optimal initial

inoculum concentration, more various range of inoculum concentration should

be examined (Figure 4.6).

- - 41

Figure 4.5. Comparison of media in various initial concentration

4.1.3 Induction of Astaxanthin Accumulation Figure 4.6. Comparison of media in various initial concentration.

1; 0.08 2; 0.15 3; 0.25 4; 0.32 g/L

initial ratio

0 1 2 3 4 5

Cel

l Con

cent

ratio

n (g

/L)

0

1

initial in proteosefinal in proteoseinitial in MBBMfinal in MBBM

- - 42

4.1.3 Induction of Astaxanthin Accumulation

4.1.3.1. Heating

H. pluvialis was cultivated in mixotrophic condition on proteose-

peptone media with optimal light intensity 60 µE/(m2·s) and combinations of

temperature 20℃ and pH 7.5 conditions. After cell was cultivated until

maximum concentration, heating cultivation was carried out in 30℃and the

other condition is same. Unfavorable condition, heating for induced

astaxanthin accumulation in cell (Figure 4.7).

Figure 4.7. Heating effect on astaxanthin accumulation

; control: grow in 20℃

; heating: transferred to 30℃ after 6 days in 20℃

CONTROL HEATING

Ast

axan

thin

con

cent

rati

on (

mg/

l)

0

5

10

15

20

25

- - 43

4.1.3.2. Light Intensity

The optimal light intensity for astaxanthin accumulation was different from

that for cell growth (Figure 4.1), suggesting that a two-stage culture may be

the best way to produce astaxanthin in commercial scale . As shown in Figure

4.4, the effect of light intensity was distinct in different light intensities. For

low light intensity of 0 ~ 30 µE/(m2·s) (�, � and q in Figure 4.5),

astaxanthin formation or accumulation was very low. For this regime of light

intensity, both the cell growth rate and astaxanthin formation rate were not

desirable. A little more statistical analysis (ANOVA) revealed that astaxanthin

accumulation rate under medium light intensity of 60 ~ 90 µE/(m2·s)

(represented by s, ¢, and £) was 6 times higher than that under low light

intensities of 15 µE/(m2·s) (� in Figure 4.5 a)). However, astaxanthin

concentration and cell concentration did not show a strong correlation,

considering this middle light intensity regime was optimal for H. pluvialis

growth as described in the previous section (refer Figure 4.4). The highest

astaxanthin concentration of 6.5 mg/L was observed at 340 hr after

inoculation under a higher light intensity (160 µE/(m2· s), marked with ¿ in

Figure 4.5) and this astaxanthin concentration was over 10 times higher than

that under 15 µE/(m2· s).

- - 44

The differences in astaxanthin contents per cell under different light

intensities were a clear indicator of the light intensity effect on H. pluvialis

cultures (Table 4.2). The astaxanthin concentration (mg/L) under 160

µE/(m2·s) was about 60% higher than that under 60 µE/(m2·s). However, this

ratio increases to over 80% on per cell basis using the data on Table 4.2. This

data also suggests that using two-stage H. pluvialis cultures would be more

practical and efficient way, since each stage can be optimized separately for

cell growth and astaxanthin production.

On the other hand, the total chlorophyll concentration (= chlorophyll a +

chlorophyll b) showed different aspects (Figure 4.5 b)). Total chlorophyll

concentrations were higher (around 4.5 mg/L) at 60 and 75 µE/(m2·s)

(represented by s and ¢, respectively) , while the chlorophyll concentration

profiles of the rest were roughly in the same range of 2.0 ~ 3.0 mg/L (15, 30,

90, and 160 µE/(m2·s), marked by �, q, £, and ¿). One interesting

observation is that the final cell concentration (Figure 1) seemed to be a close

function of the total chlorophyll (Figure 4.5 b)). The higher the cell

concentration, the higher the total amount of chlorophyll and vice versa.

However, the chlorophyll contents per cell, which can serve as a viability

parameter of microalgae, gave other information on the experiment. Though

the profiles of chlorophyll contents per cell were similar in all cases, there

- - 45

seemed to be a trend that the total astaxanthin concentration is inversely

proportional to the chlorophyll contents per cell (Figure 4.5).

- - 46

a)

b)

Figure 4.8. Comparison of per cell size distribution under various light

intensities. A. After 96 hr of cultivation; B. After 336 hr of

Volume (µm3)

10 100 1000

Dif

fere

ntia

l Cel

l Num

ber

0

100

200

300

400

500

600

700

Dark1560160

B

Volume (µm3)

10 100 1000

Dif

fere

ntia

l Cel

l Num

ber

0

50

100

150

200

250

300

350

Dark1560160

A

- - 47

cultivation.

4.1.3.3. Light Quality

The effect of colored lights on astaxanthin production by H. pluvialis was

also investigated. The algae were cultivated under 15 µE/(m2·s) light intensity

from fluorescent light with supplements of 470 or 680 nm photons from LEDs.

As shown in Figure 4.9, supplements of blue or red lights affected the cell

growth to a great extent. As expected from the results in the previous section,

the cell growth under 15 µE/(m2· s) of fluorescent light (marked by � in Figure

4.9) was not satisfactory. However, the poor growth under the low light

condition could be improved significantly by supplementing small amount of

blue (� in Figure 4.9) or red (q) light. Supplement of blue light showed

more drastic effect than the supplement of red light. Considering the even

lower intensity of supplemented colored light, 0.1 µE/(m2·s), the improvement

was much higher than expectation. The average cell concentration with blue

light supplement was almost twice than that without supplement and this

result was very stimulating in optimization of astaxanthin production by

Haematococcus.

More interesting results were obtained in astaxanthin concentration (Figure

4.10 A). Total astaxanthin concentration (mg/L) with the supplement of blue

- - 48

light (� in Figure 4.10 A) was increased over 2 times of that without

supplement. Even though the cell concentration was higher with blue light

supplement, astaxanthin contents per cell (pg/cell) with blue light

supplements were also 1.6 – 2.4 times higher than the contents without

supplement. Red light supplement also affected astaxanthin contents per cell

and astaxanthin contents with red light supplements were 1.3 ~ 1.5 times

higher than the contents without supplement. Kobayashi et. al. also reported

the enhancement of blue light on astaxanthin production using filtered

fluorescent light (did not use blue light as supplement). Our results here

showed that only a small amount of blue light was necessary to exploit the

blue light phototaxis of Haematococcu pluvialis.

The profiles of total chlorophyll concentration (mg/L) exhibited similar

trends as the total astaxanthin concentration and the profiles of chlorophyll

contents per cell (pg/cell) were again almost identical in all cases. The cell

size analysis by Coulter Counter showed similar distributions regardless of

the supplement of colored light. Figure 4.11 represented the cell size

distribution histograms at 228 hr.

As a conclusion, supplement of small amount of colored light can increase

the astaxanthin productivity to a great extent without affecting the cell cycle.

Although more detailed study is required to optimize astaxanthin production

- - 49

in H. pluvialis, the results reported in this paper is stimulating and promising

for commercialization process.

- - 50

Figure 4.9. Effect of different light sources on cell growth. Growth curves under

fluorescent lamp only (-�-); under fluorescent lamp supplemented with 470 nm (-�-

); under fluorescent lamp supplemented with 680 nm (-q-).

Time (hr)

0 100 200 300 400

Cel

l Con

cent

rati

on (

g/L

)

0.0

0.5

1.0

1.5

2.0

470 nm + FluorescentFluorescent680 nm + Fluorescent

- - 51

Figure 4.10. Pigments concentration profiles under different light sources. A.

astaxanthin under fluorescent lamp only (-�-); under fluorescent lamp supplemented

with 470 nm (-�-); under fluorescent lamp supplemented with 680 nm (-q-).

Time (hr)

0 100 200 300 400

Ast

axan

thin

Con

cent

rati

on (

mg/

L)

0.0

0.2

0.4

0.6

0.8

1.0A

- - 52

Figure 4.11. Comparison of per cell volume distribution under different light sources

at 228 hr of cultivation.

Volume (µm3)

10 100 1000

Dif

fere

ntia

l Cel

l Num

ber

0

50

100

150

200

250

300

470 nm + FluorescentFluorescent680 nm + Fluorescent

- - 53

4.1.3.4. Nutrients Deficiencies

Various combination of nutrient deficient media such as control medium,

without NaNO3, with 10 % NaNO3, without peptone, with 50 % peptone,

without NaNO3, with 50% peptone, without peptone with NaNO3, without

NaNO3 peptone were examined for effects of nutrient deficiencies such as

nitrogen and carbon source in proteose-petone medium (Table 4.3.). In control

medium, cell number was 0.98 ×106 cells/mL, cell concentration was 1.7 g/L,

astaxanthin concentration was 2.57 mg/L and astaxanthin content per cell was

1.53 mg/g cell. With no NaNO3 medium, cell number was 0.28 ×106

cells/mL, cell concentration was 0.02 g/L, astaxanthin concentration was 1.92

mg/L, and astaxanthin content per cell was 9.6 mg/g cell. In without peptone

medium, cell number was 0.51 ×106 cells/mL, cell concentration was 0.7g/L,

astaxanthin concentration was 1.99 mg/L, and astaxanthin content per cell was

2.8 mg/g cell. In withou NaNO3 and peptone medium, cell number was 0.26

×106 cells/mL, cell concentration was 0.17 g/L, astaxanthin concentration

was 0.24 mg/L, and astaxanthin content per cell was 1.4 mg/g cell (Table 4.3).

Although in nitrogen deficient media, cell growth and cell division was

inhibited compare to in control media but carotenoids formation and increase

of cell size was stimulated.

- - 54

Table 4.3. Results of nutrient deficient Media

Cell Number (cell/mL)

Cell Conc. (g/L)

Asta. Conc. (mg/L)

Asta. per Cell ( mg/g cell)

Control (proteose medium)

0.98 × 106 1.7 2.57 1.53

Without N 0.28 × 106 0.2 1.92 9.6

Without P 0.51 × 106 0.7 1.99 2.8

Without N,P 0.26 × 106 0.17 0.24 1.4

4.1.3.5. Scale up

After considerations of photobioreactor advantages and disadvantage,

bubble column photobioractor was selected reasons for easiness of operation,

scale up, weak shear stress and high cell yield. Culture condition was 400 mL

working volume, 0.2 vvm air flow, light intensity of 40, 60, 80 µE/(m2·s) and

proteose-peptone and MBBM media was tested for photobioreactor

cultivation. Optimal light intensity was 60 µE/m2·s (Figure 4.12.), astaxanthin

concentration was 2.8 mg/L and carotenoids formation was stoped after 8

days cultivation. As shown in Figure 4.12., the growth rate was increased

dramatically in bubble column photobioreactors. This enhancement was due

to better mass transer and better light delivery in bubble column

photobioreactors than those in flasks (Figure 4.13, 4.14).

Even though cultivation time was reduced of cultivation and carotenoids

- - 55

formation was increased in photobioreactors, higher density Haematococcus

pluvialis cultures must be achieved for commercialization process for

astaxanthin production. Two stage culture systems where cell growth stage

and astaxanthin formation stage are separated, could be the solution.

Figure 4.12. Designed bubble column photobioeactor

- - 56

Figure 4.13. Cell growth in photobioreactor with various light intensities

0.E+00

1.E+05

2.E+05

3.E+05

4.E+05

5.E+05

6.E+05

7.E+05

8.E+05

9.E+05

0 24 48 72 96Time(hr)

Cell

Num

ber (c

ells

/mL)

40

60

80

M60

- - 57

Figure 4.14. Astaxanthin concentration in photobioreactor under various light

intensity

0

0.5

1

1.5

2

2.5

3

0 24 48 72 96

Time(hr)

Ast

axa

nth

in C

onc. (m

g/L

)

40

60

- - 58

4.1.3.5. Astaxanthin Stability

Astaxanthin is very sensitive to heat, oxygen and light, thus, stability

process for final astaxanthin product should be examined. In this

experiment, sensitivity to light was examined under various light intensities

and light qualities with synthetic astaxanthin and natural astaxanthin.

Natural astaxanthin was found to be more stable than synthetic astaxanthin.

As shown in Figure 4.13., the degradation rates of natural astaxanthin were

relatively the same regardless of the exposed light intensity. However, the

degradation rates of synthetic astaxanthin found to be a function of exposed

light intensity. As a result, astaxanthin produced by H. pluvialis produced to

be far more stable than synthetic astaxanthin and thus biologically produced

astaxanthin has a huge advantages over synthetic one when commercialized

- - 59

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

S 0

.1

S 5

.8

S 1

5.9

S 30.9

S 56.6

S 1

63.4

S U

V C

S b

ulb

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

N 0

.1

N 5

.8

N 1

5.9

N

30.9

N

56.6

N 1

63.4

N

UV C

N b

ulb

Figure 4.15. Comparison instability to light intensities of synthetic astaxanthin and

natural astaxanthin

- - 60

4.2. Astaxanthin Analysis

4.2.1 Pigment Extraction

• Homogenization and Solvent Extraction

Because Haematococcus pluvialis has thick cell wall, extraction of

intracellular pigments is very difficult. Acetone is widely used for carotenoids

extraction and DMSO is used to increrase permeability. These two solvents

were examined for astaxanthin extraction after homogenization with specially

designed homogenizer with various homogenize time (1, 3, 5, and 10 min).

The amounts of extracted astaxanthin by acetone were higher than those by

DMSO. Besides, the extracted astaxanthin by acetone found to be more stable

(Figure 4.16.).

- - 61

Acetone ; O. D ×10 DMSO ; O.D ×100

Figure 4.16. Pigment extraction in different solvent with various time

0

2

4

6

8

10

12

14

A30V

A50V(1

)

A50V(2

)

A50V(3

)

50V(A

ver)

A90V

D30V

D50V

O.D

1

3

5

10

- - 62

4.2.2. TLC-plate and TLC-FID

Thin layer chromatography plate is well known for qualitative analysis

method. To analyze astaxanthin concentration in Haematococcus pluvialis,

TLC-plate was used. However, this TLC method was not very useful for

astaxanthin quantification. The quantification method is shown in Figure 4.17.

- - 63

• TLC plate No.: silica gel 60 F254 (Merck) ; 1.05715

Figure 4.17. Analysis method of TLC-plate and TLC-FID

Sample preparation

Separate pigments in

Ethanol:Hexane:Water

( 2 : 3 : 2 )

Apply on TLC-plate

(Acetone:Ether) or

Apply on TLC-FID (Acetone)

Collect separated pigment

Evaporation

Collect solvent layer

Purification

- - 64

Figure 4.18. Standard curve by TLC-FID

Y = 1.163 × X + 4.176

r2 = 0.945

Rf = 0.46

Astaxanthin (µg)

0.1 1 101e+3

1e+4

1e+5

1e+6

1mg Asta./1ml Acetone1mg Asta./ 1ml A2.5mg Asta./ 1ml A3mg Asta./1ml A5mg Asta./1ml A5mg Asta./ 1ml A

Plot 1 Regr

- - 65

4.2.3. Spectrophotometer

Spectrophotometer analysis method is very easy and simple. After

calculation of chlorophyll and other carotenoids in Haematococcus pluvialis,

astaxanthin equation and chlorophyll a, b equation were determined.

• Astaxanthin Assay

1) weigh approximately 30 ml of cell broth into the 50ml centrifuge tube

and record the weight.

2) add 3 g of glass beads to the tubes.

3) add 5ml of DMSO to the centrifuge tubes, place in pre-heated water

bath at 45 ~ 50℃ for 30 min. Vortex 30 sec per 10 min.

4) centrifuge at 3000 rpm for 10 min. to pellet cell material.

5) pipette the supernatant and collect in the tubes.

6) add 5ml of acetone to the centrifuge tube, vortex for 30 sec. And

centrifuge at 3,000 rpm for 10 min. to pellet cell material. transfer the

supernatant and collect in the tubes.

7) if supernatant still has color, repeat step 6

8) all supernatant is collected and bring the 25 ml acetone.

9) gently mix and centrifuge at 3000 rpm for 10 min. to pellet remainder.

10) measurement of O.D at 475 nm.

- - 66

Figure 4.19. Astaxanthin standard curve with specatrophotometer

Astaxanthin conc. = 0.0045 ×OD 475 – (calculated blank conc.)

r2 = 0.997

- - 67

4.2.5. HPLC

More quantitative and qualitative measurements of astaxanthin were

performed by a HPLC (Younglin Instrument Co., Anyang, Korea) with two

M930D pumps and a M730D photodiode array (PDA) detector. A revered-

phase C18 column (300×3.9 mm; 5µm, Waters Co., Milford, MA, USA) was

chosen for separation and analysis of extracted pigments. For better resolution

between astaxanthin and other pigments, a gradient procedure with two

solvents was introduced: solvent A (dichloromethane : methanol :

acetonitrile : water = 5 : 85 : 6 : 4 v/v) and solvent B (22 : 28 : 6 : 4 v/v).

Chromatographic peaks for astaxanthin were observed at a 480 nm as reported

earlier ([55]).

- - 68

5. Recommendation 5.1. Photobioreactor Design

Considerations of factors for photobioreactor design (Table 5.1, Table 5.2),

bubble column photobioreactor was designed and operated to solve difficulty

of mass transfer in flask cultures for high-density Haematococcus pluvialis

cultures. Designed bubble column photobioreactor was consisted of two

fluorescent light lamp as a light source, transparent glass column, and pumps

for supplying air to the column. Light intensity was about 60 µE/(m2· s) on

the surface of each column. Air was injected to the bottom of the glass

columns at a constant rate of 0.2vvm through flow meters. Temperature was

kept 20 to 25℃.

- - 69

Table 5.1. Comparison of characteristics of various algal culture systems

Capital cost Running cost Cell yield Reliability

Shallow ponds Medium Low Low Variable

Raceways High Higher Higher Good

Cascade systems Medium Medium Higher Good

Tubular systems High-Very high High High-Very

high Very good

Fermenters Very high Very high Very high Very good

- - 70

Table 5.2. Astaxanthin yields obtained by various authors

Yield Author

(pg/cell) (mg/L) (mg/g DCW) Notes

Lee and Soh 1991 - - 40-43 Chemostat Yong and Lee 1991 - 7-8 -

Boussiba and Vonshak 1991

60-100 - 17-19

Kobayashi et al. 1992 - 7.5 10-15 Grung et al. 1992 - - 7

Kobayashi et al. 1993 40-50 - - Zlotnik et al. 1993 77 30 days

Tjahjono et al. 1994 200-250 - - Elevated temperature

Lee and Ding 1995 30.6 3.9 7.3 Fan et al. 1995 - - 32 Chaumont and Thepenier 1995

- - 13.8 Tubular PBR

Harker et al. 1996 - 27.5 - PBR, 80 days Chumpolkulwong et

al. 1997 100 - - Compactin mutant

Kobayashi et al. 1997c

30 9 - Light independence

Grunewald et al. 1997

40-70 - - In flagellates

- - 71

Figure 5.1. Recommended two stage photobioreactor

N rich 5% CO2 W/o N

- - 72

5.2. Process Design for Astaxanthin Production

Factors considerations for astaxanthin production systems such as

inoculation system, vegetative growth systems, astaxanthin accumulation

systems, harvesting systems, drying systems and product storage systems and

economic factors for commercialization.

- - 73

Table 5.3. Advantage and disadvantage of different harvesting methods[2]

Method Reliability Energy requirement Quality for conversion

Centrifugation good high good

Chemoflocculation good high poor

Sandfiltration fair low poor

Ultrafiltration good high good

Microstraining poor low poor

Bioflocculation poor low good

- - 74

Vegetative cell

aplanospore

for astaxanthin

Figure 5.2. Proposed flow chart of astaxanthin production system

Inoculation system

First Stage Photobioreactor

Vegetative cell cultures

( high density algal cultures )

Second Stage

Photobioreactor

Astaxanthin accumulation

Cell Separation

Harvest System Drying

Cracking System Extraction

Purification

Feeds, Nutraceuticals Pharmaceuticals,

Foods, Cosmetics

- - 75

6. CONCLUSIONS 6.1. Conclusions

This study has dealt with optimal cultivation of Haematococcus pluvialis

for astaxanthin production and the specific conclusions drawn in this study are

as follows;

• Haematococcus pluvialis has 4 stages cell cycle and dramatic

morphological change.

• pH 7 and temp 20 ~ 25℃was optimal culture condition for

Haematococcus pluvialis growth.

• proteose-peptone medium was superior medium.

• 60 µE/(m2· s) light is optimal light intensity for cell growth.

• heating cultivation transfer to 30℃, stimulate astaxanthin

accumulation 183%.

• higher light intensity stimulate carotenoids formation for cell

protection.

• blue light 470 nm, LEDs stimulate cell growth and astaxanthin

formation.

• nutrient deficient media such as carbon and nitrogen, astaxanthin

content per cell was increase.

- - 76

• bubble column photobioreactor reduced time of vegetative cell

cultivation and carotenoids formation by rapid mass transfer rate.

- - 77

6.2. Future Work

• complete pigments separation

• rise astaxanthin stability

• quantitative analysis

• separated astaxanthin purification

• design and operation of large scale photobioreactor

• genetic engineering

- - 78

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