Haematococcus pluvialis is a microalgae that is one of the best natural sources of the carotenoid astaxanthin. It can be cultivated using different systems, including outdoor ponds or indoor photobioreactors. H. pluvialis is usually cultivated using a two-phase system, with a green growth phase under normal conditions followed by a red stressed phase to induce astaxanthin accumulation. Photobioreactors allow for better control of cultivation parameters but have higher costs than open ponds. Astaxanthin is then extracted from the microalgae biomass using organic solvents.

1. Astaxanthin

2. Source of Astaxanthin
• The natural sources of astaxanthin are algae, yeast, salmon, trout,
krill, shrimp and crayfish. The commercial astaxanthin is mainly from
Phaffia yeast, Haematococcus and through chemical synthesis.
Haematococcus pluvialis is one of the best sources of natural
astaxanthin.
• Concentrated biomass of the yeast Phaffia
rhodozyma (ATCC 74219), killed,
containing at least 4.0g astaxanthin per
kilogram.
Phaffia rhodozyma

3. Sources Astaxanthin (%) on the Dry Weight Basis
Chlorophyceae
Haematococcus pluvialis 3.8
Haematococcus pluvialis (K-0084) 3.8
Haematococcus pluvialis (Local isolation) 3.6
Haematococcus pluvialis (AQSE002) 3.4
Haematococcus pluvialis (K-0084) 2.7
Chlorococcum 0.2
Chlorella zofingiensis 0.001
Neochloris wimmeri 0.6
Ulvophyceae
Enteromorpha intestinalis 0.02
Ulva lactuca 0.01
Florideophyceae
Catenella repens 0.02
Alphaproteobacteria
Agrobacterium aurantiacum 0.01
Paracoccus carotinifaciens (NITE SD 00017) 2.2
Tremellomycetes
Xanthophyllomyces dendrorhous (JH) 0.5
Xanthophyllomyces dendrorhous (VKPM Y2476) 0.5
Labyrinthulomycetes
Thraustochytrium sp. CHN-3 (FERM P-18556) 0.2
Malacostraca
Pandalus borealis 0.12
Pandalus clarkia 0.015
Microorganism sources of astaxanthin

4. Haematococcus pluvialis Chlorococcum Chlorella zofingiensis Enteromorpha intestinalis
Ulva lactuca Catenella repens Xanthophyllomyces dendrorhous Pandalus borealis

5. Cultivation of Haematococcus pluvialis (1)
• H. pluvialis is mainly cultured in two-phase cultivation systems.
• The first phase, known as the green phase or growth phase, is
optimised for vegetative growth to achieve a high cell density. In
suspended cultivation, a maximum light intensity of 150 µmol
photons m-2 s-1 should not be exceeded in order to maintain cell
growth and divisions, and environmental parameters such as
temperature, carbon dioxide (CO2 ) levels, and pH need to be closely
monitored.
• As the required biomass is attained, the second phase, known as the
stressed or red phase, is switched on to stimulate astaxanthin
accumulation.

6. Cultivation of Haematococcus pluvialis (2)
• In the two-phase system, each growth phase requires different cultivation
conditions and technologies, high energy consumption, and prolonged cultivation
time.
• Currently, suspended cultivation of H. pluvialis is more common for the
production of astaxanthin at the commercial scale. Suspended cultivation is
applied in open ponds or closed photobioreactors.
• Open-pond cultivation is utilised only for the stressed phase with a short
cultivation time (4-6 days) to minimise contamination and apply stressed
conditions.
• The closed photobioreactor can minimise contamination and control culture
parameters better but it has drawbacks such as of high assembly and
maintenance cost. Moreover, suspended systems have very low biomass
concentration (0.05-0.06% of cultivated liquid) and the harvest of algae thus
demands additional costs of energy and labour.

7. System Strain Medium
Temp
(°C)
CO2 (%)
Light
condition
(µmol photon
m
-2
s
-1
)
Stress factor
Cultivation
period (green
phase + red
phase) (days)
Astaxanthin
content
(% dried
biomass)
Astaxanthin
productivity
(mg l
-1
day
-1
)
Astaxanthin
productivity (mg
m
-2
day
-1
)
Dried biomass
productivity (g
m
-2
day
-1
)
Outdoor tube (50 l) Isolated BG11 25
For controlling
pH
Sunlight
400-1600 Intense light 4 (Red phase) 3.6 7.2 136.8 3.8
Outdoor open pond ZY-18 NIES-N 28 None Sunlight
Max. 1000
Intense light
+ N limited
20 (Green phase
+ red phase)
1.7 -/- 40 2.34
Indoor open pond 26 BG11 20
For controlling
pH
20-350
14/10 hour Intense light
12 (Green phase
+ red phase) 2.79 4.3 61 2.2
Indoor bubble
column
ZY-18 NIES-N 28 None 250
Continuous
Intense light
+ N limited
12 (Green phase
+ red phase)
3.6 -/- 237.6 6.6
Indoor bubble
column (0.5 l) K-0084
Modified
BG11 25 1.5
350
Continuous
Intense light
+ N limited 5 (Red phase) 4.0 11.5 528 13.2
Indoor closed
container (10 l)
HB
(isolated)
Modified
RM
25
For controlling
pH 85
16/8 hour
Intense light, N
limited, high
C/N, +
bicarbonate
30 (green phase)
+ 3 (Red phase)
4.88 2.75 92 1.88
Indoor bubble
column (5 l) -/- RM 25 40 ml/min
60
16/8 hour
N limited,
High C/N
22 (Green phase
+ red phase) -/- 0.009 0.264 -/-
Indoor immobilised
biofilm (0.08 m
2
) NIES-144 NIES-N 25 None
150
Continuous N limited
12 (Green phase
+ red phase) 1.3 -/- 65.8 3.7
Indoor immobilised
biofilm (0.08 m
2
) SAG 34-1b BG11 25 1.5
100
Continuous
N limited or
exhausted
7 (Green phase +
red phase) 2.2 -/- 143 6.5
Indoor immobilised
biofilm (0.05 m
2
)
CCAC
0125
Modified
BG11 26 1
650
14/10 hour
Intense light
+ N, P limited
7 (Green phase +
red phase) 3.5 -/- 371 10.6
Indoor angled
immobilised biofilm
(0.05 m
2
)
CCAC
0125
Modified
BG11
26
For controlling
pH 600-700
14/10 hour
Intense light
+ N, P limited
10 (Green phase
+ red phase)
3.0 7.2 360 12
Indoor angled
immobilised biofilm
(2 m
2
)
CCAC
0125
Modified
BG11 26
For controlling
pH
600-700
14/10 hour
Intense light
+ N, P limited
10 (Green phase
+ red phase) 2.8 6.3 315 11.25
Comparison of H. pluvialis cultivation results on an angled biofilm-based photobioreactor system with other cultivation system based on surface area.

8. Pathway of (3S-3′S)-astaxanthin biosynthesis in H.pluvialis.

9. Culture Conditions and Requirements for
Cell Growth and Astaxanthin Formation
• Optimization of the various culture parameters, such as growth medium composition, light, pH, temperature
etc. is necessary to achieve high biomass and astaxanthin production.
• Various types of growth media are used for cultivation of H.pluvialis. The most commonly used media are
BG-11, BBM, OHM; KM1-basal medium with organic carbon sources in the form of sodium acetate, and their
modifications.
• Sodium nitrate was found to be the most optimal inorganic nitrogen source, alternatively an organic source
such as urea can be used.
• The suitable temperature for the growth and astaxanthin accumulation of H.pluvialis is between 20 and
28◦C.
• In terms of biomass and astaxanthin production optimal pH is within the range of 7.00–7.85.
• The typical irradiation for H.pluvialis cultivation ranges between 40 and 50 μmol photons m−2s−1
• During vegetative stage cultivation of H.pluvialis, the regular cycles of alternating light and dark 12:12 or
16:8 h are often used.
• Regarding the type of illumination, the highest carotenoid content was obtained by using a continuous PAR
lighting

10. Culture Systems
• Astaxanthin-producing H.pluvialis is capable of growing in
photoautotrophic, heterotrophic, or mixotrophic growth conditions in
indoors, open raceway ponds or closed photobioreactors in batch, fed
batch, or continuous modes.

11. Photoautotrophic Culture
• Photoautotrophic culture of H. pluvialis is mainly carried out in open raceway ponds or
closed photobioreactors. Typical photobioreactors used for its cultivation include tubular,
bubble column and airlift photobioreactors.
• As the culture conditions for maximum growth and maximum astaxanthin content are
mutually exclusive, a two-step cultivation strategy is commonly adopted for the
commercial production.
• The first step, green vegetative growth phase (“green stage”) is to promote algal growth
under favorable culture conditions (e.g., low light and nitrogen-replete).
• When high cell density is reached, the culture enters into the second step, reddish
inductive production phase (“red stage”), where algal cells are subjected to stress
conditions such as high light intensity and nitrogen depletion, excess acetate addition, pH
or salt stress, phosphate deficiency, or the addition of specific cell division inhibitors.
• The reported biomass productivities in green stage and red stage ranged from 0.01
to 0.5 g L−1 d−1 and 0.01 to 4.8 g L−1 d−1
• Astaxanthin productivity and astaxanthin content ranged from 0.44 to 21 mg L−1 d−1
and 0.8 to 4.8% of DW

12. Heterotrophic and Mixotrophic Culture (1)
• Under heterotrophic growth conditions light is not needed as organic substrates
serve as carbon and energy sources for growth and synthesis of secondary
metabolites.
• Various types of organic carbon sources have been used for heterotrophic
cultivation and induction using acetate has been found effective for
Haematococcus encystment and initiation of astaxanthin production.
• However, unlike many microalgae in which oversupply of easily accessible carbon
in combination with nitrogen limitation yields diversion of the carbon flux toward
lipid accumulation H. pluvialis grows at a relatively low rate (0.22 d−1) and
accumulates negligible amount of astaxanthin, too low to be considered for
commercial scale production if single step cultivation is considered.
• Additionally, heterotrophic cultivation of Haematococcus increases the risk of
bacterial or fungal contamination .

13. Heterotrophic and Mixotrophic Culture
• H. pluvialis can be also produced indoors mixotrophically employing an organic acid (e.g.,
acetate) or carbohydrates as an additional carbon and energy source.
• Studies have shown that both growth and astaxanthin production can be enhanced
under mixotrophic culture conditions. A final cell density of 0.9–2.65 g L−1 and a
maximum astaxanthin content of 1–2% DW were obtained from mixotrophic cultures of
H. pluvialis
• Heterotrophic culture was used in the green stage to produce algal biomass,
while astaxanthin production was induced in photoautotrophic culture conditions. The
induction of astaxanthin accumulation was performed under nitrogen deprivation
conditions and whilst using bicarbonate or CO2 as carbon sources. As a result, a very high
cellular astaxanthin content of 7% (DW) was achieved, 3.4-fold higher than
heterotrophic induction whilst astaxanthin productivityof 6.25mg L−1 d−1 was obtained.
• Photoautotrophic induction of astaxanthin production in H. pluvialis is more effective
than heterotrophic one. But, heterotrophic and mixotrophic culture modes are less cost-
effective than the photoautotrophic one for Haematococcus mass culture.

14. Summary of various methods of H. pluvialis biomass cultivation and corresponding astaxanthin productivities.

15. Summary of astaxanthin extraction methods from H. pluvialis.

16. Scheme of two stage cultivation H. pluvialis biorefinery producing high value compound
astaxanthin and either edible oil or biofuel compound-biodiesel.

17. Company Name Country Brand Name Product particulars
Cyanotech Corporation (www.cyanotech.com) USA BioAstin® Astaxanthin extract packaged in soft gel, beadlets; dietary supplement
NaturoseTM Algae meal; pigmentation source for ornamental fish and animals
Mera Pharmaceutigals Inc. (www.merapharma.com) USA AstaFactor® Astaxanthin packaged as soft gel; dietary supplement
Stazen Inc. (www.stazen.com) USA Stazen® Dietary supplement containing algae crushed and dried algae meal
Valensa International (www.valensa.com) USA Zanthin® Astaxanthin extract, soft gel, beadlets
AIgatechnologies Ltd. (www.algatech.com) Israel AstaPureTM Dry algal biomass, astaxanthin beadlets, and oleoresin
Fuji Chemical Industry Co. Ltd. (AstaReal Co Tld)
(www.fujichemical.co.jp; www.astareal.com)
Japan, Sweden, USA AstaREAL® Astaxanthin oleoresin products, water dispersible, and soluble powders
BioReaI (Sweden) AB (subsidiary of Fuji Chemical)
(www.bioreal.se)
Sweden AstaXine®
AstaCaroxe®
Dietary supplement containing algae crushed, and dried algae meal
AstaEquus® Astaxanthin extract feed supplement for horses
Novaasta® Astaxanthin extract feed supplement for animals
Britannia Health Products Ltd. (www.britanniahealth.co.uk) UK Britaxan® Astaxanthin complex with other carotenoids packaged as capsule- dietary supplement
Supreme Biotechnologies NZ Ltd.
(www.supremebiotech.com)
New Zealand AstaSupreme® Algal biomass, Oleoresins, beadlets, and soft gels of Astaxanthin
Atacama Bio Natural (www.atacamabionatural.com) Chile Supreme Asta OilTM
Supreme Asta powderTM
Oleoresin for food, nutraceutic, and cosmetic products, and powder for animal feed
supplement
Jingzhou Natural Astaxanthin Inc. (www.asta.cn) China NaturAstaTM Dry algal biomass and astaxanthin soft gel
Kunming Biogenic Co. Ltd. (www.bgenic.com) China AstaBio® Algal biomass, oleoresins, beadlets, and soft gels of Astaxanthin
Beijing Ginko Group (BGG) Biological Technology Co. Ltd.
(www.gingkogroup.com.cn)
China AstaZine® Astaxanthin oil, powder, and beadlets
Wefirst Biotechnology Co. Ltd. (www.astawefirst.com) China AstaFirstTM Dried algal powder, Astaxanthin oleoresin, and soft gel
Algaetech International SDN BHD (www.algaetech.com.my) Malaysia AstaxanthinPremia-Ex Algae biomass, oleoresin, and soft gel
Parry Nutraceuticals Ltd. (EID Parry) (www.eidparry.com/) India Zanthin® Astaxanthin oleoresins, beadlets, and soft gel
Leading commercial companies and their H.pluvialis-derived astaxanthin and related products in the world market.

18. Indoor, Controlled Cultivation System
www.algalif.com

19. Astaxanthin Production Process
www.algamo.cz

20. References
• Ambati, R. R., Phang, S. M., Ravi, S., & Aswathanarayana, R. G. (2014). Astaxanthin: sources,
extraction, stability, biological activities and its commercial applications--a review. Marine drugs,
12(1), 128–152. https://doi.org/10.3390/md12010128
• Tran, H., Do, T., Le, T., Nguyen, M., Pham, C., & Melkonian, M. (2019). Cultivation of
Haematococcus pluvialis for astaxanthin production on angled bench-scale and large-scale
biofilm-based photobioreactors. Vietnam Journal Of Science, Technology And Engineering, 61(3),
61-70. Retrieved from https://vietnamscience.vjst.vn/index.php/VJSTE/article/view/248
• Shah MMR, Liang Y, Cheng JJ and Daroch M (2016) Astaxanthin-Producing Green Microalga
Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Front. Plant Sci.
7:531. doi: 10.3389/fpls.2016.00531
• European Commission (2002) Report of the Scientific Committee for Animal Nutrition on the use
of astaxanthin-rich Phaffia rhodozyma in feedingstuffs for salmon and trout. Available at:
<https://ec.europa.eu/food/sites/food/files/safety/docs/animal-feed_additives_rules_scan-
old_report_out76.pdf>

21. • Process System Engineering,
• Process Synthesis and Design,
• Process Modelling and Simulation,
• Process Integration and Pinch Analysis,
• Process Optimization,
• Techno-Economic Analysis,
• Sustainability Assessment,
• Data Analytics,
• Machine Learning