Taxonomy of Haematococcus pluvialis

Haematococcus pluvialis Flotow Bainbridge Island, Washington, USA: birdbath.

Scientific Classification

The name H. pluvialis might have been commonly attached to any red-colored palmelloid
Haematococcus (Klochkova et al., 2013), while, coccus is the morphology of this cells. Pluvialis
is the latin word which mean produced by rain.

Green algae are part of a eukaryotic kingdom called the Plantae (Cavalier-Smith, 1998) or the
Archeoplastida (Adl et al., 2005), that is strongly supported in most phylogenies. This clade
originated with the primary endosymbiotic event between a protist and a cyanobacterium that
introduced photosynthesis to the Eukarya (Keeling et al., 2005) and displays impressive diversity
in cellular structure, physiology and ecology. The microalga H. pluvialis Flotow (Chlorophyceae,
order Volvocales) is the best source of natural astaxanthin (Margalith, 1999). To be simplified
the classification of this microalga as follow:
Empire Eukaryota

Kingdom Plantae

Phylum Chlorophyta

Class Chlorophyceae

Order Chlamydomonadales

Family Haematococcaceae

Genus Haematococcus

Species pluvialis (www.algaebase.org)

Physical Characteristic

H. pluvialis live in freshwater environment. Usually found in temperate regions around

the world. Their resting cysts are often responsible for the blood-red colour seen in the bottom of
dried out rock pools and bird baths. This colour is caused by astaxanthin which is believed to
protect the resting cysts from the detrimental effect of UV-radiation, when exposed to direct
sunlight (Dore and Cysewski, 2003).

The cells of H. pluvialis range from spherical to elliptical and are surrounded by a cell
wall, having inner and outer diameters of approximately 34 µm and 37.5 µm, respectively
(Iyengar & Desikachary 1981). The cells are composed of starch and haematochrome, and the
cells contain cup-shaped chloroplasts. This Chlorophycean has a thick trilaminar cell wall
composed of cellulose and sporopollenin (Mendes et al., 2001; Aflalo et al., 2007; Montsant et
al., 2001). The thick cell wall of the aplanospore of H. pluvialis could resist against mechanical
and chemical attack. The composition of its cell wall, similar to that of spores, makes this
microalga less permeable and extremely resistant to mechanical treatments (Hagen et al., 2002).
Other than that, algaenan has been identified in the akinetes of H. pluvialis (Blokker, 2000). This
species was grown commercially for astaxanthin and other carotenoids (Guiry, 2013). In natural
environment, accumulation of the astaxanthin is an adaptation to habitats that exhibit strong
radiation, in addition to the formation of cysts having rigid cell walls (Hagen et al. 1994, 2002,
Montsant et al. 2001).
The extracellular matrix of a young (1-week old) flagellate of H. pluvialis (Hagen C. et al. 2002)
CP (reticulate chloroplast), MI (mitochondria), PL (plasmalemma), TCL (intermediate tripartite
crystalline, W1 (inner), W7 (outer).

Astaxanthin (3,3'-dihydroxy-β, β-carotene-4,4'-dione) is a fat-soluble compound

classified into xanthophylls that are oxygenated derivatives of carotenoids (Akira Satoh et al.
2009). Beta Immune (2004) stated that astaxanthin is an oxygenated carotenoid pigment that is
a fat-soluble nutrient with a molecular weight of 596.8 Da. The astaxanthin which is a red
ketocarotenoid in H. pluvialis microalgae occurs in the esterified form, which is more stable than
the free astaxanthin form (http://www.algatech.com). Lorenz & Cysewski (2000) stated that H.
pluvialis is one of the natural sources of highly concentrated astaxanthin and having high
antioxidant, containing 1.5-3.0% astaxanthin by dry weight.

Life Cycle of Haematococcus Pluvialis

The life cycle of H. pluvialis consists of four cell stages, vegetative cell growth,
ecystment, maturation and germination (Kobayashi et al. 1997). The duration of the life cycle is
two weeks. Each algal stage could be distinguised by the ration of pigments
(carotenoid/chlorophyll) and the intracellular pprotein content.
 Schematic diagram of the model life cycle of H. pluvialis (Kobayashi et al. 1997)

Margalith (1999) stated that during the vegetative cell growth stage, green flagellate cells
are mobile in a growth medium and reproduction occurs by cell division. Under adverse
environmental conditions such as high irradiance and nitrogen deprivation, cells are immobilized
forming non-motile resting cells by shedding their flagella and green vegetative cells are
transformed into brown immature cyst cells throughout the encystment stage (Fabregas et al.
2001; Kobayashi et al. 1997; Margalith 1999; Olaizola 2000). The cyst cells are called
aplanospores or hypnoblasts (Ettl 1988). During the maturation stage, the brown aplanospores
are enlarged with a thicker cell wall and the aplanospores color changes to red, accumulation of
secondary carotenoids (Boussiba 2000). This red color signifies the accumulation of astaxanthin
(Margalith 1999). At the end of the maturation stage, over 99% of the carotenoids in the
aplanospores are composed of astaxanthin. Kobayashi et al. (1997) and Margalith (1999)
reported that mature cysts release daughter cells and leave behind the cell wall throughout the
germination stage in a fresh medium. From here, the vegetative cells regenerated from the
daughter cells grew mixotrophically.

In addition, Hagen et al. (2001) stated that flagellates are formed by germination of
resting cells when environmental conditions become favourable. The flagellates exhibit a
voluminous, transparent and gelatinous-looking cell wall characteristic of volvocalean motile
cells. Two-three weeks after flagellates had emerged from resting cells, morphogeniesis into
aplanospores with a rounding off of the protoplasts and continued by loss of motility and
formation of new rigid cell wall within the former flagellate extracellular matrix. The dry matter
of the perchlorate-soluble part of flagellate extracellular matrix consisted of 19% carbohydrates
(18% hexoses) and 75% proteins and contained neither cellulose nor acetolysis-resistant material.
In flagellates older than 2 weeks, vesicles filled with electron-dense granular material having
changes where formation of a new two-layered amorphous primary wall by secretion into
innermost fibrous layer of inner flagellate extracellular matrix.

During the life cycle of the alga, vegetative cells contained high levels of chlorophyll an
dprotein but had very low carotenoid contents whereas encystment was accompanied by the
degradation of chlorophyll and protein. The maturation of cyst cells was accompanied by
enhanced carotenoid biosynthesis and accelerated protin degradation. Germination coincided
with chlorophyll and protein syntheses and carotenoid degradation (Kobayashi et al. 1997).

Effect of Environment Towards Haematococcus Pluvialis

 Nutrient Conditions

H. pluvialis is an organism living in freshwater environment. Under optimal growth

condition, vegetative cells of the algae persist and the alga possesses carotenoids normally found
in the Chlorophyta such as β-carotene, lutein, neoxanthin, violaxanthin and zeaxanthin (Mark
Harker et al. 1995). However, exposing H. pluvialis towards growth-limiting condition could
stimulate the vegtative cells to synthesize astaxanthin. Such growth-limiting condition is the
nutrient condition. Phosphate starvation has been reported previously to act as a trigger for the
accumulation of astaxanthin (Boussiba & Vonshak 1991) However, Borowitzka et al. (1991)
suggested that high amount of phosphate will stimulate the production of astaxanthin within
algal cells. Experiment done by Mark Harker et al. (1995) showed that highr concentrations of
phosphate will increase the cell number of H. pluvialis yet produce low astaxanthin. In contrast
for nitrate, Boussiba et al. (1992) reported that nitrogen is an important requierement for
astaxanthin synthesis. Mark Harker et al. (1995) did an experiment where the results are in
agreement with Spencer (1989) and Goodwin & Jamikorn (1954). It showed that at lower nitrate
concentrations, algal growth was limited severely while the astaxanthin produce is high
accumulated in the surviving cells. While presence of ferous in low levels of amount produced
little change of cell growth and astaxanthin. Kobayashi et al.(1991) studies showed that
astaxanthin formation elevated by increased in iron concentrations. In addition of Kobayashi et
al. (1991), the formation of astaxanthin in H. pluvialis stimulated by four active oxygen species
such as O2, H2O2, peroxyl radical and superoxide anion radical.

 Salinity

Salinity is the saltiness or dissolved salt content of a body of water. Such compound that
contribute to salinity are sodium chloride, magnesium sulfate, potassium nitrate and sodium
bicarbonate. Mark Harker et al. (1995) experiment showed that increases in the salinity by
addition of sodium chloride and potassium chloride resulted in initiation of astaxanthin
formation in the alga which at 100 mM sodium chloride produce highest level of astaxanthin.
The exposure of H. pluvialis to increased salinity has been reported to induce astaxanthin
formation in the alga (Borowitzka et al. 1991; Boussiba & Vonshak 1991; Spencer 1989).
However, increasing in salinity were accompanied by high rates of cell mortality.

 Temperature

In the cultivation of H. pluvialis, temperature is one important and basic factor

influencing biomass concentration and astaxanthin content due to seasonal and diurnal
fluctuations ( Borowitzka et al., 1991, Fan et al., 1994, Harker et al., 1995 and Tripathi et al.,
2002). It has been widely reported that the suitable temperature for the astaxanthin accumulation
of H. pluvialis was between the 20 °C and 28 °C ( Fan et al., 1994 and Jiang et al., 2005; Kang et
al., 2005). Based on the experiment of effect of temperature towards production of astaxanthin
(Minxi Wan et. al 2014), the highest net biomass and astaxanthin productivities were 0.12 g/L/d
and 5.4 mg/L/d at 28 °C was the highest, followed by that at 23 °C, and biomass decreased with
the lowering temperature. Yet, it was reported that H. pluvialis can accumulate astaxanthin at
35 °C ( Tjahjono et al., 1994). In this study of Minxi Wan et. al (2014), the cell was lysed
gradually, and was proved to be death at 33 °C may be because of the inter-specific difference.

 Light Intensity

Light intensity is the most important factor when considering the physiological and
metabolic activities of this microalga (A. Monsant 2001). Thus, light is needed by H. pluvialis to
increase its biomass and the astaxanthin production. Yet, if the amount of light is insufficient, H.
pluvialis will turn to heterotroph mode where it gets energy by eating other organisms instead of
photosynthesis process. Mark Harker et al. (1995) showed that high light intensities caused large
quantities of astaxanthin to be accumulated in H. pluvialis though it resulted in high rates of cell
mortality. Kobayashi et al. (1992) reported that astaxanthin production could be enhanced when
alga grownunder blue light as opposed to white or red light. Furthermore, continuous
illumination rather than light or dark illumination cycles are more favourable for astaxanthin
formation.As other the experiment done by Tomohisa Katsuda et. al (2008), with continuous
light, the astaxanthin produce by H. pluvialis increases more linear before reaching plateau
compared to flashing light. The amount of flashing light could affect too as lower in the intensity
of flashing light will give low amount of astaxanthin produce by H. pluvialis. For instance,
formation of a trilaminar sheath containing sporopollenin-like material (algaenan) in the resting
cell state might be related to an increased resistance to ultraviolet radiation (Kobayashi & Okada
2000)
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