REVIEW

published: 28 April 2016

doi: 10.3389/fpls.2016.00531

Astaxanthin-Producing Green
Microalga Haematococcus pluvialis:
From Single Cell to High Value
Commercial Products
Md. Mahfuzur R. Shah 1 , Yuanmei Liang 1 , Jay J. Cheng 1, 2 and Maurycy Daroch 1*
1
School of Environment and Energy, Peking University, Shenzhen Graduate School, Shenzhen, China, 2 Department of
Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC, USA

Many species of microalgae have been used as source of nutrient rich food, feed,
and health promoting compounds. Among the commercially important microalgae,
Haematococcus pluvialis is the richest source of natural astaxanthin which is considered
as “super anti-oxidant.” Natural astaxanthin produced by H. pluvialis has significantly
greater antioxidant capacity than the synthetic one. Astaxanthin has important
applications in the nutraceuticals, cosmetics, food, and aquaculture industries. It is
now evident that, astaxanthin can significantly reduce free radicals and oxidative
stress and help human body maintain a healthy state. With extraordinary potency
Edited by:
Flavia Vischi Winck,
and increase in demand, astaxanthin is one of the high-value microalgal products of
University of São Paulo, Brazil the future.This comprehensive review summarizes the most important aspects of the
Reviewed by: biology, biochemical composition, biosynthesis, and astaxanthin accumulation in the
Rosana Goldbeck,
cells of H. pluvialis and its wide range of applications for humans and animals. In
Universidade Estadual de Campinas,
Brazil this paper, important and recent developments ranging from cultivation, harvest and
Xianhai Zeng, postharvest bio-processing technologies to metabolic control and genetic engineering
Xiamen University, China
are reviewed in detail, focusing on biomass and astaxanthin production from this
*Correspondence:
Maurycy Daroch
biotechnologically important microalga. Simultaneously, critical bottlenecks and major
m.daroch@pkusz.edu.cn challenges in commercial scale production; current and prospective global market
of H. pluvialis derived astaxanthin are also presented in a critical manner. A new
Specialty section:
This article was submitted to
biorefinery concept for H. pluvialis has been also suggested to guide toward economically
Plant Biotechnology, sustainable approach for microalgae cultivation and processing. This report could serve
a section of the journal as a useful guide to present current status of knowledge in the field and highlight key
Frontiers in Plant Science
areas for future development of H. pluvialis astaxanthin technology and its large scale
Received: 31 October 2015
Accepted: 04 April 2016 commercial implementation.
Published: 28 April 2016
Keywords: Haematoccoccus pluvialis, astaxanthin, nutraceuticals, algae cultivation and processing, biorefinery
Citation:
Shah MMR, Liang Y, Cheng JJ and
Daroch M (2016)
Astaxanthin-Producing Green
INTRODUCTION
Microalga Haematococcus pluvialis:
From Single Cell to High Value
“Green microalgae” comprise more than 7000 species growing in a variety of
Commercial Products. habitats. Haematococcus pluvialis (Chlorophyceae, Volvocales) is unicellular fresh water
Front. Plant Sci. 7:531. microalga distributed in many habitats worldwide. It is considered as the best natural
doi: 10.3389/fpls.2016.00531 source of astaxanthin and the main producing organism of this commercial product

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Shah et al. Astaxanthin Production from H. pluvialis

(Lorenz, 1999; Ranga Rao et al., 2010). Astaxanthin (3,3′ - from H. pluvialis. Various cultivation and astaxanthin production
dihydroxy-ß-carotene-4,4′ -dione) is a bright red secondary methods in photoautotrophic, heterotrophic, and mixotrophic
carotenoid from the same family as lycopene, lutein, and growth conditions, both indoors; in open raceway ponds or
β-carotene, synthesized de novo by some microalgae, plants, closed photobioreactors using batch or fed-batch approach have
yeast, bacteria, and present in many of our favored seafood been reported (Kang et al., 2005, 2010; Kaewpintong et al.,
including salmon, trout, red sea bream, shrimp, lobster, and 2007; Ranjbar et al., 2008; García-Malea et al., 2009; Issarapayup
fish eggs (Higuera-Ciapara et al., 2006; Ranga Rao et al., et al., 2009; Zhang et al., 2009; Li et al., 2011; Han et al.,
2014). Astaxanthin contains two chiral centers and can exist 2013; Wang et al., 2013a,b). The most recent advances in
in three different stereoizomers, (3S, 3′ S); (3R, 3′ S), and (3R, H. pluvialis cultivation for astaxanthin production include a
3′ R). The ratio of 1:2:1 of these isomers is obtained during two-stage mixotrophic culture system (Park et al., 2014) and
chemical synthesis of this compound. H. pluvialis biosynthesizes an “attached cultivation” system using the immobilized biofilm
predominantly the 3S, 3′ S stereoisomer, the most valuable one (Zhang et al., 2014). Along with the cultivation process, the
(Yang et al., 2013; Al-Bulishi et al., 2015). Astaxanthin synthesis induction of carotenoid synthesis in H. pluvialis has a direct
in H. pluvialis is directly correlated in space and time with correlation with the astaxanthin content of cells and total
deposition of cellular reserves in lipid droplets under conditions astaxanthin productivity. The accumulation of astaxanthin is
of cellular stress (Solovchenko, 2015). From biochemical affected by both environmental factors such as light (Saha
perspective astaxanthin is synthesized through carotenoid et al., 2013; Park et al., 2014); temperature (Yoo et al., 2012);
pathway from glyceraldehyde-3-phosphate and pyruvate. Both pH (Hata et al., 2001); salt concentration (Kobayashi et al.,
of these compounds are products of photosynthesis and/or 1993); and nutritional stresses (Boussiba et al., 1999; Chekanov
glycolysis depending on cultivation conditions. These two key et al., 2014), as well as various plant hormones and their
metabolic intermediates then enter non-mevalonate (MEP) derivatives (Yu et al., 2015). Attempts were made to genetically
pathway to generate IPP—key intermediate for the synthesis of enhance the capacity of H. pluvialis to produce astaxanthin
all carotenoids including astaxanthin. Astaxanthin has a wide using both classical mutagenesis (Hu et al., 2008), and more
range of applications in the food, feed, cosmetic, aquaculture, recently genetic engineering (Sharon-Gojman et al., 2015).
nutraceutical, and pharmaceutical industries because of its free Once biomass is successfully grown and achieved high cell
radical scavenging capacity. In terms of antioxidant activity density, efficient harvesting, cell disruption, dehydration, and
astaxanthin is 65 times more powerful than vitamin C, 54 recovery/extraction of astaxanthin from H. pluvialis biomass
times stronger than β-carotene, 10 times more potent than are important factors. Harvesting have been so far achieved
β-carotene, canthaxantin, zeaxanthin, and lutein; and 100 times through a combination of passive settling and subsequent
more effective than α-tocopherol (Miki, 1991; Borowitzka, 2013; centrifugation (Han et al., 2013; Pérez-López et al., 2014), or
Koller et al., 2014; Pérez-López et al., 2014; Cyanotech, 2015). flotation and centrifugation (Panis, 2015). Mechanical processes
Currently, over 95% of the astaxanthin available in the market such as expeller pressing and bead milling are commonly used
is produced synthetically; while H. pluvialis derived natural cell disruption methods to enhance recovery of astaxanthin from
astaxanthin corresponds to <1% of the commercialized quantity H. pluvialis (Razon and Tan, 2011). For the dehydration of
(Koller et al., 2014). Synthetic astaxanthin, synthesized from H. pluvialis biomass, spray drying (Li et al., 2011; Panis, 2015),
asta-C15 -triarylphosphonium salt and the C10 -dialdehyde in freeze drying, lyophilization, and cryodesiccation (Milledge,
a Wittig reaction (Krause et al., 1997), has 20 times lower 2013) methods have been utilized. There are several methods
antioxidant capacity than its natural counterpart and to date of astaxanthin extraction utilizing solvents, acids, edible oils,
has not been approved for human consumption (Lorenz and enzymes, pressurized liquids (Jaime et al., 2010; Zou et al.,
Cysewski, 2000; Koller et al., 2014). Moreover, there are concerns 2013; Dong et al., 2014), supercritical carbon dioxide (SC-CO2 )
about the safety of using synthetic astaxanthin for direct human (Wang et al., 2012; Reyes et al., 2014), and liquefied dimethyl
consumption due to both different stereochemistry and potential ether (DME) (Boonnoun et al., 2014). Although many studies
carryover of synthesis intermediates. These concerns make have explored various methods of extraction and downstream
natural astaxanthin from H. pluvialis a preferred choice for high- processing of astaxanthin from H. pluvialis biomass, more
end markets (Li et al., 2011). In addition, H. pluvialis has been comprehensive investigation is required to take an advantage
already approved as a color additive in salmon feeds and as a of the biological potential of this microalga and its highly
dietary-supplement ingredient for human consumption in the valuable product. Since astaxanthin has a great potential in
USA, Japan, and several European countries (Yuan et al., 2011). the global market (280 mt, $447 million in 2014 for both
Nevertheless, there is no EFSA (European Food Safety Authority) synthetic and natural astaxanthin) and high market value
approval for the therapeutic application so far. Supercritical CO2 ($2500–7000/kg); (Koller et al., 2014; Pérez-López et al., 2014;
extracts from H. pluvialis have been granted “novel food” Status Industry Experts, 2015), in depth investigation of H. pluvialis
by the UK Food Standards Agency (FSA), whilst US FDA (Food biology, physiology, efficient culture techniques, downstream
and Drug Administration) granted astaxanthin from H. pluvialis bioprocessing, and product formation are highly desired for
“GRAS” status (Generally Recognized As Safe) (Grewe and further development of this sector. Even though currently
Griehl, 2012; Capelli and Cysewski, 2013). several commercial companies (Cyanotech Corporation, Mera
The increasing demand for natural astaxanthin and its high Pharmaceuticals Inc, AIgatechnologies, Fuji Chemical Industry
price raises interest in efficient systems to produce astaxanthin Co. Ltd etc.) are involved in large scale production of H. pluvialis

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Shah et al. Astaxanthin Production from H. pluvialis

and astaxanthin, the production capacity is far beyond the global emerging from anterior end, and a cup-shaped chloroplast with
demand of natural astaxanthin. numerous, scattered pyrenoids (Figure 1A). The macrozooid
This review summarizes both classical knowledge and cells are between 8 and 20 µm long with a distinct gelatinous
most recent advances in the cell biology, physiological, and extracellular matrix of variable thickness. Numerous contractile
biochemical characteristics, responses to environmental vacuoles are irregularly distributed near the protoplast surface
stresses, and their effect on astaxanthin accumulation, genetic of the cell (Hagen et al., 2002). These flagellated fast-growing
engineering, growth conditions, and different cultivation vegetative cells predominate under favorable culture conditions
techniques, harvesting, and post harvest downstream in the early vegetative growth stage (Figure 1A) Macrozooids
bioprocessing of H. pluvialis. The biorefinery concept, may divide into 2–32 daughter cells by mitosis (Wayama
global market potential, challenges, and future direction for et al., 2013) (Figures 2A,B). Under unfavorable environmental
development of H. pluvialis and astaxanthin production in or culture conditions, macrozooids start losing flagella, and
commercial scale also are discussed. expand their cell size. They form an amorphous multilayered
structure in the inner regions of the extracellular matrix
BIOLOGY OF H. PLUVIALIS or the primary cell wall as they develop into non-motile
“palmella” and become resting vegetative cells (Hagen et al.,
Taxonomy and Occurrence 2002) (Figure 1B). With the continued environmental stress (i.e.,
The freshwater unicellular biflagellate green microalgae nutrient deprivation, high light irradiance, high salinity) and
H. pluvialis Flotow belongs to the class Chlorophyceae, order cessation of cell division, “palmella” transform into the asexual
Volvocales and family Haematococcaseae (Bold and Wynne, “aplanospores” (Figures 1C,D). At this stage, cells contain two
1985; Eom et al., 2006). It is also known as Haematococcus distinct structures, a thick and rigid trilaminar sheath, and
lacustris or Sphaerella lacustris. Haematococcus was first secondary cell wall of acetolysis-resistant material. Such cells
described by J. Von Flotow in 1844 and later in 1899 Tracy become resistant to prevailing extreme environmental conditions
Elliot Hazen extensively presented its biology and life cycle (Santos and Mesquita, 1984; Boussiba and Vonshak, 1991).
(Hazen, 1899; Leonardi et al., 2011). H. pluvialis is common Mature aplanospores; accumulate large amounts of secondary
in small transient freshwater bodies and widely distributed in carotenoids, particularly astaxanthin, in lipid droplets deposited
many habitats worldwide. It occurs primarily in temporary water in the cytoplasm, which results in a characteristic bright red
bodies like ephemeral rain pools, artificial pools, natural and color of these cells (Hagen et al., 2002). Some H. pluvialis
man-made ponds, and birdbaths (Czygan, 1970; Burchardt et al., strains are reported to be capable of accumulating astaxanthin
2006). This microalga can be usually found in temperate regions without forming aplanospores (Brinda et al., 2004). Once
around the world and has been isolated from Europe, Africa, environmental or culture conditions return to optimal, red
North America, and Himachal Pradeslv India (Pringsheim, 1966; aplanospores germinate to form flagellated zoospores to initiate
Suseela and Toppo, 2006). It has been also found across diverse a new vegetative growth cycle (Figure 1A). In some cases,
environmental and climate conditions: in brackish water on gametogenesis may occur in aplanospores (Figures 3A–D). Such
the rocks on the seashore (Chekanov et al., 2014); freshwater process requires an exposure to extreme adverse conditions
basin in the rock filled with melted snow on Blomstrandhalvøya (freezing, desiccation, or nutrient starvation) followed by
Island (Norway) (Klochkova et al., 2013); dried fountain near return to favorable culture conditions. During gametogenesis,
Rozhen, Blagoevgrad in Bulgaria Gacheva et al., 2015, freshwater aplanospore cells can produce up to 64 gametes which are known
fishpond in Bihor, Romania (Dragos et al., 2010); rooftop surface as microzooids. The microzooids are smaller in size (<10 µm)
of a building of KIOST in Seoul Korea (Kim et al., 2015). It compared to the zoospores (20–50 µm), and exhibit high-speed
is well suited for survival under conditions of expeditious and motility after their release from gametocysts. Sexual reproduction
extreme in light, temperature, and salt concentration that would is rarely observed in H. pluvialis, and has been largely replaced by
be deleterious to many other microalgae, due to its ability to vegetative reproduction (Triki et al., 1997).
encyst (become enclosed by thick membrane) in a rapid manner
(Proctor, 1957).
Ultrastructural Changes of H. pluvialis
Cellular Morphology and Life Cycle during the Life Cycle
Cellular structure of H. pluvialis is similar to most of other In H. pluvialis cells, large amount of ultrastructural changes
members of volvocalean unicellular green algae. The life cycle occurs during their life cycle which is frequently associated
of H. pluvialis consists of four types of distinguishable cellular with responses to stress conditions in the environment. In the
morphologies: macrozooids (zoospores), microzooids, palmella, green vegetative palmella cells, a thick cell wall surrounds the
and hematocysts (aplanospores) (Hazen, 1899; Elliot, 1934). cell, and two layers of extracellular matrix present near the cell
Macrozooids (zoospores), microzooids, and palmella stages wall (Figure 4A). Nucleus is located in the center of the cell,
are usually called “green vegetative phase” (Figures 1A,B). and highly developed chloroplasts are located at the periphery
Hematocysts (aplanospores) are referred as “red nonmotile (Figure 4A). Few astaxanthin granules are present which located
astaxanthin accumulated encysted phase” of the H. pluvialis life around the nucleus (Figure 4A). Conspicuous pyrenoids with
cycle (Figures 1C,D). Macrozooids (zoospores) are spherical, electron-dense matrix can be found in the stroma (Figures 4A,B)
ellipsoidal, or pear-shaped cells with two flagella of equal length (Wayama et al., 2013). During the starting of encystment process,

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Shah et al. Astaxanthin Production from H. pluvialis

FIGURE 1 | Light microscopic images of H. pluvialis cells in life cycle. (A) Green vegetative motile cell; (B) Green vegetative palmella cell; (C) Astaxanthin
accumulating palmella cell in transition to aplanospore; (D) Astaxanthin accumulated aplanospore cell. Scale bar: 10 µm.

FIGURE 2 | Life cycle of H. pluvialis. (A) Fluorescence microscopy images, showing the 1- to 32-cell stages, and the flagellated stage. DIC, differential interference
contrast image; SYBR, SYBR Green I-stained cells (green); CHL, chlorophyll autofluorescence (red); and Overlay, overlaid images of SYBR and CHL. (B) Illustration of
life cycle of H. pluvialis. Refresh, when old cultures are transplanted into fresh medium, coccoid cells undergo cell division to form flagellated cells within the mother
cell wall. Germination, Flagellated cells settle and become coccoid cells. Continuous and/or strong light accelerate the accumulation of astaxanthin during encystment
(red arrows). Figure reproduced from Wayama et al. (2013) distributed under the terms of the Creative Commons Attribution License.

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Shah et al. Astaxanthin Production from H. pluvialis

Haematococcus turned into greenish-orange cells (with some 2013). With the gradual accumulation of astaxanthin chloroplast
astaxanthin accumulation) which can be referred as intermediate reduce in volume but photosynthetic activity remains. In the
stage cells. In this stage, conspicuous pyrenoids with electron- aplanosopre or cyst stage, astaxanthin accumulates and cells form
dense matrix located in the stroma, remain surrounded by thick cysts. Oil droplets and astaxanthin accumulation patterns may
starch capsules and many starch grains are located around the differ among cyst cells. For example, electron-dense astaxanthin
pyrenoids (Figure 5). Round oil droplets with various sizes, granules in oil droplets (Figures 6A,B) occurred in some cells. In
containing astaxanthin located around the nucleus (Figure 5). At other cells, relatively large oil droplets occurred throughout the
this stage, thylakoids become partial degraded (Wayama et al., cell (Figures 6C,D). Chloroplasts are degenerated and localized
in the interspace between oil droplets (Wayama et al., 2013).

BIOCHEMICAL COMPOSITION OF
H. PLUVIALIS
Because of the unique life cycle of H. pluvialis, cellular
composition of this microalga varies tremendously between its
“green” and “red” stages of cultivation (Table 1).

Protein
In green stage, during favorable growth conditions most H.
pluvialis strains are rich in protein (29–45) (Table 1), lower
protein content (23.6%) have been however observed in a
Bulgarian strain Haematococcus cf. pluvialis Rozhen-12 during
green stage cultivation (Gacheva et al., 2015). It was estimated
that proteins contribute to 21 (Kim et al., 2015) and 23% (Lorenz,
1999) of cellular content during red stage cultivation of H.
pluvialis. Amino acid composition of proteins in the red stage
indicated that proteins were mainly composed of aspartic acid,
glutamic acid, alanine, and leucine with total amino acid content
of 10.02/100 mg, 46.0% of which belonged to essential amino
acids (Lorenz, 1999; Kim et al., 2015).
FIGURE 3 | Gametogenesis in H. pluvialis. (A) C, cyst; G, gametocyst; S, a
sporocyst; (B) vegetative zoospore, F, flagella (indicated by arrow); (C): Carbohydrates
gametocyst before releasing gametes; (D): release of gametes from In green stage, carbohydrate content approximates to 15–17%,
gametocyst. Reproduced with permission from Triki et al., 1997, Phycologia, about a half of the red stage (Table 1). In the red stage, under
Allen Press Publishing Services. Copyright (1997) Allen Press Publishing
conditions of stress (e.g., nutrient starvation, light stress, high
Services.
acidity, temperature variations etc.), H. pluvialis accumulates

FIGURE 4 | Transmission electron micrographs of green vegetative cells of H. pluvialis. (A) General ultrastructure. The cell wall is surrounded by extracellular
matrix (arrowheads). Arrows indicate astaxanthin granules. (B) Chloroplast and pyrenoid. C, chloroplast; CW, cell wall; N, nucleus; P, pyrenoid. Scale bars in (A,B): 5
and 1 µm, respectively. Figure reproduced from Wayama et al. (2013) distributed under the terms of the Creative Commons Attribution License.

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Shah et al. Astaxanthin Production from H. pluvialis

the suitable profile of its fatty acids indicate a possibility of

biodiesel production from this microalga (Damiani et al., 2010;
Saha et al., 2013). The massive astaxanthin accumulation in
H. pluvialis is a cellular response to stress conditions and is
accompanied by the enhanced biosynthesis of triacylglycerols
(TAG) (Zhekisheva et al., 2002, 2005; Cerón et al., 2007), and the
reduction in photosynthetic activity of PSII, loss of cytochrome
f, and subsequent reduction in electron transport, and increased
respiration rate (Boussiba, 2000). During transition from green
vegetative cells to red aplanospores after exposure to stress
conditions astaxanthin start to accumulate as fatty acid mono- or
diesters in cytoplasmic lipid droplets (LD) (Aflalo et al., 2007). As
cells undergo transition to red stage, both chlorophyll and protein
contents drop.

Carotenoid
The carotenoid fraction of green vegetative cells consists
of mostly lutein (75–80%), β-carotene (10–20%) and others,
including chlorophyll a and b, primary carotenoids, violaxanthin,
FIGURE 5 | Transmission electron micrograph of intermediate neoxanthin, lactucaxanthin, and zeaxanthin (Renstrøm et al.,
H. pluvialis cell (general ultrastructure). C, chloroplast; CW, cell wall; N, 1981; Harker et al., 1996a). In the red stage, the total carotenoid
nucleus; OD; oil droplet; P, pyrenoid; SC, starch capsule; SG, starch grain.
content is markedly enhanced, and the characteristic primary
Scale bar: 5 µm. Figure reproduced from Wayama et al. (2013) distributed
under the terms of the Creative Commons Attribution License.
carotenoid pattern of vegetative stage is replaced by secondary
carotenoids, mainly astaxanthin (80–99% of total carotenoids)
(Harker et al., 1996a; Dragos et al., 2010). The ratio of carotenoids
to chlorophylls is about 0.2 in the green stage and increases in
higher content of carbohydrates (starch), for example 38 (Lorenz, the red stage by an order of magnitude and reaches about 2–9.
1999), 60 (Recht et al., 2012), and 74% (Boussiba and Vonshak, The majority of astaxanthin is not deposited in its free form but
1991). Under prolonged stress conditions starch is consumed in it exists within the cell as fatty acid esters of astaxanthin, usually
the cell. mono- or diesters of palmitic (16:0), oleic (18:1), or linoleic (18:2)
acids. This type of modification is required for the deposition
Lipid of this highly polar molecule within non-polar matrix of lipid
In green stage, total lipid content varies from 20 to 25%, droplets. Approximately 70% monoesters, 25% diesters, and only
with approximately 10% lipids composed predominantly of 5% of the free ketocarotenoid is present in the mature “red”
short (C16, C18) polyunsaturated fatty acids deposited in the cells of H. pluvialis (Zhekisheva et al., 2002; Solovchenko, 2015).
chloroplasts. Neutral lipids are predominant lipid class in both Under certain conditions of stress H. pluvialis has been shown
green and red cells (Table 1). In red stage, prolonged stress to accumulate up to 3–5% DW of astaxanthin (Han et al., 2013;
conditions direct larger flux toward the synthesis of neutral Chekanov et al., 2014).
lipids—triacylglycerols (TAG). Red stage cells can accumulate up
to 40% of their cell weight as cytoplasmic lipid droplets (LD),
and considerable amount of secondary metabolites including H. PLUVIALIS-DERIVED ASTAXANTHIN
up to 4% of the ketocarotenoid astaxanthin (Boussiba et al.,
1992, 1999; Saha et al., 2013). The phospholipid content does H. pluvialis as a Major Source of
not change compared to the green stage, while the glycolipid Astaxanthin
fraction nearly doubles in red cells when compared with green H. pluvialis can accumulate up to 5% DW of astaxanthin
vegetative cells (Damiani et al., 2010). The total fatty acid profile and is considered as the best natural source of this high-
of H. pluvialis is relatively complex. Palmitic (16:0), linoleic value carotenoid pigment (Wayama et al., 2013). Dietary
(18:2), and linolenic (18:3) acids are predominant components supplements containing Haematococcus astaxanthin has proved
of the profile with highly polyunsaturated species also present to be safe to humans and widely used for over 15 years as
in considerable amounts (Table 2). Based on the comparative a nutraceutical supplement with no adverse side-effects of its
studies on fatty acids profile of two different H. pluvialis, it supplementation (Capelli and Cysewski, 2013; Yang et al., 2013).
was revealed that both strains varied in composition, especially Natural astaxanthin from H. pluvialis or krill oil is available in
of palmitic (16:0), oleic (18:1), linoleic (18:2), and linolenic the market as a dietary supplement in dosages from 3.8 to 7.6 mg
(18:3) acids. This variation might be associated with several per day due to potential health benefits (Yang et al., 2013). As
factors such as culture environment, stress conditions, culture societies nowadays are looking toward “green” solutions, natural
parameters, variation of strain origin, nutrient etc. Higher lipid astaxanthin form H. pluvialis seems to be more favorable than its
content of H. pluvialis grown under nutrient starvation and synthetic counterpart due to structure, function, application, and

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Shah et al. Astaxanthin Production from H. pluvialis

FIGURE 6 | Transmission electron micrographs of H. pluvialis cyst cells. (A) General ultrastructure of cyst cells, showing small granules that contain
astaxanthin. (B) General ultrastructure of a cyst cell, showing astaxanthin accumulation in oil droplets. (C) General ultrastructure of a cyst cell, showing large oil
droplets. Chloroplasts localize in the interspace between oil droplets (arrows). (D) Some oil droplets are fused. C, chloroplast; N, nucleus; OD, oil droplet. Scale bars in
(A–D): 5 µm. Figure reproduced from Wayama et al. (2013) distributed under the terms of the Creative Commons Attribution License.

security (Choubert and Heinrich, 1993; Capelli and Cysewski, the mevalonate pathway (MVA) catalyzing the formation of
2013; Pérez-López et al., 2014). isopentenyl pyrophosphate (IPP) from acetoacetyl-CoA (Gwak
et al., 2014). There has been numerous evidence of the full
Biosynthesis of Astaxanthin in H. pluvialis set of enzymes required for the conversion of photosynthesis-
Biosynthesis of astaxanthin in H. pluvialis is a complex process derived products i.e., pyruvate and glyceraldehyde-3-phosphate
that is highly up-regulated in conditions of stress and which into isopentenyl pyrophosphate through DOXP pathway inside
coincides with the accumulation of triacylglycerols (TAGs). H. pluvialis chloroplasts (Gwak et al., 2014). It makes it the
Both compounds are deposited in the cytosolic lipid bodies most likely source of IPP in H. pluvialis cells. The process
during the “red” stage of H. pluvialis cultivation. Astaxanthin of astaxanthin biosynthesis is presented on Figure 7. IPP
belongs to carotenoids, a C40 tetraterpenes, synthesized from derived from DOXP pathway is an initial building block of
isoprene units. Isopentenyl pyrophosphate (IPP) is a key astaxanthin synthesis. In the subsequent step the IPP undergoes
intermediate of carotenoid synthesis. In principle, IPP can isomerization to dimethylallyl diphosphate (DMAPP). It has
originate from two dissimilar pathways: mevalonate pathway been long assumed that this conversion was catalyzed exclusively
(MVA) located in cytosol and non-mevalonate (MEP) located by isopentenyl pyrophosphate isomerase (IPI) (Sun et al., 1998;
in the chloroplast (Lichtenthaler et al., 1997; Lichtenthaler, Lichtenthaler, 1999). However, recent transcriptomic studies
1999; Eisenreich et al., 2001). Alternative name for MEP is suggest that neither of the two ipi genes of H pluvialis (ipi1
DOXP, due to the formation of 1-deoxy-D-xylulose-5-phosphate and ipi2IPI2) are up-regulated during cellular accumulation of
in the first stage of the pathway. Comparative transcriptomic astaxanthin (Gwak et al., 2014). Suggestions have been made
analysis of astaxanthin biosynthesis in H pluvialis have shown that another enzyme of similar activity, 4-hydroxy-3-methylbut-
that the key intermediate-IPP is most likely synthesized using 2-enyl diphosphate reductase (HDR) was more likely to be
the DOXP pathway. H. pluvialis lacks three key enzymes of responsible for catalyzing interconversion between IPP and

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Shah et al. Astaxanthin Production from H. pluvialis

TABLE 1 | Typical composition of H. pluvialis biomass in green and red TABLE 2 | Comparison of fatty acid composition (%) of two different
cultivation stages. H. pluvialis strains.

Composition content (% of DW) Green stage Red stage Fatty acids Haematococcus sp. H. pluvialis
KORDI03 (Kim et al., (Lorenz, 1999)
Proteins 29–45 17–25 2015)
Lipids (% of total) 20–25 32–37
C12:0 lauric N/A 0.1
Neutral lipids 59 51.9–53.5
C14:0 myristic 0.1 0.5
Phospholipids 23.7 20.6–21.1
C15:0 pentadecanoic acid 0.1 N/A
Glycolipids 11.5 25.7–26.5
C16:0 palmitic 13.7 29.0
Carbohydrates 15–17 36–40
C16:1 palmitoleic 0.5 0.6
Carotenoids (% of total) 0.5 2–5
C16:2 0.4 N/A
Neoxanthin 8.3 n.d
C16:3 3.5 N/A
Violaxanthin 12.5 n.d
C16:4 3.3 N/A
β -carotene 16.7 1.0
C17:0 margaric N/A 0.2
Lutein 56.3 0.5
C17:1 margaroleic N/A 1.3
Zeaxanthin 6.3 n.d
C18:0 stearic 0.7 2.1
Astaxanthin (including esters) n.d 81.2
C18:1 oleic 4.9 25.9
Adonixanthin n.d 0.4
C18:2 linoleic 24.9 20.8
Adonirubin n.d 0.6
C18:3 linolenic 39.7 12.8
Canthaxanthin n.d 5.1
C18:4 octadecatetraenoic 5.8 1.4
Echinenone n.d 0.2
C20:0 arachidic N/A 0.6
Chlorophylls 1.5–2 0
C20:1 gadoleic 0.5 0.3
Adapted from Grewe and Griehl (2012). n.d., no data. C20:2 eicosadenoic N/A 1.2
C20:3 eicosatrienoic gamma N/A 0.5
DMAPP (Hoeffler et al., 2002; Rohdich et al., 2002; Gwak et al., C20:4 arachidonic 0.9 1.4
2014). Further studies are required to assess the contribution of C20:5 eicosapentaenoic 0.6 0.6
these three enzymes to this key biosynthetic step of astaxanthin C22:0 behenic N/A 0.4
formation. Elongation of the isoprenoid chain is initiated with C24:0 lignoceric 0.3 0.2
a molecule of DMAPP and a subsequent linear additions of C24:1 nervonic acid 0.1 0.1
three molecules of IPP catalyzed by an enzyme geranylgeranyl P
SFAs 15.0 33.2
pyrophosphate synthase (GGPS) (Britton, 1993; Cunningham P
MUFAs 6.0 28.1
and Gantt, 1998). The final step of this process is the formation P
PUFAs 79.1 38.7
of a C20 compound, geranylgeranyl pyrophosphate (GGPP), a Total 100.0 100.0
shared precursor with other isoprenoids. The first committed
step of carotenoid synthesis is catalyzed by phytoene synthase
(PSY) and results in a head-to-tail condensation of two GGPP cyclases (LCY-e and LCY-b). Cyclization is a branching point
molecules to form a C40 compound—phytoene that serves as a of the carotenoid biosynthesis in most organisms, yielding α-
precursor for astaxanthin and other carotenoids (Cunningham carotene (precursor of lutein) and β-carotene (precursor of
and Gantt, 1998). The expression of the phytoene synthase gene other carotenoids including astaxanthin). In H. pluvialis vast
(psy) was up-regulated in Haematococcus cells stressed with high majority of the carbon flux is directed into the latter (Gwak
light and undergoing transformation from “green” to “red” stage et al., 2014), and high level of LCY-b transcripts have been
(Steinbrenner and Linden, 2001; Vidhyavathi et al., 2008; Gwak observed under stress conditions (Lorenz and Cysewski, 2000;
et al., 2014). The subsequent formation of highly unsaturated Gwak et al., 2014). Final two oxygenation steps catalyzed by
compound—lycopene proceeds through four desaturation steps β-carotene ketolase (BKT) and β-carotene hydroxylase (CrtR-
catalyzed by two phytoene desaturases (PDS) and a ζ-carotene b) are rate limiting steps of astaxanthin synthesis (Linden,
desaturase (ZDS) with two plastid terminal oxidase (PTOX 1, 1999; Steinbrenner and Linden, 2001; Vidhyavathi et al., 2008).
PTOX 2) acting as co-factors for electron transfer between Although in principle the reactions catalyzed by these two
C40 carotenoid intermediates, plastoquinone and final electron enzymes can proceed in any order, higher substrate specificity
acceptor—oxygen (Li et al., 2010; Nawrocki et al., 2015). Of the of BKT toward β-carotene than zeaxanthin favors initial
two, PTOX 1 was found to be co-regulated with astaxanthin addition of keto group before enantio-selective hydroxylation of
synthesis in H. pluvialis (Wang et al., 2009; Nawrocki et al., canthaxanthin to astaxanthin is catalyzed by CrtR-b (Lotan and
2015). Desaturation reactions increase the number of conjugated Hirschberg, 1995). Enantioselectivity of astaxanthin synthesis is
carbon-carbon double bonds that form the chromophore in of primary importance for the nutraceuticals market and the
carotenoids and convert a colorless molecule of ζ-carotene to major advantage of H. pluvialis astaxanthin over its synthetic
a pink colored lycopene (Cunningham and Gantt, 1998). Both counterpart. Since astaxanthin has two identical chiral centers
termini of lycopene undergo cyclization catalyzed by lycopene at the positions of 3 and 3′ it can exist in four different

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Shah et al. Astaxanthin Production from H. pluvialis

configurations which yield three different isomers: (3R, 3′ S); 2008; Radakovits et al., 2010; Forján et al., 2015). In majority
(3R, 3′ R); (3S, 3′ S) depending on the spatial orientation of of these reports however, only a transient transgene expression
the hydroxyl (OH) groups in chiral carbon. During chemical has been achieved and a desired, stable, hereditary, and efficient
synthesis these isomers are present in the ratio of 2:1:1, genetic modification existed only for model species such as
respectively, yielding only 25% of the naturally occurring (3S, Chlamydomonas reinhardtii and Volvox carteri. Due to these
3′ S) isoform. H. pluvialis synthesizes the (3S, 3′ S) stereoisomer constrains genetic improvement of H. pluvialis strains have
of astaxanthin and is therefore a much sought-off product in the been long limited to classical mutagenesis. Combination of
nutraceutical market. mutagenic treatment and various screening methods resulted in
the development of numerous interesting mutants of H. pluvialis
Effect of Small Molecules on Astaxanthin including those of higher astaxanthin accumulation capacity
(Tjahjono et al., 1994a; Chumpolkulwong et al., 1997; Tripathi
Synthesis et al., 2001; Chen et al., 2003; Hu et al., 2008; Hong et al.,
Astaxanthin is a secondary metabolite, a carotenoid synthesized
2012; Gomez et al., 2013). Various mutagenic treatments have
by H. pluvialis in a response to stress conditions such as high
been tested, and can be broadly divided into UV and chemical
light, salinity, or carbon to nitrogen ratio (Gao et al., 2012a).
mutagenesis. Chemical mutagenesis has been generally found
Regulation of these pathways can be affected by numerous
to be more suitable for H. pluvialis due to alga’s intrinsic
small molecules like plant hormones or their analogs. An array
capacity to limit the damage from light. Typical chemicals
of such molecules has been explored to modulate astaxanthin
used for mutagenesis include ethyl methanesulphonate (EMS)
accumulation by H. pluvialis. Plant hormones that are usually
and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). In all
associated with stress response mechanisms e.g., abscisic
studies their concentrations were adjusted to target 85–95%
acid (ABA), jasmonic acid (JA), methyl jasmonate (MJ) or
cell mortality. Successful mutants are usually screened using a
growth regulators like gibberellic acid (GA3), salicylic acid
combination of factors that promote identification of mutants
(SA) or brassinosteroid—2, 4-epibrassinolide (EBR) were found
capable of high astaxanthin accumulation. Typically, herbicides
particularly promising in increasing astaxanthin accumulation
that affect carotenoid synthesis pathway such as norflurazon,
in H. pluvialis (Kobayashi et al., 1997b; Gao et al., 2012a,b,
fluridone, nicotine, compactin, or diphenylamine are used
2013a,b; Yu et al., 2015). It was found that these compounds
(Tjahjono et al., 1994a; Chumpolkulwong et al., 1997; Tripathi
affect numerous genes involved in astaxanthin biosynthesis
et al., 2001; Chen et al., 2003; Gomez et al., 2013). Screening
and result in their even six to 10 fold up-regulation. All of
relies on identifying colonies that are capable of surviving and/or
these compounds were tested in various concentrations and
growing well in the presence of inhibitory concentrations of these
the highest improvement of astaxanthin accumulation was
compounds and high illumination. Surviving strains should
achieved with salicylic acid. At relatively low concentrations
in principle exhibit better capacity to synthesize carotenoids
of the hormone 50 mg L−1 and low light 25 µmol photons
and divert larger, or more efficient carbon flux toward these
m−2 s−1 the content of astaxanthin raised seven fold from
compounds. A number of successful mutants have been isolated
0.391 mg L−1 to 2.74 mg L−1 . Higher levels of these hormones
this way and typical improvement of astaxanthin accumulation
had however deleterious effect on both growth and astaxanthin
ranges from several percent (Gomez et al., 2013) to two or
accumulation (Gao et al., 2012a). Correlation between mRNA
three fold improvement (Hu et al., 2008). In former case the
transcript levels of five key enzymes of astaxanthin synthesis
mutated strain was tested in commercial scale cultivation system
pathway (ipi, psy, pds, crtO, and crtR-b encoding respectively
(120,000 L) and retained the improved capacity for astaxanthin
isopentenyl-diphosphate δ-isomerase (IPI), phytoene synthase
production. An alternative approach to strain improvement
(PSY), phytoene desaturase (PDS), β-carotene oxygenase
relying on selection of photosensitive mutants was recently
(CrtO), and β-carotene hydroxylase (CrtR-b) with alga growth
attempted (Hong et al., 2012). Photosensitive mutant with an
and astaxanthin production suggested a complex, multiple
ability to grow under hetero or mixotrophic conditions should
regulatory mechanisms at transcriptional, translational, and
be in principle advantageous over wild type strains due to faster
post-translational levels controlling entire process of carotenoid
growth rates and more efficient stress trigger. Screening for
synthesis in H. pluvialis (Li et al., 2010). Small molecules can
successful mutants was performed in a three stage process. Since
have multiple effects on the regulation of each of these genes and
photosensitivity is connected with impaired photosynthesis,
more detailed investigation of the molecular responses to their
these impaired mutants were selected in the first screening.
application is required for both understanding gene regulation
Secondary screening tested for the ability of heterotrophic
in H. pluvialis and enhancing its capacity as commercial
growth of these photosensitive isolates. Tertiary screening
astaxanthin producer.
involved mixotrophic conditions with moderate illumination to
obtain mixotrophic photosensitive strain that accumulated 4.7%
Genetic Improvement of H. pluvialis for (w/w) of astaxanthin under much shorter induction time (Hong
Astaxanthin Production et al., 2012). The mutated strain was stable for at least 1.5 year
Green eukaryotic microalgae are among the organisms that are and is an interesting example of using classical mutagenesis for
notoriously difficult to genetically engineer. In principle, genetic improvement of H. pluvialis. Mutagenic strain improvement
engineering of microalgae has been reported for over 30 strains can be expanded by breeding or creating hybrid strains from
of various species, including Haematococcus (Rosenberg et al., previous genetic improvement efforts. Technique of protoplast

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Shah et al. Astaxanthin Production from H. pluvialis

FIGURE 7 | Pathway of (3S-3′ S)-astaxanthin biosynthesis in H. pluvialis. Major carbon flux during the red stage of H. pluvialis cultivation is indicated with thick
arrows, minor products are indicated with thin arrows. Major intermediates of biosynthesis are indicated in large fonts, minor intermediates in small fonts. Enzyme
abbreviations are as follows: IPI, Isopentenyl pyrophosphate isomerase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; GGPS, geranylgeranyl
pyrophosphate synthase; PSY, phytoenesysthase; PDS, phytoenedesaturase; ZDS, ζ-carotene desaturase; LCY-b, lycopene β-cyclase; LCY-e, lycopene ε-cyclase;
BKT, β-carotene ketolase; CrtR-b, β-carotene 3,3′ -hydroxylase; Intermediates: Phytofluene, Neurosporene, γ-carotene, β-Cryptoxanthin, Adonixanthin, Echinenone,
Adonirubin.

fusion has been successfully applied to H. pluvialis (Tjahjono emerged and stable transformations of H. pluvialis chloroplast
et al., 1994a). Two mutagenized strains, norflurazon-resistant (Gutiérrez et al., 2012) and nuclear genomes (Sharon-Gojman
and nicotine-resistant have been fused to create a hybrid et al., 2015) were achieved. Most recent nuclear transformation
containing genetic material of two initial strains and showed 30% vectors are capable to transform one or two transgenes into the
higher astaxanthin accumulation than the initial wild type strain, nuclear genome either 5′ or 3′ of the endogenous dominant
when neither of the fused strains showed such characteristics selection marker, in the absence of any additional antibiotic
(Tjahjono et al., 1994a). Till very recently H. pluvialis was one resistance genes. The selection marker used in this system is a
of the organisms in which genetic engineering of its nuclear phytoene desaturase (pds) variant that confers resistance to a
genome was considered difficult due to lack of suitable shuttle herbicide norflurazon due to a single point mutation (L504A).
vectors and satisfactory transformation efficiencies (Sharon- Successful transformation of H. pluvialis was obtained with
Gojman et al., 2015). A number of unsuccessful approaches particle bombardment and numerous constructs based on
have been tried that included various transformation methods pds selection marker were delivered and incorporated to the
(particle bombardment, electroporation, Agrobacterium), genome showing stability of integration for over 16 months of
vectors, promoters, and strains (Sharon-Gojman et al., 2015). To subculturing (Sharon-Gojman et al., 2015). Genetic engineering
address these limitations and open a new array of possibilities of chloroplast genome of H. pluvialis have been also achieved
in H. pluvialis and astaxanthin biology and technology new relatively recently (Gutiérrez et al., 2012). So far these studies are
developments were required. In recent years these developments limited to expressing exogenous antibiotic resistance gene (aadA

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Shah et al. Astaxanthin Production from H. pluvialis

cassette) between Internal Transcribed Spacer region and 16S major neurodegenerative diseases (Alzheimer’s, Huntington’s,
gene of H. pluvialis chloroplast genome, but this technique may Parkinson’s, and amyotrophic lateral sclerosis-ALS). Diets high
in the near future have significant impact on protein production in antioxidants offer the potential to lower the associated
in H. pluvialis (Gutiérrez et al., 2012) as higher protein yields are risks (Ferrante et al., 1997). Natural astaxanthin can cross the
generally obtained during chloroplast expression of transgenes in blood-brain barrier in mammals and can extend its antioxidant
other microalgal strains (Li et al., 2015). These new developments benefits beyond that barrier. Therefore, astaxanthin can help to
in genetic engineering of H. pluvialis can open a new chapter for alleviate the effects of Alzheimer’s disease and other neurological
the development of this organism as both industrial astaxanthin diseases. Astaxanthin can improve respiratory and sympathetic
producer and an interesting model for carotenoid synthesis and nervous system activities (Nagata et al., 2006), inhibit the
accumulation studies. growth of fibrosarcoma, breast, and prostate cancer cells and
embryonic fibroblasts (Palozza et al., 2009); cell death, cell
proliferation and mammary tumors (Nakao et al., 2010).
APPLICATIONS OF H. PLUVIALIS Astaxanthin supplementation can help to protect against UV-
ASTAXANTHIN induced photooxidation; as an oral sun-protectant; can prevent
skin thickening and reduce collagen reduction against UV
Astaxanthin in Human Health and as induced skin damage (Ranga Rao et al., 2013) and can improve
Nutraceutical skin condition across its layers i.e., corneocyte, epidermis,
Astaxanthin possesses various human health benefits and basal, and dermis by combining oral supplementation and
nutraceutical applications and plenty of published information topical treatment (Seki et al., 2001; Yamashita, 2002; Tominaga
available with evidences, mainly from in vitro and animal et al., 2012). Results have shown that semen quality, pregnancy
models (Guerin et al., 2003; Chew et al., 2004; Higuera- rate and sperm velocity in human subject can be improved
Ciapara et al., 2006; Palozza et al., 2009; Yuan et al., 2011). (Elgarem et al., 2002; Comhaire et al., 2005) whereas idiopathic
The effect of Haematococcus derived astaxanthin on various infertility can be decreased by astaxanthin (Andrisani et al.,
physiological systems in animal and human subject is presented 2015).
in Table 3. Astaxanthin is considered as “super anti-oxidant”
which possesses one of the strongest known antioxidant effects.
Its unique structure allows it to span biological membranes
and act as an antioxidant by reducing and stabilizing free Astaxanthin in Aquaculture and Poultry
radicals (Hussein et al., 2006; Liu and Osawa, 2007; Ranga Industry
Rao et al., 2010). It is very good at protecting membrane During last 20 years, synthetic astaxanthin has been widely used
phospholipids and other lipids against peroxidation (Naguib, for pigmentation of fish. Haematococcus astaxanthin has great
2000). There are several studies which showed high antioxidant potential in aquaculture industry, due to increasing consumer
activity of astaxanthin from H. pluvialis in rats supplemented demands for natural products and ability of Haematococcus
with diet (Kamath et al., 2008; Ranga Rao et al., 2010, 2013; astaxanthin to provide necessary supplementation for adequate
Augusti et al., 2012). Astaxanthin can terminate the induction growth and reproduction of commercially valuable fishes
of inflammation in biological systems. It can help to fight (Salmonid, Red sea bream), rainbow trouts, and shrimps.
symptoms of ulcer disease from Helicobacter pylori (Liu and Microalgae- derived astaxanthin has been demonstrated as safe
Lee, 2003); protect against gastric lesions (ulcers), improve and effective compound for flesh pigmentation of these fish
gastrointestinal health (Nishikawa et al., 2005; Kamath et al., (Torrissen and Naevdal, 1984; Tolasa et al., 2005). Utilization
2008); and treat gastrointestinal discomfort (Andersen et al., of H. pluvialis meal for pigmenting has resulted in significant
2007; Kupcinskas et al., 2008). Astaxanthin offers protection astaxanthin deposition in flesh and skin, flesh coloration
against free radical damage to preserve immune-system defenses. enhancement, enhanced antioxidant system, fish egg quality,
The immunomodulating capacity of astaxanthin has been better growth and survival of fry of Salmonid, sea bream,
found to be superior to that of β-carotene and canthaxanthin and rainbow trout (Arai et al., 1987; Sommer et al., 1991;
(Chew and Park, 2004). Astaxanthin has shown significant Choubert and Heinrich, 1993; Sheikhzadeh et al., 2012a,b),
effect on immune function in a number of in vitro and in ornamental fishes (Ako and Tamaru, 1999), and shrimp (Arai
vivo assays using both animal models (Chew et al., 2004) et al., 1987; Parisenti et al., 2011). A recent study indicated
and humans (Park et al., 2010). Astaxanthin is a potential that diets supplemented with H. pluvialis can improve large
therapeutic agent against atherosclerotic cardiovascular disease yellow croaker fish growth more than diets supplemented with
(Fassett and Combes, 2011). Astaxanthin supplementation can synthetic astaxanthin (Li et al., 2014). H. pluvialis-derived
be beneficial for people with enhanced risk for heart attacks. It natural astaxanthin has shown to be efficient in pigmentation
is carried by VLDL, LDL, and HDL (high-density lipoprotein) of egg yolks, egg production (Elwinger et al., 1997) in hen and
in human blood and protects LDL-cholesterol against oxidation breast muscle tissue improvement and higher feed efficiency
(Iwamoto et al., 2000); has a role in the reduction of blood in broiler chicken (Inborr and Lignell, 1997; Inbbor, 1998).
plasma level (Karppi et al., 2007); and increases basal arterial It has also been proved to improve health and fertility of
blood flow (Miyawaki et al., 2008). Oxidative stress is a chicken and to decrease their mortality (Lignell and Inborr, 1999,
causative or at least ancillary factor in the pathogenesis of 2000).

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Shah et al. Astaxanthin Production from H. pluvialis

TABLE 3 | Effect of H. pluvialis-derived astaxanthin on various physiological systems in human and animal subjects.

Physiological System Subject Effect followed Main outcome References

Anti-oxidation Rabbits Thioredoxin reductase; Paraoxonase Enhanced; No effect Augusti et al., 2012
activity
Rats Hepatoprotective and antioxidant Improved Ranga Rao et al., 2015
activity
Rats Antioxidant enzymes, catalase, Increased Ranga Rao et al., 2010,
superoxide dismutase, peroxidase, 2013
and lipid peroxidation in plasma and
liver
Men (bilateral cataract) Antioxidative effects through changes Enhanced; Suppressed Hashimoto et al., 2013
in superoxide scavenging activity, and
hydroperoxides production in
aqueous humor

Eye function 18 healthy men Deep vision Improved Sawaki et al., 2002
10 healthy men Eye function Improved Iwasaki and Tawara, 2006
40 asthenopia patients Eye accommodation power Improved Kenji et al., 2005
49 healthy men Uncorrected far visual acuity Improved Nakamura et al., 2004
87 men (visual display Eye accommodation amplitude (the Improved; Nagaki et al., 2002
terminal workers) adjustment in the lens of the eye that
allows it to focus);
Eye soreness, dryness, tiredness, and Reduced Nagaki et al., 2006
blurred vision

Skin Healthy female or male Skin wrinkle, corneocyte layer, Improved Tominaga et al., 2012
epidermis, and dermis
46 healthy women Skin elasticity and moisture Improved Seki et al., 2001;
Yamashita, 2002

Immune response 14 healthy women Oxidative stress and inflammation Reduced; Improved Park et al., 2010
markers; Immune response
Inflammation Rats Gastrointestinal health Improved Nishikawa et al., 2005
Gastric ulcer H. pylori-infected mice Bacterial load Gastric inflamation Reduced Liu and Lee, 2003
Rats Gastric ulcer markers Reduced Kamath et al., 2008
44 patients with functional Inflammatory markers; No effect; No effect Andersen et al., 2007;
dyspepsia gastrointestinal discomfort Kupcinskas et al., 2008

Cardiovascular system 20 adult men Blood flow time Improved Miyawaki et al., 2008
Men Blood plasma levels Reduced Karppi et al., 2007

Muscle endurance 16 non-trained men Lactic acid accumulation after run Reduced Sawaki et al., 2002
19 non-trained men Respiratory and sympathetic nervous Improved Nagata et al., 2006
system activities
20 non-trained men Strength/explosiveness test; No effect; Improved Lignell, 2001
strength/endurance test
20 resistance-trained men Markers of skeletal muscle injury No effect Bloomer et al., 2005

Cancer Rats Growth of colon cancer cells Inhibited Palozza et al., 2009

Central nervous system Healthy mice Memory Improved Zhang et al., 2007
10 healthy men (50–69 Response time and accuracy of Improved Satoh et al., 2009
years) several tasks
Middle aged/elderly men Cog Health battery scores Improved Katagiri et al., 2012
and women (Nuropsychological memory test)

Male fertility 20 sub-fertile men Semen quality, pregnancy rate Improved Elgarem et al., 2002
30 sub-fertile men Sperm velocity; oxidation markers; Improved; Reduced; Comhaire et al., 2005
pregnancy rate Improved
24 healthy men Idiopathic infertility Decreased Andrisani et al., 2015

Metabolic Syndrome (MS) Obese rats Body weight; adipose tissue weight; Reduced; Reduced; Ikeuchi et al., 2007
MS markers Improved

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Shah et al. Astaxanthin Production from H. pluvialis

CULTIVATION AND PROCESSING OF The increased temperature is likely to affect the synthesis of
H. PLUVIALIS FOR ASTAXANTHIN astaxanthin through stimulation of oxygen radicals formation
and their higher reactivity (Tjahjono et al., 1994b). It is preferred
PRODUCTION
that the temperature change takes place gradually, allowing
Culture Conditions and Requirements for better acclimation to the new conditions (Hata et al., 2001).
Cell Growth and Astaxanthin Formation pH can also significantly affect the cell growth and synthesis
Optimization of the various culture parameters, such as growth of chlorophyll and carotenoids in H. pluvialis. In terms of
medium composition, light, pH, temperature etc. is necessary biomass and astaxanthin production optimal pH is within the
to achieve high biomass and astaxanthin production. Most of range of 7.00–7.85 (Hata et al., 2001; Sarada et al., 2002a). The
these parameters have different optima for biomass accumulation typical irradiation for H. pluvialis cultivation ranges between
and astaxanthin production. For carotenogenesis induction, the 40 and 50 µmol photons m−2 s−1 (Hata et al., 2001; Chekanov
stronger exposure to stress conditions, the higher astaxanthin et al., 2014; Park et al., 2014). Optimal irradiation to achieve
accumulation. The origins of this stress can be diverse and a high growth rates tend to be higher, namely 70 (Zhang
successful astaxanthin accumulation has been induced with both, et al., 2014), 80 (Saha et al., 2013), 90 (Fan et al., 1994), or
high levels of one stressor, or from a combination of multiple even up to 177 µmol photons m−2 s−1 (Domínguez-Bocanegra
stress factors. In some cases, if cells are exposed to strong stress, et al., 2004). These different optimal values may be caused
cells growth completely ceases and cells begin to die in a relatively by other cultivation parameters such as media composition,
short time (Su et al., 2014). Various types of growth media are temperature, or the strain of H. pluvialis. During vegetative
used for cultivation of H. pluvialis. The most commonly used stage cultivation of H. pluvialis, the regular cycles of alternating
media are BG- 11 (Rippka et al., 1979), BBM (Bischoff and Bold, light and dark 12:12 or 16: 8 h are often used (Saha et al.,
1963), OHM (Fábregas et al., 2000); KM1-basal medium with 2013; Park et al., 2014), but the higher density cultures are
organic carbon sources in the form of sodium acetate (Kobayashi achieved with continuous illumination (Domínguez-Bocanegra
et al., 1993), and their modifications. An ideal composition of the et al., 2004). The best practice to date appears to be white or
medium to achieve high growth rate and biomass accumulation blue LED lighting (Saha et al., 2013) or the mixture of both
is different from ideal composition for high accumulation of at the ratio of 3:1 at 7000 lx (∼95 µmol photons m−2 s−1 ).
astaxanthin. Sodium nitrate was found to be the most optimal These conditions promote morphologic changes from green
inorganic nitrogen source (Sarada et al., 2002a), alternatively vegetative cells to red cyst cells (Sun et al., 2015). Carotenogenesis
an organic source such as urea can be used. When culture is induced in cells upon exposure to higher light intensity
is subjected to nutrient deficiency, it leads to accumulation than the corresponding light saturation point (LSP). However,
of astaxanthin within the cells (Saha et al., 2013). Nitrogen specific optimum value of LSP differ between studies. The lowest
limitation leads to approximately twice the rate of astaxanthin intensities that have been reported utilized irradiation of around
production than the limitation of phosphorus. It can be due to 100–150 µmol photons m−2 s−1 (Zhang et al., 2014) followed
higher cellular damage resulting from a lack of nitrogen, which by 240 (Saha et al., 2013), 345 (Domínguez-Bocanegra et al.,
manifests in significant degradation of chlorophyll, compared to 2004), and 480 µmol photons m−2 s−1 (Chekanov et al., 2014).
the phosphorus starvation (Boussiba et al., 1999). Micronutrients Lower optimal irradiation was found to be influenced by other
such as selenium and chromium result in an increased biomass stress conditions, such as deficiency of nutrients (Saha et al.,
and astaxanthin production (Tripathi et al., 1999; Fábregas 2013; Zhang et al., 2014) or elevated temperature (Tjahjono et al.,
et al., 2000; Domínguez-Bocanegra et al., 2004). Formation of 1994b). It proves that for effective induction of carotenogenesis
astaxanthin can also be induced by adding NaCl (0.25–0.5% w/v) excessive irradiation may not be necessary if other stressors are
to the media. Also, when NaCl is added together with 2.2 present. With the reduced requirements of light for cultivation
mM sodium acetate, astaxanthin accumulation can be increased in photobioreactors, the costs of cultivation can be minimized
(Sarada et al., 2002b). Addition of 0.45 mM Fe2+ in the form which is essential for astaxanthin production in an industrial
of ferrous sulfate may significantly increase the biosynthesis scale. Regarding the type of illumination, the highest carotenoid
of carotenoids in cysts due to formation of hydroxyl radicals. content was obtained by using a continuous PAR lighting (Saha
(Kobayashi et al., 1997b). This effect may be enhanced by et al., 2013). An interesting alternative to an immediate change
combining Fe2+ treatment with an addition of sodium acetate in the radiation intensity to induce the transition from the
and high temperature exposure (Kobayashi et al., 1993; Tjahjono vegetative phase to carotenoid production is gradually increasing
et al., 1994b). According to most studies, the suitable temperature the level of lighting. Gradual increase of light intensity can
for the growth and astaxanthin accumulation of H. pluvialis result in gradual transformation of cells to cysts and can also
is between 20 and 28◦ C (Fan et al., 1994; Hata et al., 2001; contribute to better accumulation of astaxanthin, because the
Lababpour et al., 2005; Kang et al., 2010; Yoo et al., 2012; cells are capable to cope with increasing higher levels of stress
Wan et al., 2014a). However, temperature above 30◦ C induces a (Park et al., 2014).
transition from green vegetative stage to red stage and formation
of red cysts can be observed within 2 days. This transition Culture Systems
is combined with a significant slowdown in growth, while Astaxanthin-producing H. pluvialis is capable of growing
astaxanthin accumulation is 2–3 times higher than at 20◦ C. in photoautotrophic, heterotrophic, or mixotrophic growth

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Shah et al. Astaxanthin Production from H. pluvialis

conditions in indoors, open raceway ponds or closed phase of cultivation. Under the optimized conditions, biomass,
photobioreactors in batch, fed batch, or continuous modes. and astaxanthin productivities in the attached cultivation system
were 2.8 (3.7 g m−2 d −1 ) and 2.4-fold (65.8 mg m−2 d−1 )
Photoautotrophic Culture higher than those of the suspended bioreactor, respectively (Wan
Photoautotrophic culture of H. pluvialis is mainly carried out et al., 2014b). Other studies that used similar approach have
in open raceway ponds or closed photobioreactors. Typical reported higher astaxanthin productivities of 124 mg m−2 d−1
photobioreactors used for its cultivation include tubular, bubble (Yin et al., 2015) and 164.5 mg m−2 d−1 (Zhang et al., 2014).
column and airlift photobioreactors. As the culture conditions Attached cultivation approach is superior to suspended induction
for maximum growth and maximum astaxanthin content are in several aspects such as, lower water consumption and smaller
mutually exclusive, a two-step cultivation strategy is commonly risk of contamination. This indicates that attached induction
adopted for the commercial production. The first step, green approach can provide a promising way to boost economic
vegetative growth phase (“green stage”) is to promote algal benefits and considerably reduce production cost of astaxanthin
growth under favorable culture conditions (e.g., low light and from H. pluvialis (Zhang et al., 2014; Wan et al., 2014b). Recently,
nitrogen-replete) (Boussiba, 2000; Aflalo et al., 2007; Del Rio Park et al. (2014) established a two-stage “perfusion culture”
et al., 2007). When high cell density is reached, the culture enters system for H. pluvialis combining it with stepwise increase of
into the second step, reddish inductive production phase (“red light irradiance. Approach is based on repeated replacement of
stage”), where algal cells are subjected to stress conditions such the growth medium. Cells are grown in a photobioreactor and are
as high light intensity and nitrogen depletion, excess acetate periodically retained in the cell settling chamber whilst growth
addition, pH or salt stress, phosphate deficiency, or the addition medium is being replaced in the photobioreactor. Cells are later
of specific cell division inhibitors. These stress factors (either recycled to the bioreactor and can efficiently utilize fresh growth
one or combination of more) induce the astaxanthin production medium which is free of inhibitory metabolic by-products.
in H. pluvialis (Fábregas et al., 2001; Torzillo et al., 2003; Perfusion culture can provide high biomass productivities of
Orosa et al., 2005; He et al., 2007; Hu et al., 2008; Li et al., 0.18 g L−1 d−1 . Under stepwise increased light irradiance (150–
2010; Choi et al., 2011). Therefore, carotenoid induction method 450 µE/m2 /s), cellular density of 12.3 g L−1 of have been obtained
has a direct correlation with both the astaxanthin content and which is 3.09 and 1.67 times higher than batch and fed-batch
total astaxanthin productivity. The optimal environmental and processes, respectively whilst the productivity of astaxanthin
nutritional conditions for each stage are quite different (Del reached 602 mg L−1 (Park et al., 2014).
Rio et al., 2007). The reported biomass productivities in green
stage and red stage ranged from 0.01 to 0.5 g L−1 d−1 and Heterotrophic and Mixotrophic Culture
0.01 to 4.8 g L−1 d−1 , respectively. Astaxanthin productivity High light irradiance is often employed for enhancing
and astaxanthin content ranged from 0.44 to 21 mg L−1 d−1 astaxanthin formation in H. pluvialis cultures. However,
and 0.8 to 4.8% of DW, respectively (Table 4). Astaxanthin light absorption and scattering caused by mutual shading of cells
can be also produced efficiently by H. pluvialis using a simpler in large-scale cultures severely affects the productivity and quality
“one-step strategy.” This strategy involves the administration of algal biomass and products. The high cost of illumination
of nitrate starvation and specific average irradiance in the is another problem hindering the commercialization of
culture medium, resulting in simultaneous algal cell growth Haematococcus products. To overcome this drawback,
and astaxanthin accumulation (Del Río et al., 2005; Del Rio heterotrophic culture approach may be considered. Under
et al., 2007; Del Río et al., 2008; García-Malea et al., 2009). At heterotrophic growth conditions light is not needed as organic
the laboratory scale and under continuous illumination, mean substrates serve as carbon and energy sources for growth
astaxanthin productivity of 20.8 mg L−1 d−1 has been reported and synthesis of secondary metabolites. Also, since lipid
for the one-step method (Del Río et al., 2008). The technical accumulation and astaxanthin biosynthesis are connected in
feasibility of this approach at a pilot scale was demonstrated in an space and time the effect of carbon source on lipid accumulation
outdoor tubular photobioreactor, which resulted in biomass and can have significant effect on overall productivity. It has been
astaxanthin productivities of 0.7 g L−1 d−1 and 8 mg L−1 d−1 , shown in Haematococcus and other microalgae lipid content
respectively (García-Malea et al., 2009). One-stage cultivation and lipid profiles of microalgae are dependent on the cultivation
seems attractive, since it is less complicated than the two- conditions with various stress factors such as starvation or
stage process and the production of astaxanthin takes place salt stress are efficient triggers of lipid accumulation, and can
in a continuous mode. It has however two serious drawbacks. result in the alteration of fatty acid profiles due to cellular
First, the actual astaxanthin production is significantly lower adjustment to particular stressor (Damiani et al., 2010; Lei
compared to the two-stage approach. Second, this cultivation et al., 2012; Saha et al., 2013; Chen et al., 2015). Various types
is unsuitable for outdoor setting, since it requires incessant of organic carbon sources have been used for heterotrophic
illumination during night as well what makes the process cultivation and induction using acetate has been found effective
too expensive (Aflalo et al., 2007). An “attached cultivation” for Haematococcus encystment and initiation of astaxanthin
approach was successfully applied in the induction of H. pluvialis production (Kobayashi et al., 1991; Kakizono et al., 1992; Orosa
for astaxanthin production. In this method green cells are et al., 2000; Hata et al., 2001; Kang et al., 2005). However, unlike
cultured in the conventional water column and then deposited on many microalgae in which oversupply of easily accessible carbon
the membrane to increase light stress surface area in the second in combination with nitrogen limitation yields diversion of

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 TABLE 4 | Summary of various methods of H. pluvialis biomass cultivation and corresponding astaxanthin productivities.

PBRs type Outdoor/Indoor Mode Culture medium* Biomass Biomass productivity Astaxanthin content Astaxanthin References
Shah et al.

productivity in in Red stage (g L−1 (%, DW) productivity (mg L−1

Green stage (g d−1 ) d−1 )
L−1 d−1 )

Airlift column (30 L) Indoor Batch Modified Bold’s 0.03 0.01 2.7 0.44b Harker et al., 1996b
Basal medium
Tubular /open pond Outdoor – Modified Bold’s 0.036–0.052 N/A 2.8–3.0 N/A Olaizola, 2000
(25,000 L) Basal Medium
Tubular (50 L) Indoor Semi continuous BG-11 medium N/A 0.05 3.6 7.2c,d Torzillo et al., 2003
Bubbling column (1.8) Indoor Batch Basal inorganic N/A 0.6 0.8 5.6a Del Río et al., 2005
culture medium

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Airlift Tubular (55 L) – Inorganic medium N/A 0.41 1.1 4.4a López et al., 2006
free of acetate
Bubbling column Indoor Batch BG-11 medium 0.5 0.21 4 11.5b Aflalo et al., 2007
(0.5 L)
Tubular (200 L) Outdoor Batch BG-11 medium 0.37 0.21 3.8 10.1b,c Aflalo et al., 2007
Bubbling column Indoor Batch Basal inorganic N/A 1.9 1.1 21a Del Río et al., 2008
(1.8 L) culture medium
Bubbling column (1 L) Indoor Batch Standard inorganic 0.36 0.14 3.6 12b Ranjbar et al., 2008
medium
Tubular (1.8 L), Outdoor Continuous Standard inorganic N/A 0.7 1 8a García-Malea et al., 2009
outdoor, medium

15
Open pond Indoor Batch BG-11 medium N/A 0.15 2.79 4.3a Zhang et al., 2009
Flat type (1 L) Indoor Fed batch NIES-C medium 0.33 0.44 4.8 14b Kang et al., 2010
Airlift column Indoor Batch Haematococcus N/A 0.14 N/A 3.3a Choi et al., 2011
medium (OHM)
Bubbling column (6 L) Indoor Batch NIES-C medium N/A 0.047 N/A 1.4a Yoo et al., 2012
Bubbling column Outdoor Batch BG-11 medium N/A 0.58 2.7 17.1b,d Wang et al., 2013a
(0.6 L)
Bubbling column Outdoor Batch BG-11 medium N/A 0.30 3.8 16.0b,d Wang et al., 2013b
(0.6 L)

a Obtained from one-step culture process. b Obtained from two-step culture process. Productivity value was calculated based on total time required by the “green stage” and “red stage” of cultivation. c Induction of astaxanthin was

performed outdoors; d Obtained from a two-step process in which astaxanthin productivity was calculated based on time spent on the “red stage” only.
*Detail medium composition can be found in relevant references.
Astaxanthin Production from H. pluvialis

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Shah et al. Astaxanthin Production from H. pluvialis

the carbon flux toward lipid accumulation (Miao and Wu, agents) (Carney and Lane, 2014). Recently, several patent
2006; Jia et al., 2014), H. pluvialis grows at a relatively low rate applications relating to control of fungus P. sedebokerenses have
(0.22 d−1 ) and accumulates negligible amount of astaxanthin, been developed in the USA and China to protect production
too low to be considered for commercial scale production if losses in commercial Haematococcus culture facilities across
single step cultivation is considered (Kobayashi et al., 1992, the world (McBride et al., 2013; Zhang et al., 2013; Carney and
1997a; Moya et al., 1997). Additionally, heterotrophic cultivation Sorensen, 2015).
of Haematococcus increases the risk of bacterial or fungal
contamination (Hata et al., 2001; Olguín et al., 2012). H. pluvialis Harvesting
can be also produced indoors mixotrophically employing an Harvesting remains one of the most challenging issues and
organic acid (e.g., acetate) or carbohydrates as an additional a limiting factor for commercial algal biomass production.
carbon and energy source (Kobayashi et al., 1993). Studies Harvesting of H. pluvialis refers to the selection of appropriate
have shown that both growth and astaxanthin production can techniques to recover the “red” biomass, after the accumulation
be enhanced under mixotrophic culture conditions. A final of astaxanthin in the cells and also that can facilitate cost-
cell density of 0.9–2.65 g L−1 and a maximum astaxanthin efficient astaxanthin extraction in the extraction phase. For the
content of 1–2% DW were obtained from mixotrophic cultures large scale harvesting of H. pluvialis centrifugation is the most
of H. pluvialis (Chen et al., 1997; Zhang et al., 1999; Wang common method and combined with other processes. Usually
et al., 2003). A sequential, heterophotric-photoautotrophic haematocysts are separated from the water through passive
culture mode was also explored. Heterotrophic culture was settling and subsequently concentrated with centrifugation
used in the green stage to produce algal biomass, while (Lorenz and Cysewski, 2000; Olaizola, 2000; Li et al., 2011; Han
astaxanthin production was induced in photoautotrophic culture et al., 2013; Pérez-López et al., 2014). Through the combination of
conditions. The induction of astaxanthin accumulation was these processes total suspended solid of 13.5% in the algal cake is
performed under nitrogen deprivation conditions and whilst achieved (Li et al., 2011). Flotation and disk-stack centrifugation
using bicarbonate or CO2 as carbon sources. As a result, a very have been also reported as another alternative for H. pluvialis
high cellular astaxanthin content of 7% (DW) was achieved, harvest. Both showed more than 95% biomass recovery efficiency
3.4-fold higher than heterotrophic induction whilst astaxanthin (Panis, 2015).
productivity of 6.25 mg L−1 d−1 was obtained (Kang et al., 2005).
Results indicate that photoautotrophic induction of astaxanthin Cell Disruption
production in H. pluvialis is more effective than heterotrophic Different techniques have been developed in order to disrupt
one. Based upon the information obtained thus far, heterotrophic the algal cell and recover the intracellular metabolites. The
and mixotrophic culture modes are less cost-effective than the most appropriate cell disruption methods to enhance recovery
photoautotrophic one for Haematococcus mass culture. of astaxanthin from H. pluvialis at a commercial scale involve
mechanical processes and more specifically expeller pressing
and bead milling (Lorenz and Cysewski, 2000; Olaizola, 2003;
Microbial Contamination and Possible Mercer and Armenta, 2011; Razon and Tan, 2011). During
Control Measures pressing (pulverization) microalgae cells are squeezed under
Since interest in commercial microalgae cultivation is high pressure in order to rupture the thick sporopollenin wall.
increasing, microbial contaminants that hamper production Main advantage of expeller pressing is simple operation and
by resulting in reduced biomass yield and quality received minimization of contamination from external sources. Algal oil
great attention recently. Mass culture of H. pluvialis is reported recovery efficiency of 75% can be achieved in a single step. Bead
to be contaminated by fungal parasites and zooplanktonic milling utilizes vessels filled with tiny glass, ceramic or steel
predators (e.g., amoebas, ciliates, and rotifers), as well as other beads that are agitated at high speeds. The dried biomass is fed
microalgae and cyanobacteria (Han et al., 2013). A parasitic in these vessels, where continuous exposure of biomass to the
chytrid/blastoclad fungus Paraphysoderma sedebokerenses is grinding media (beads) leads to cell-wall rupture, and subsequent
found to be responsible for reduced astaxanthin productivity release of intracellular compounds. This method is most effective
and frequent culture collapses in commercial Haematococcus when biomass concentration in the algal cake after harvesting is
cultivation facilities (Hoffman et al., 2008; Strittmatter et al., between 100 and 200 g/l (Greenwell et al., 2010). Both methods
2015). Detection of contaminants is prerequisite for preventing are reliable and widely applied for the H. pluvialis cells disruption
and controlling of microbial contamination in mass microalgal at a commercial scale.
culture. Methods of detection usually include microscopy
and staining, flow cytometry, molecular based detection Dehydration
and monitoring. In order to cope with microalgae culture In commercial scale astaxanthin production, dehydration
contamination, the techniques that are generally used include (drying) ensures the quality of the pigment and leads to the
abiotic stresses such as NO− 3 limitation, pH stress, temperature formulation of the final product (Mata et al., 2010; Li et al.,
stress, light stress, toxic substances, and shear forces. There are 2011). After algal cell walls have been disrupted, biomass must
some other techniques for parasite removal including salvage be processed rapidly within few hours to avoid spoilage. Thus,
harvest, chemical agents (abscisic acid, copper sulfate), physical dehydration is a process applied prior to recovery of the desired
methods, biological methods (selective breeding and biological metabolite, in order to extend the shelf-life of the algal biomass

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Shah et al. Astaxanthin Production from H. pluvialis

(Mata et al., 2010). The most known dehydration techniques retains the sample in an oxygen-free and light-free environment
that have been employed on microalgae are solar drying, spray in contrast to traditional organic solvent extraction (Jaime et al.,
drying, and freeze drying (Molina Grima et al., 2003; Brennan 2010). Recently, a simple method for the direct extraction of
and Owende, 2010; Milledge, 2013). Spray drying has been lipids from high moisture H. pluvialis microalgae was successfully
considered as the most appropriate method to dry high-value achieved using liquefied dimethyl ether (Boonnoun et al., 2014).
microalgal products including H. pluvialis astaxanthin (Leach
et al., 1998; Brennan and Owende, 2010; Li et al., 2011; Han
et al., 2013; Milledge, 2013; Panis, 2015). The recovery efficiency BIOREFINERY APPROACH FOR
of dry biomass (in powder) using this method exceeds 95% and H. PLUVIALIS
in some occasions may approach 100% (Leach et al., 1998). After
spray drying, the moisture content in “red” biomass is lowered to Microalgae have been often proposed as third generation
about 5% (Pérez-López et al., 2014). The main drawbacks of spray feedstock for biofuel production that does not compete
drying include high operational costs and the risk of microalgae for freshwater or land resources (Daroch et al., 2013a).
pigments deterioration (Molina Grima et al., 2003). Freeze drying However, despite significant advances in recent years it
(lyophilization or cryodesiccation), involves the freezing of algal becomes apparent that cultivation of microalgae for the sole
cake, the technique causes less damage than spray drying, but it is purpose of biofuel production is unlikely to be possible
even more expensive, especially on a commercial scale (Milledge, unless a major low-energy breakthrough technologies in algae
2013). cultivation, dewatering, and harvesting are developed (Li et al.,
2015). In the meantime, microalgae are extremely important
Recovery of Astaxanthin producers of many high-value nutraceutical compounds such
Once the cell wall is disrupted and the biomass is fully dried, as polyunsaturated fatty acids or astaxanthin that can justify
the recovery of the desired product is possible. Astaxanthin is high cost of microalgae cultivation and processing technologies.
a lipophilic compound and can be dissolved in solvents and Integration of simultaneous production of numerous compounds
oils. There is an abundance of astaxanthin extraction methods within one system maximizing the benefits and limiting the costs
from H. pluvialis utilizing solvents, acids, edible oils, supercritical is called biorefining (Li et al., 2015). Taking into consideration
carbon dioxide (SC-CO2 ) as well as microwave-assisted and these findings H. pluvialis emerges as a very useful organism
enzyme-assisted approaches. Among the recovery methods used for the development of a dedicated microalgal biorefinery. It
solvent extraction and supercritical carbon dioxide (SC-CO2 ) fits numerous requirements of for the development of first
extraction are considered as the most efficient, compatible, and microalgal biorefineries especially the “high value product first”
widely used methods for astaxanthin extraction from H. pluvialis. principle (Li et al., 2015). First, H. pluvialis is the best-known
The summary of various extraction methods of astaxanthin producer of astaxanthin-high value product worth in excess of
from H. pluvialis with recent updates is presented in Table 5 several thousand US $ per kilogram. This product itself can
Supercritical carbon dioxide (SC- CO2 ) extraction has been easily justify expensive cultivation systems required for this
widely used for industrial applications due to its many processing organism. Second, H. pluvialis grown under nutrient starvation
advantages. Due to low critical temperature of carbon dioxide, conditions induces both carotenogenesis (astaxanthin formation)
the SC- CO2 system can be operated at moderate temperatures, and deposition of storage materials (triglycerides). It has been
preventing the degradation of valuable substances (Machmudah shown that these two responses are closely related and coincide in
et al., 2006). Several studies have reported experiments on both space and time and triglycerides are essential for deposition
supercritical CO2 extraction for the recovery of astaxanthin of astaxanthin inside lipid bodies to confer its protective function
from H. pluvialis. Considering astaxanthin quality as the most (Solovchenko, 2015). In traditional approaches of microalgae to
important criterion, supercritical CO2 extraction is the most biofuels starvation-induced lipid accumulation is considered as
favorable option. Supercritical CO2 provides shorter extraction significant challenge for commercialization of these systems as
time and limits the use of toxic organic solvents. By contrast the overall lipid productivity of culture can drop significantly due
to most solvents, CO2 is relatively cheap, chemically inert, to impaired growth rates under starvation conditions (Daroch
non-toxic, and stable (Guedes et al., 2011). Supercritical fluid et al., 2013b). In case of high value product like astaxanthin
extraction has also been tested with Haematococcus, aiming at this drop becomes much less of the burden as the high value
improving the extraction efficiency. For instance, supercritical of the main product will compensate for this delay in final
carbon dioxide (SC- CO2 ) coupled with ethanol or vegetable oil product formation. Due to the coexistence of astaxanthin and
as a co-solvent can further increase the extraction efficiency of triglycerides in space and time it is possible to simultaneously
astaxanthin (80–90%) (Nobre et al., 2006; Krichnavaruk et al., obtain high value product (astaxanthin) and a biofuel feedstock
2008). There is an array of alternative approaches that can (triglycerides) from a single algal feedstock. Since fatty acid
assist astaxanthin extraction from H. pluvialis such as solvents, content in the astaxanthin-containing ‘red’ cells can be as high
acids, edible oils, enzymes, or pressurized liquids (Sarada et al., as 30–60% of algae dry weight (Solovchenko, 2015) making
2006; Kang and Sim, 2008; In, 2009; Jaime et al., 2010; Zou H. pluvialis a very good candidate for biorefining strain. The
et al., 2013; Dong et al., 2014) Pressurized liquid extraction fatty acid profiles of the alga have been evaluated by several
has several advantages over traditional solvent extraction. PLE studies and are summarized in Table 2, indicating that fatty
requires shorter time, can be automated, uses less solvent, and acid profiles of the algae are suitable for biodiesel production

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Shah et al. Astaxanthin Production from H. pluvialis

TABLE 5 | Summary of astaxanthin extraction methods from H. pluvialis.

Astaxanthin Extraction/Purification method Astaxanthin yield/Extraction efficiency References

SC-CO2 at 20 MPa, 55◦ C and13% (w/w) ethanol for 120 min of extraction time. 83% recovery Reyes et al., 2014
CO2 expanded ethanol (50% %w/w ethanol), 7 MPa, 45◦ C, 120 min of extraction time. 124.2% recovery Reyes et al., 2014
SC- CO2 at at 20 MPa, 60◦ C, 2 ml of ethanol for 1 h of extraction time 2.45 mg/g DW Fujii, 2012
SC- CO2 , co-solvent 0.154–1% (v/v) ethanol, 7–34 MPa, 30–80◦ C, 0–100 min 74% recovery Pan et al., 2012
SC- CO2 , co-solvent 1.25–8.75% (v/v) ethanol, 30–50 MPa, 35–75◦ C, 210 min 87.4% recovery Wang et al., 2012
SC- CO2 , co-solvent 0–12% (v/v) vegetable oils, 30–50 MPa 50–80◦ C 300 min 51% recovery Krichnavaruk et al., 2008
SC- CO2 , co-solvent 30–50 MPa 40–80◦ C 60–240 min 84% recovery Thana et al., 2008
SC- CO2 , co-solvent,1.67–7.5% (v/v) ethanol 20–55 MPa 40–80◦ C, 240 min 80% recovery Machmudah et al., 2006
SC- CO2 , co-solvent 0, 10% (v/v) ethanol 20–30 MPa, 40–60◦ C 90% recovery Nobre et al., 2006
SC- CO2 , co-solvent 0, 9.4% (w/w) ethanol, 30 MPa at 60◦ C 97% recovery Valderrama et al., 2003
Cell germination (12 h), Ionic liquid (1-ethyl-3- methylimidazolium ethylsulfate) (24 h), 32.5 pg/cell Praveenkumar et al., 2015
Direct extraction using liquefid dimethyl ether (DME) at 0.59 MPa and 25◦ C, without 1 (mg/g cell) Boonnoun et al., 2014
drying, cell disruption, or heating,
HCl:acetone (5:5), 70◦ C, 20 min 19.8 mg/g cell Dong et al., 2014
Ultrasound in solvent (EtOH and EA),16 min, 41◦ C, 40 kHz, 200 W, EtOH: ethyl acetate 28 (mg/g) Zou et al., 2013
(20:1)
Grinding three repetitions, pressurized hexane (10.3 Mpa) 35 (mg/g cell) Jaime et al., 2010
Treating with enzymes (Viscozyme, Alcalase) at 50◦ C, 2 h 2649 ± 359 µg/g cell In, 2009
Dodecane mixing 48 h, saponification with methanolic NaOH (0.02 M), sedimentation in 85% efficiency Kang and Sim, 2008
darkness at 4◦ C, 12 h.
Acid digestion, 2 N HCl, 70◦ C. Acetone extraction for 1 h 87% efficiency Sarada et al., 2006
NaOH 30 min, Acetone (16 h) 7 (mg/g cell) Mendes-Pinto et al., 2001
40% (v/v) acetone for 2 min at 80◦ C, followed by lyophilization or treatment with specific 70% recovery Kobayashi et al., 1997c
lytic enzymes

SC, supercritical; EA, ethyl acetate.

(Damiani et al., 2010). Third, H. pluvialis have been found producing high value product (astaxanthin) and biofuel molecule
to be a mixotrophic alga what is highly advantageous for (biodiesel and/or biogas). The proposed biorefinery scheme
development of microalgae biorefineries. H. pluvialis is capable is presented on Figure 8 and employs a classical two stage
of utilizing carbon dioxide, carbonates, and carbohydrates as cultivation of H. pluvialis in green and red stage.
carbon sources, this opens a possibilities of lowering cultivation
costs and/or speeding up the cultivation of the strain through
using various waste streams like flue gasses or waste streams CURRENT GLOBAL MARKET AND
containing carbon and nutrient compounds (Wu et al., 2013). MARKET PLAYERS OF H. PLUVIALIS
Auto-, hetero-, and mixo-trophic cultivation modes require ASTAXANTHIN
energy and nutrients, both of which can be to an extent
recycled from anaerobic digestion process. Carbon sources vary Synthetic astaxanthin dominates current commercial market, of
depending on cultivation mode. Photoautotrophic cultivation the total value exceeding $200 million, corresponding to 130
requires CO2 that can be recycled from energy production metric tons of product per year (Li et al., 2011). According
at anaerobic digestion stage. Heterotrophic cultivation requires to recent reports, microalgae-derived astaxanthin corresponds
reduced carbon source—such as carbohydrates or acetate which to less than 1% of the commercialized quantity due to much
need to be supplied from alternative source. These compounds lower price of synthetic astaxanthin and technological problems
can also originate from waste streams. For example, food associated with large-scale algae cultivation (Koller et al., 2014;
industry is rich in carbohydrate-rich waste streams that can be Pérez-López et al., 2014). In recent years, there has been
used in heterotrophic cultivation of H. pluvialis (Wang, 2014). a growing trend toward using natural ingredients in food,
Mixotrophic cultivation can take advantage of both sources nutraceutical, and cosmetic markets, resulting from increasing
of carbon. After simultaneous extraction of both high value concerns for consumer safety and regulatory issues over the
product-astaxanthin and biofuel product-triglycerides algal cake introduction of synthetic chemicals into the human food chain.
composed of residual biomass can be utilized as a supplementary The demand for natural astaxanthin derived from H. pluvialis
feedstock for biogas production using anaerobic digestion that in the global market has been “sky-rocketing” in recent years
would further assist in extraction of residual energy from this owing to increasing consumer awareness of its health benefits.
integrated bioprocess. These three features of H. pluvialis make Global market for both synthetic and natural source astaxanthin
it a suitable strain for the development of algal biorefineries in aquaculture feed, nutraceuticals, cosmetics, and food and

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Shah et al. Astaxanthin Production from H. pluvialis

FIGURE 8 | Scheme of two stage cultivation H. pluvialis biorefinery producing high value compound astaxanthin and either edible oil or biofuel
compound-biodiesel. Green stage of cultivation can be performed using either photoautotrophic cultivation (deep green section) or hetero/mixotrophic cultivation
(pale green section) systems. Red stage cultivation (red section) takes place after green stage of H. pluvialis cultivation and is aimed to maximize astaxanthin content
Recycling of waste is performed through anaerobic digestion process. Following annotations are used: solid arrows—subsequent steps; dashed arrows—optional
steps; double lines—final products; double arrows—inputs; dotted lines—opportunities for recycling resources.

beverages is estimated at 280 metric tons valued at $447 million products is presented in Table 6. The size of the nutraceutical
in 2014. It is further projected to reach 670 metric tons astaxanthin market is growing day by day and this market is
valued at $1.1 billion by 2020 (Industry Experts, 2015; Panis, very attractive to Haematococcus astaxanthin producers since
2015). Synthetic astaxanthin, astaxanthin rich Phaffia yeast, and the price of these products is significantly higher than those
Paracoccus bacteria are predominantly used in the aquaculture of feed applications. Haematococcus producers need to invest
sector, while the astaxanthin derived from H. pluvialis is the their attention for increasing astaxanthin production capacity to
main source for human applications such as dietary supplements, meet the global demand. It is worthy to mention that several
cosmetics, and food and beverages. Nowadays, the estimated manufacturers already have doubled the cultivation capacities
market value of astaxanthin depending on products’ purity in recent years. Apart from an increase in existing players’
varies from $2500–7000/kg to about $15,000/kg pigment from capacities, new producers, such as BGG in China, have entered
H. pluvialis in some cases (Borowitzka, 2013; Koller et al., 2014; the market with significant production capacities.
Pérez-López et al., 2014; Industry Experts, 2015), while the
production cost is estimated at about $1000 per kg of astaxanthin
from H. pluvialis (Li et al., 2011). Natural astaxanthin is three MAJOR CHALLENGES FOR THE
to four times more valuable than the synthetic alternative in IMPROVEMENT OF H. PLUVIALIS
nutraceutical and pharmaceutical markets (Han et al., 2013).
BIOMASS AND ASTAXANTHIN
Since there is growing market demand for natural astaxanthin
for specific commercial applications (e.g., the nutraceuticals PRODUCTION
market) to replace the synthetic astaxanthin, mass cultivation of
There are many challenges and problems for the development
H. pluvialis in industrial scale has great potential and attractive
of large scale production of biomass and astaxanthin from
business opportunity. However, current market demand for
H. pluvialis. Due to these obstacles the productivity can be
natural astaxanthin is not met. It is expected that in the
hampered and in some cases a failure of the production system
foreseeable future after the optimization of the production
can make the production process economically unsustainable.
technology, the production costs of the natural astaxanthin from
Following issues are considered as most important challenges
H. pluvialis should be more competitive to these of the synthetic
for the development of H. pluvialis astaxanthin production
alternative (Pérez-López et al., 2014). Since the mid 1990’s, several
process:
leading companies are successfully producing H. pluvialis at
commercial scale and marketing natural astaxanthin from H. • Lack of effective solution to prevent or treat microbial
pluvialis worldwide. The list of the leading companies with their contaminations of mass cultures in a commercial scale.

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Shah et al. Astaxanthin Production from H. pluvialis

TABLE 6 | Leading commercial companies and their H. pluvialis-derived astaxanthin and related products in the world market.

Company Name Country Brand Name Product particulars

Cyanotech Corporation USA BioAstin® Astaxanthin extract packaged in soft gel,

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

• Slow cell growth rate, sensitivity of the cells to hydrodynamic CONCLUSION AND PERSPECTIVES
stress, and changes in cell morphology under various
environmental conditions. This review provides an insight about the latest scientific
• Inadequate and cost ineffective cultivation, drying, and and technological advancements in various aspects of
astaxanthin extraction technologies at the commercial scale. astaxanthin-producing microalga H. pluvialis such as cell
• Unavailability of genetically improved/engineered strains of biology, reproduction, biosynthesis pathway, stress mechanism,
H. pluvialis and genetic transformation tools for engineering biomass production, and downstream processing. It also
astaxanthin biosynthesis pathways in this organism for contemplates a broader image including potential benefits,
improved astaxanthin production. global market opportunities and integration of astaxanthin
• Lack of sufficient number of skilled workers in production production into biorefining. In recent years there is an
farms and insufficient collaboration between universities and increased interest for natural astaxanthin from green microalga
commercial enterprises. H. pluvialis. Wide ranges of scientific improvements have been
• Lack of adequate scientific research on the economic achieved during the last decade in terms of productivity and
performance and viability of commercial scale astaxanthin bioprocessing in order to obtain a refined astaxanthin product.
production process. Yet its commercial production, especially for low-end markets is

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Shah et al. Astaxanthin Production from H. pluvialis

too expensive for mass adoption of natural astaxanthin over its bead milling are the most common described methods for
synthetic counterpart. H. pluvialis has been shown to be cultured commercial scale astaxanthin production from H. pluvialis. After
in photoautotrophic, heterotrophic or mixotrophic growth cell walls disruption, biomass is usually processed by spray
conditions in various culture systems. Research have been drying or freeze drying. A number of astaxanthin extraction
conducted on the optimization of the various culture parameters, methods such as (solvents, acids, edible oils, supercritical carbon
such as growth medium composition, light, pH, temperature dioxide, microwave-assisted, and enzyme-assisted approaches
etc. to achieve high biomass and astaxanthin production. Most have been reported for H. pluvialis and supercritical carbon
of these parameters have been optimized and found different dioxide (SC- CO2 ) extraction has been widely used for industrial
for biomass accumulation and astaxanthin production. Little applications. Two recently developed methods allow efficient
can be done to address this limitatiation as it is funadamentally extraction of astaxanthin-containing lipids from wet biomass
connected with the life cycle of this microalgae. We believe there at yields comparable to conventional drying-solvent extraction
exist three key areas where further improvements are required method. Efficient extraction of astaxanthin from wet H. pluvialis
and interesting novel approaches have been recently developed: biomass was achieved with liquefied dimethyl ether (Boonnoun
cultivation efficiency and cost; good cultivation practice and et al., 2014) and also cell germination process in conjunction with
predator control; and astaxanthin isolation and purification. ionic liquids treatment (Praveenkumar et al., 2015).
First, due to complex life-cycle of H. pluvialis it is important Despite significant advances in research and development
to maximase cell densities of alga at “green stage” of cultivation to of H. pluvialis astaxanthin production is still in laboratory
maximize astaxanthin yield from the “red stage.” We think that stage and often faces difficulties to become implemented
a number of recent developments can make significant impact in large-scale commercial production. There is a number
in maximizing cell densities. Especially attached cultivation of other areas of improvement that will contribute to the
approach and a two-stage “perfusion culture” system can be expansion of Haematococcus production capacity, lowering
considered most promising due to the capability of producing the production cost, and increasing market penetration at
several fold higher biomass and astaxanthin productivity and low end applications. These include: next-generation culture
some other benefits such as lower water consumption and smaller systems along with advanced management practices; better
risk of contamination. These improvements may boost economic understanding of astaxanthin biosynthesis, metabolic pathways
benefits and reduce production cost of astaxanthin from H. and their regulation, genetic engineering, and omics-scale
pluvialis (Park et al., 2014; Wan et al., 2014b; Zhang et al., 2014). understanding of astaxanthin accumulation; development of
Alternatively, utilization of supplementary carbon source and genetic manipulation toolbox; exploration of integration of
adoption a two-stage sequential heterotrophic-photoautotrophic H. pluvialis cultivation with other processes. Yet, we firmly
approach could improve biomass and astaxanthin production. believe that three key areas of focus should be: cultivation
Especially utilization of waste carbon and nutrient sources efficiency and cost; good cultivation practice and predator
in biorefinery setup could help to decrease cultivation costs. control; and astaxanthin isolation and purification. Further
Unfortunately, these researches are still in laboratory stage and developments in these fields can have a profound effect on the
need to be tested in large-scale commercial production for further commercial deployment of H. pluvialis astaxanthin products and
validation. can act as a catalyst for the development of an entire microalgae
Second, control of contaminants, parasites, and predators industry in the near future.
remains to be primary concern for Haematococcus growers and
major issue in culture stability and astaxanthin productivity. AUTHOR CONTRIBUTIONS
Since there is very little that can be done once contamination
takes place it is important to limit the possibility of such MS, collected data, participated in preparation of draft
disruption and identify it as soon as possible, and avoid spreading manuscript, participated in assembly and editing of the final
to other parts of culture. Traditional detection methods such as manuscript; YML, collected data, participated in preparation of
microscopy and staining can be used to visualize algal parasites, draft manuscript; JJC, participated in assembly and editing of the
however this technique may be too labor intensive to perform final manuscript; MD, collected data, participated in preparation
on a routine basis for most commercial operations. For routine of draft manuscript, participated in assembly and editing of the
detection, more automated systems such as flow cytometry final manuscript.
would be ideal. Alternatively, molecular-based techniques that
are considered as the most informative and sensitive for the FUNDING
detection and identification of parasites. Following techniques
are worth further exploring DNA sequencing (Sanger, shotgun, Authors would like to acknowledge the support of
or next generation) and then monitoring for these specifically National Natural Science Foundation of China for Young
using qPCR or phylochip technology. Decreasing costs of next International Scientists Grant no. 31450110424 and
generation DNA sequencing can make DNA sequencing for 31550110497, Shenzhen Municipal Government for Special
culture diagnostic purposes more accessible in the near future. Innovation Fund for Shenzhen Overseas High-level Personnel
Third, combination of low cell densities and robust trilayer KQCX20140521150255300 and Shenzhen Knowledge and
cell walls of astaxanthin-containing aplanospores make isolation Innovation Basic Research Grant JCYJ20150626110855791,
of astaxanthin difficlut and expensive. Currentlly harvesting by State Ocean Administration Grant 201305022, and National
centrifugation, cell wall disruption by expeller pressing and Thousand People Plan Grant of Jay Jiayang Cheng.

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Shah et al. Astaxanthin Production from H. pluvialis

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Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) Conflict of Interest Statement: The authors declare that the research was
under nitrogen starvation or high light is correlated with that of conducted in the absence of any commercial or financial relationships that could
astaxanthin esters. J. Phycol. 38, 325–331. doi: 10.1046/j.1529-8817.2002. be construed as a potential conflict of interest.
01107.x
Zhekisheva, M., Zarka, A., Khozin-Goldberg, I., Cohen, Z., and Boussiba, S. Copyright © 2016 Shah, Liang, Cheng and Daroch. This is an open-access article
(2005). Inhibition of astaxanthin synthesis under high irradiance does not distributed under the terms of the Creative Commons Attribution License (CC BY).
abolish triacylglycerol accumulation in the green alga Haematococcus pluvialis The use, distribution or reproduction in other forums is permitted, provided the
(Chlorophyceae). J. Phycol. 41, 819–826. doi: 10.1111/j.0022-3646.2005.05015.x original author(s) or licensor are credited and that the original publication in this
Zou, T. B., Jia, Q., Li, H. W., Wang, C.-X., and Wu, H.-F. (2013). Response journal is cited, in accordance with accepted academic practice. No use, distribution
surface methodology for ultrasound-assisted extraction of astaxanthin from or reproduction is permitted which does not comply with these terms.

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