H. pluvialis cell has a 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 conditions, H. Pluvialis cells are green without astaxanthin accumulation (Shar, 2016). Under unfavorable conditions, these cells transform 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).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 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. (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).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 (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).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 (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).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 (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. 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 (Kang & Sim, 2007b; Chemat et al., 2012).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 Shimadzuhigh-performance liquid chromatography system (Shimadzu, Japan) (Kang et al.
, 2007a). The absorbance of the oil extract (top layer) was scanned (400–700 nm) (Britton et al. 2004). The peaks of astaxanthin were determined at 480 nm compared with an authentic standard (Sigma, USA) (Yuan & Chen, 1998, 2000; Šesták, Britton, Liaaen-Jensen, & Pfander, 2004; Kang et al., 2005).
ResultsOne recent study of a science group (Kang et al., 2007a) researched relationship between C/N ratio and astaxanthin accumulation in aplanospore H. Pluvialis cells stated the results that encystment production at zero extra nitrates 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 (Kang et al., 2007a) 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 (Zhang, Wang, Hu, Sommerfeld, & Han, 2016).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 Haematoccocusastaxanthin (Lorenz & Cysewski, 2000; Hussein, Sankawa, Goto, Matsumoto, Watanabe, 2006). The result from the experiment of Kang and Sim (2007a) 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.
