H. pluvialis cell has unique life cycle consisting

pluvialis cell has unique
life cycle consisting of two main types of distinct cellular morphologies:
“green motile phase” and “red nonmotile phase” (Hazen, 1899; Elliot, 1934).
Under optimal growing cocitions,
H. pluvialis cells are green without astaxanthin
accumulation (Shar, 2016).
Under unfavorable conditions, these
cells transforms from the
green vegetative stage to the aplanospore stage as astaxanthin
synthesized encysted phase, which is the red color. (Boussiba & Vonshak, 1991; Kobayashi et al., 1991, 1993,
1997a, 1997b; Harker, Tsavalos,
& Young, 1996;
Fábregas, Otero, Maseda, &
Domínguez, 2001; Margalith, 1999; Hata et al., 2001; Sarada, Tripathi, & Ravishankar,
2002; Domínguez-Bocanegra, Legarreta, Jeronimo, & Campocosio, 2003; Wang & Zarka, 2003).

There are many induction methods such as lack of nitrogen,
salt stress-inducing, strong light intensity, surplus acetate, phosphate
limitation or the adding inhibitors to synthesize high astaxanthin contents from the green cells to red cysts
of cultivation. (Boussiba & Vonshak, 1991; Kobayashi et al., 1993, 1997a,
1997b; Harker et al., 1996; Fábregas et al., 2001; Margalith 1999; Hata et al.
2001; Sarada et al., 2002; Wang & Zarka, 2003).

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Each of manners has a specific
operation. However, they all begin from the same principle of promoting the
accelerated cell morphology changes by stress-inducing conditions. In a
previous study, preventing heterotrophic contamination from the addition of
carbon sources as using acetate, Kang, Lee, Park, and Sim (2005) found that
utilizing CO2 gas supplemented with strong light intensity during photoautotrophic induction was more
efficient for H. pluvialis
astaxanthin accumulation than heterotrophic induction. Increasing the light
intensity from 200 to 300 ?mol photon m-2 s-1 boosted astaxanthin quantity at
low C/N ratio. A science group consisted of Kang, Lee, Park, and Sim (2007a)
indicated that during photoautotrophic induction, the light intensity was more necessary than C/N ratio to enhance H. pluvials astaxanthin synthesis with continuous supplies CO2 and

Many extraction methods, such as
cryogenic grinding, enzyme lysis, spray drying, mechanical disruption, and acid
or base substances, have been commonly utilized to isolate astaxanthin from red
cysts. (Kobayashi et al. 1997b; Mendes-Pinto,
Raposo, Bowen, Young, & Morais, 2001; Machmudah, Shotipruk, Goto, Sasaki, & Hirose,
2006; Sarada et al., 2006). However, these methods consume high energy and
undergo numerous steps. Moreover, using petroleum-derived solvents for
extraction astaxanthin causes not only toxic-related health problems but also
environmentally unfriendly issues. The
direct extraction of astaxanthin from Haematococcus
by vegetative oils but for a
cell harvest process step made downstream processing much easier than the other
methods as a simple and green extraction technique (Kang & Sim, 2007a; Chemat, Vian, & Cravotto, 2012).

pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH

operating 250 ml Erlenmeyer ?asks
containing 130 ml medium aerated with5% CO2 in air at 65 ml min-1 (Hata et al.,
2001; Kang et al., 2007a). The flasks were incubated in a photoincubator
(Vision Scientific, Korea) at 150 rpm and 23°C (Fig. 1) (Kang et al. 2007a).
The cool white fluorescent lamps contributed light at 50 ?mol photon m-2 s-1   with a dark/light cycle of 12:12 h. When a
culture grasped the immobile stage due to nitrogen source exhaustion. The cells
began the cellular morphology transformation from the green motile phase to the
red nonmotile phase that accumulated high astaxanthin contents by a
high-intensity photoincubator. The culture was further incubated under
unsynchronized illumination with 200 ?mol photon m-2 s-1
of light for 7 days. (Kang & Sim, 2007a).

pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH

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