H. for H. pluvialis astaxanthin accumulation than heterotrophic induction.

H.pluvialis cell has uniquelife 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 astaxanthinaccumulation (Shar, 2016).Under unfavorable conditions, thesecells transforms from thegreen vegetative stage to the aplanospore stage as astaxanthinsynthesized 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, phosphatelimitation or the adding inhibitors to synthesize high astaxanthin contents from the green cells to red cystsof cultivation. (Boussiba & Vonshak, 1991; Kobayashi et al.

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, 1993, 1997a,1997b; Harker et al., 1996; Fábregas et al., 2001; Margalith 1999; Hata et al.

2001; Sarada et al., 2002; Wang & Zarka, 2003).Each of manners has a specificoperation. However, they all begin from the same principle of promoting theaccelerated cell morphology changes by stress-inducing conditions.

In aprevious study, preventing heterotrophic contamination from the addition ofcarbon sources as using acetate, Kang, Lee, Park, and Sim (2005) found thatutilizing CO2 gas supplemented with strong light intensity during photoautotrophic induction was moreefficient for H. pluvialisastaxanthin accumulation than heterotrophic induction. Increasing the lightintensity from 200 to 300 ?mol photon m-2 s-1 boosted astaxanthin quantity atlow 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 andlight.Many extraction methods, such ascryogenic grinding, enzyme lysis, spray drying, mechanical disruption, and acidor base substances, have been commonly utilized to isolate astaxanthin from redcysts.

(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 andundergo numerous steps.

Moreover, using petroleum-derived solvents forextraction astaxanthin causes not only toxic-related health problems but alsoenvironmentally unfriendly issues. Thedirect extraction of astaxanthin from Haematococcusby vegetative oils but for acell harvest process step made downstream processing much easier than the othermethods as a simple and green extraction technique (Kang & Sim, 2007a; Chemat, Vian, & Cravotto, 2012).H.pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH7.5)operating 250 ml Erlenmeyer ?askscontaining 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 aculture grasped the immobile stage due to nitrogen source exhaustion. The cellsbegan the cellular morphology transformation from the green motile phase to thered encysted phase that accumulated high astaxanthin contents by ahigh-intensity photoincubator. The culture was further incubated underunsynchronized illumination with 200 ?mol photon m-2 s-1of light for 7 days.

(Kang & Sim, 2007a).H.pluvialis NIES-144 was cultured photoautotrophically in NIES-C medium (pH7.5)operating 250 ml Erlenmeyer ?askscontaining 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 aculture grasped the immobile stage due to nitrogen source exhaustion. The cellsbegan the cellular morphology transformation from the green motile phase to thered nonmotile phase that accumulated high astaxanthin contents by ahigh-intensity photoincubator. The culture was further incubated underunsynchronized illumination with 200 ?mol photon m-2 s-1of light for 7 days. (Kang & Sim, 2007a).After ten-day red cysts cultured ininduction conditions with low C/N ratio and high-intensity light from 200 to300 ?mol ?mol photon m-2 s-1 separated by a single unified process to collect astaxanthin contents(Kang & Sim, 2007a; Kang et al.

, 2007a; Cuellar-Bermudez, 2014). 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. Redaplanospore cells were disrupted for isolation of the astaxanthin-containingoil extract during the forceful stirring of the mixture. Under gravity and water-hating interaction allowed vegetative oils to separate fromthe culture media containing the cell fragments at room temperature. In general astaxanthin extraction method, cell intake and extraction collaborated into aunit process (Kang & Sim, 2007b; Chemat et al., 2012).Using ion chromatography analyzedinorganic compounds in the culture medium after filtration through a membranefilter. A DIONEX 500 IC system (Dionex, USA) quantified nitrate.

Astaxanthinconcentrations were evaluated by a Shimadzuhigh-performance liquidchromatography system (Shimadzu, Japan) (Kang et al., 2007a). The absorbance ofthe oil extract (top layer) was scanned (400–700 nm) (Britton et al. 2004). Thepeaks of astaxanthin were determined at 480 nm compared with an authenticstandard (Sigma, USA) (Yuan & Chen, 1998, 2000; Šesták, Britton, Liaaen-Jensen, & Pfander, 2004;Kang et al., 2005).ResultsOnerecent study of a science group (Kang et al.

, 2007a) researched relationshipbetween C/N ratio and astaxanthin accumulation in aplanospore H. pluvialis cells stated the resultsthat encystment production at zero extra nitrate was more than that of in thenitrate addition culture; it only rose again at lower C/N ratios added 1.0 and2.0 mM nitrate.

However, productive astaxanthin at 2.0 mM nitrate addition wasless than at infinite C/N ratio as at no extra nitrate medium. Red cystformation appeared after 2 days in lower C/N ratio conditions determined bymicroscopic examination (Fig. 4).As shown in Fig, 6 (Kang et al.,2007a) in the decreasing C/N ratios as increasing concentration of nitrate inthe 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.

Duringthe second 9-day period, under supplemental light, astaxanthin productiveconcentration was remarkably enhanced to 313 mg l-1 in high-densitycultures with both the low C/N ratio and continuous input of both CO2–airmixture (Zhang, Wang, Hu,Sommerfeld, & Han, 2016).As a result, 85 mg productive astaxanthin l-1from the red aplanospore cells was extracted into each of the preparedvegetative 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 ofHaematoccocusastaxanthin (Lorenz & Cysewski,2000; Hussein, Sankawa, Goto, Matsumoto, Watanabe, 2006). The result from theexperiment of Kang and Sim (2007a) shown that the color intensity depended onthe isolated astaxanthin quantity that was deeper redness in high concentration(Figs. 1 and 2) after derivation for 48h with 87.

5% yields (table 1) whichindicated that astaxanthin was absorbed at 480 nm.