H. the light intensity was more necessary

H.
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, the
green cells transform

 to the aplanospore stage as
astaxanthin synthesized encysted phase, which is the red color.

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.

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,  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  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 light.

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. 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

H.
pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH
7.5)

operating 250 ml Erlenmeyer ?asks
containing 130 ml medium aerated with5% CO2 in air at 65 ml min-1 The flasks
were incubated in a photoincubator (Vision Scientific, Korea) at 150 rpm and
23°C (Fig. 1) 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 encysted 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.

H.
pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH
7.5)

operating 250 ml Erlenmeyer ?asks
containing 130 ml medium aerated with5% CO2 in air at 65 ml min-1 The flasks
were incubated in a photoincubator (Vision Scientific, Korea) at 150 rpm and
23°C (Fig. 1) 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 non-motile 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.

After ten-day red cysts cultured in
induction conditions with low C/N ratio and high-intensity light from 200 to
300 ?mol ?mol photon m-2 s-1 separated by a single unified process to collect astaxanthin contents .Without a cell harvest step, the induced cyst culture broth
was straightforwardly  blended  with a commercial vegetable oil such as
soybean oil, olive oil, corn oil and
grape oil. Red aplanospore cells were disrupted for isolation of the
astaxanthin-containing oil extract during the forceful stirring of the mixture.
Under gravity and water-hating
interaction allowed vegetative oils
to separate from the culture media containing the cell fragments at room temperature. In general
astaxanthin extraction method,
cell intake and extraction
collaborated into a unit process

Using ion chromatography analyzed
inorganic compounds in the culture medium after filtration through a membrane
filter. A DIONEX 500 IC system (Dionex, USA) quantified nitrate. Astaxanthin
concentrations were evaluated by a Shimadzu

high-performance liquid
chromatography system (Shimadzu, Japan) The absorbance of the oil extract (top
layer) was scanned (400–700 nm) The peaks of astaxanthin were determined at 480
nm compared with an authentic standard (Sigma, USA)

Results

One
recent study of a science group researched relationship between C/N ratio and
astaxanthin accumulation in aplanospore H.
pluvialis cells stated the results that encystment production at zero extra
nitrate was more than that of in the nitrate addition culture; it only rose
again at lower C/N ratios added 1.0 and 2.0 mM nitrate. However, productive
astaxanthin at 2.0 mM nitrate addition was less than at infinite C/N ratio as
at no extra nitrate medium. Red cyst formation appeared after 2 days in lower
C/N ratio conditions determined by microscopic examination (Fig. 4).

As shown in Fig, 6 in the
decreasing C/N ratios as increasing concentration of nitrate in the initial
stage, the biomass of the culture increased from 2.75 to 4.73g l-1.
However, astaxanthin quantity was retained 60 mg g-1 biomass. During
the second 9-day period, under supplemental light, astaxanthin productive
concentration was remarkably enhanced to 313 mg l-1 in high-density
cultures with both the low C/N ratio and continuous input of both CO2–air
mixture

As a result, 85 mg productive astaxanthin l-1
from the red aplanospore cells was extracted into each of the prepared
vegetative oils after the cyst cells had been broken into cell detritus.
Accumulated astaxanthin contents contained 70% monoesters, 25% diesters, and 5%
free forms tended to combine with or dissolve in lipids or fats of
Haematoccocus

astaxanthin. The result from the
experiment of  shown that the color
intensity depended on the isolated astaxanthin quantity that was deeper redness
in high concentration (Figs. 1 and 2) after derivation for 48h with 87.5%
yields (table 1) which indicated that astaxanthin was absorbed at 480 nm.