H. a specific operation. However, they all

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 negative conditions, thegreen cells transform  to the aplanospore stage asastaxanthin 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, phosphatelimitation or the adding inhibitors to synthesize high astaxanthin contents from the green cells to red cystsof cultivation. Each of manners has a specificoperation.

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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,  foundthat utilizing 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  indicated that during photoautotrophicinduction, the light intensity wasmore necessary than C/N ratio to enhanceH. pluvials astaxanthin synthesiswith continuous supplies CO2 and light.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. However, these methods consume high energy and undergo numerous steps.

Moreover, using petroleum-derived solvents for extraction astaxanthin causesnot only toxic-related health problems but also environmentally unfriendlyissues. The direct extractionof astaxanthin from Haematococcus by vegetative oils but for a cellharvest process step made downstream processing much easier than the othermethods as a simple and green extraction technique 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 The flaskswere incubated in a photoincubator (Vision Scientific, Korea) at 150 rpm and23°C (Fig. 1) The cool white fluorescent lamps contributed light at 50 ?molphoton 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. 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 The flaskswere incubated in a photoincubator (Vision Scientific, Korea) at 150 rpm and23°C (Fig. 1) The cool white fluorescent lamps contributed light at 50 ?molphoton 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 non-motile 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. 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 .Without a cell harvest step, the induced cyst culture brothwas straightforwardly  blended  with a commercial vegetable oil such assoybean oil, olive oil, corn oil andgrape oil. Red aplanospore cells were disrupted for isolation of theastaxanthin-containing oil extract during the forceful stirring of the mixture.Under gravity and water-hatinginteraction allowed vegetative oilsto separate from the culture media containing the cell fragments at room temperature. In generalastaxanthin extraction method,cell intake and extractioncollaborated into a unit process 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) The absorbance of the oil extract (toplayer) was scanned (400–700 nm) The peaks of astaxanthin were determined at 480nm compared with an authentic standard (Sigma, USA) ResultsOnerecent study of a science group researched relationship between C/N ratio andastaxanthin accumulation in aplanospore H.pluvialis cells stated the results that encystment production at zero extranitrate was more than that of in the nitrate addition culture; it only roseagain at lower C/N ratios added 1.0 and 2.0 mM nitrate. However, productiveastaxanthin at 2.

0 mM nitrate addition was less than at infinite C/N ratio asat no extra nitrate medium. Red cyst formation appeared after 2 days in lowerC/N ratio conditions determined by microscopic examination (Fig. 4).As shown in Fig, 6 in thedecreasing C/N ratios as increasing concentration of nitrate in the initialstage, 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 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. The result from theexperiment of  shown that the colorintensity depended on the isolated astaxanthin quantity that was deeper rednessin 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. 

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