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MediumOptimizationandInducingConditionsOptimizationofHaematococcuspluvialisLIUKehuan1,BAIHuan2,MALi1etal (1.CollegeofLifeSciences,SichuanUniversity,Chengdu,Sichuan610064;2.ElectricPowerResearchInstituteofSichuanElectricPowerCompany,Chengdu,Sichuan610072)Abstract [Objective]MediumforculturingHaematococcuspluvialisoriginatedfromlaboratorywasscreened,accumulationconditionsofastaxanthininHaematococcuspluvialiswereoptimizated.[Method]SevenculturemediumswereusedtocultivatefourkindsofHaematococcuspluvialis,4kindsofmediumswereobtained.AstaxanthinaccumulationinHaematococcuspluvialiswereexposedunderlightinduced,highsalt,andnitrogendeficientrespectively.[Result]BiomassaboutfourstrainsalgaeintheformulaAmediumwas165-7.30timesofthatintheothersixmediums,HP3gainedthelargestbiomassinformulaA,themaximumbiomasswasupto7.13×106cell/mL.Undernitrogendeficiencycondition,theastaxanthinproductionwasupto21.05mg/L,thisnumberwas1.07timesofthebestastaxanthinyieldobtainedbylight,and1.64timesofthemaximumastaxanthinyieldobtainedbyhighsalt.[Conclusion]ThemostsuitableHaematococcuspluvialismediumwasformulaAandtheregnantastaxanthinaccumulationconditionwas10%inthisstudy.Keywords Haematococcuspluvialis;Astaxanthin;Mediumoptimization;Stressconditions
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[1]FASSETTRG,COOMBESJS.Astaxanthinincardiovascularhealthanddisease[J].Molecules,2012,17(2):2030-2048.
[2]HANDX,LIYT,HUQ.Astaxanthininmicroalgae:Pathways,functionsandbiotechnologicalimplications[J].Algae,2013,28(2):131-147.
[3]AMBATIRR,PHANGSM,RAVIS,etal.Astaxanthin:Sources,extraction,stability,biologicalactivitiesanditscommercialapplications:Areview[J].Marinedrugs,2014,12(1):128-152.
[4]P?REZL?PEZP,GONZ?LEZGARC?AS,JEFFRYESC,etal.Lifecycleassessmentoftheproductionoftheredantioxidantcarotenoidastaxanthinbymicroalgae:Fromlabtopilotscale[J].Journalofcleanerproduction,2014,64:332-344.
[5]LIJ,ZHUDL,NIUJF,etal.AneconomicassessmentofastaxanthinproductionbylargescalecultivationofHaematococcuspluvialis[J].Biotechnologyadvances,2011,29(6):568-574.
[6]KOLLERM,MUHRA,BRAUNEGGG.Microalgaeasversatilecellularfactoriesforvaluedproducts[J].Algalresearch,2014,6:52-63.
[7]MILLEDGEJJ.Commercialapplicationofmicroalgaeotherthanasbiofuels:Abriefreview[J].Reviewsinenvironmentalscience&bio/technology,2011,10(1):31-41.
[8]PANISG,CARREONJR.CommercialastaxanthinproductionderivedbygreenalgaHaematococcuspluvialis:Amicroalgaeprocessmodelanda
technoeconomicassessmentallthroughproductionline[J].Algalresearch,2016,18:175-190.
[9]BARROSMP,POPPESC,SOUZAJUNIORTP.Putativebenefitsofmicroalgalastaxanthinonexerciseandhumanhealth[J].Revistabrasileiradefarmacognosia,2011,21(2):283-289.
[10]YINSC,WANGJF,CHENL,etal.ThewaterfootprintofbiofilmcultivationofHaematococcuspluvialisisgreatlydecreasedbyusingsealednarrowchamberscombinedwithslowaerationrate[J].Biotechnologyletters,2015,37(9):1819-1827.
[11]DRAGO爦N,BERCEAV,BICAA,etal.Astaxanthinproductionfromanewstrainofhaematococcuspluvialisgrowninbatchculture[J].Annalsoftheromaniansocietyforcellbiology,2010,15(2):353.
[12]SOLOVCHENKOAE,CHIVKUNOVAOB,MASLOVAIP.Pigmentcomposition,opticalproperties,andresistancetophotodamageofthemicroalgaHaematococcuspluvialis,cultivatedunderhighlight[J].Russianjournalofplantphysiology,2011,58(1):9-17.
[13]CHOIYE,YUNYS,PARKJM,etal.DeterminationofthetimetransferringcellsforastaxanthinproductionconsideringtwostageprocessofHaematococcuspluvialis,cultivation[J].Bioresourcetechnology,2011,102(24):11249-11253.
[14]UìV
,Wo¿
,2@X
,°
.ÛYZ[\.]Û%^__`3ôab
&cd
[J].IJef
,2016(8):29-32.[15]
ùgò
,#D
,hij
,°
.k-
、l-
、mnH-9
pH5io\p_
�¹.�F
[J].ioRqr
,2001,32(1):15-18.[16]CORDEROBF,COUSOI,LE?NR,etal.Enhancementofcarotenoids
biosynthesisinChlamydomonasreinhardtiibynucleartransformationusingaphytoenesynthasegeneisolatedfromChlorellazofingiensis[J].Appliedmicrobiology&biotechnology,2011,91(2):341-351.
[17]DEMMIGADAMSB,ADAMSW W.Antioxidantsinphotosynthesisandhumannutrition[J].Science2002,298(5601):2149-2153.
[18]fs
,'>½
,tBu
,°
.]Û%^_vw^xNOyz&].{|
R\}~�
[J].)ÛÛ�CD
,2012,36(4):640-645.[19]KIMJH,AFFANA,JANGJ,etal.Morphological,molecular,andbio
chemicalcharacterization ofastaxanthinproducing green microalgaHaematococcussp.KORDI03(Haematococcaceae,Chlorophyta)isolatedfromKorea[J].JMicrobiolBiotechnol,2015,25(2):238-246.
[20]�'ä
,ù��
,�s
,°
.mnH-9k-5]Û%^_Û¹
、Z[\
3�Â�R���.ßà
[J].IJioBCCD
(��iCj
),2016,46(8):28-36.
[21]���
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[J].��iÅ
,2013,38(1):17-20.[22]LEMOINEY,SCHOEFSB.Secondaryketocarotenoidastaxanthinbiosyn
thesisinalgae:Amultifunctionalresponsetostress[J].Photosynthesisresearch,2010,106(1/2):155-177.
[23]WAYAMAM,OTAS,MATSUURAH,etal.Threedimensionalultrastructuralstudyofoilandastaxanthinaccumulationduringencystmentinthegreenalga,Haematococcuspluvialis[J].PLoSOne,2013,8(1):1-9.
[24]BOROWITZKAMA,HUISMANJM,OSBORNA.CultureoftheastaxanthinproducinggreenalgaHaematococcuspluvialis1.Effectsofnutrientsongrowthandcelltype[J].Journalofappliedphycology,1991,3(4):295-304.
[25]öD�
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[J].¡Û�C¢D
,2007,34(2):256-260.[26]MARKOUG,NERANTZISE.Microalgaeforhighvaluecompoundsand
biofuelsproduction:Areviewwithfocusoncultivationunderstressconditions[J].Biotechnologyadvances,2013,31(8):1532-1542.
[27]TOUCHETTEBW,BURKHOLDERJM.Reviewofnitrogenandphosphorusmetabolisminseagrasses[J].Journalofexperimentalmarinebiology&ecology,2000,250(1):133-167.
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