自养_异养和混养下小球藻的生长及生化成分_英文_
第32卷第5期2004年5月
华南理工大学学报(自然科学版)JournalofSouthChinaUniversityofTechnology
(NaturalScienceEdition)
Vol.32 No.5
May 2004
ArticleID:1000-565X(2004)05-0047-04
GrowthandBiochemicalComponentsofChlorellaUnderAutotrophic,Heterotrophicand3
WangHai2ying GuoSi22zhi
(ResearchInstituteofLight,SouthChinaUniv.ofTech.,
,uangdong,China)
Abst:paperarethegrowthandbiochemicalcomponents,suchassolubleprotein,soluble
sugar,andfattyacidcompositionofChlorellavulgaris,underautotrophic,heterotrophicandmixotrophiccultivations.TheresultsshowthatthebiomassofChlorellavulgarisunderheterotrophicandmixotrophiccultivationsismuchhigherthanthatunderautotrophiccultivation,thatthespecificgrowthratesarerespectively2.13and3timesthatunderautotrophicgrowth,andthatthegrowthofcellsundermixotrophiccultivationisaffectedbyilluminence,buttheeffectisnotsignificantattheilluminancesof2.5and4.0klx,especiallyinthestationaryphase.Comparedwithautotrophicgrowth,thecontentoffatunderheterotrophiccultivationobviouslyincreases,whilethecontentsofsolubleproteinandsaccharidedecrease,especiallychlorophyll.
Heterotrophiccultivationishelpfultothe
accumulationoflinolenicacid.Theadoptionoflightmakessolubleproteins,saccharidesandchlorophyllincreaseundermixotrophicgrowth,andtheilluminencealsohaseffectonthebiochemicalcomponentofChlorellavulgaris.Keywords:Chlorellavulgaris;biochemicalcomponent;growth;autotrophiccultivation;heterotrophiccultivation;
mixotrophiccultivation
CLCnumber:Q815 Documentcode:A
0 Introduction
Microalgaehavefoundwidepracticalapplicationinbiologicalengineering,suchashealthyfood,agriculture,wastewatertreatment,aquacultureandfoodadditives.Theyarealsoofgreatvaluetothesourcesofchemicalsandpharmaceuticals
[1]
vitalfunctionsandgreatcommercialvalue.Itisoneoftypicalmicroalgaestrainsthatarebothphoto2autotrophicandheterotrophicnutrition.Inthispaper,C.vulgariswasusedasanexperimentalstraintoinvestigateitsgrowthandbiochemicalcomponentsunderdifferentcultivations,suchasautotrophiccultivation(AC),heterotrophicculti2vation(HC)andmixo2trophiccultivation(MC).
.
Chlorellavulgaris(C.vulgaris),akindofunicel2
lulargreenalga,whichcontainshighlynutritioussub2stances,isofvariousbiologicaleffectsaswellasalotof
1 MaterialsandMethods
1.1 StrainandItsCultivation
ThestrainofC.vulgariswassuppliedbythefer2mentationlabintheCollegeofFoodandBiologicalEngi2neering,SouthChinaUniv.ofTech.,withBa2salmedi2umused[1].ForAC,thecellswereillumina2tedwithflo2
rescentlamps(4.0klxonthebottlesurface).Themassconcentrationofglucoseasheterotrophiccarbonsourceinthedarkwas10g/L.ForMC,thecellswereincubatedinheterotrophicme2diumwithillumination.Illuminence
Receiveddate:October29,2003
3Foundationitem:SupportedbytheKeyResearchGrantfrom
ScienceandTechnologyCommitteeofGuangdongProvince(99M01409G)
Biography:WangHai2ying(bornin1974),female,Ph.D.
candidate,mainlyresearchesonmicroalgaebiologicaltechnolo2gy.E2mail:[email protected]
48JournalofSouthChinaUniversityofTechnology(NaturalScienceEdition)Vol.32
wasdeterminedbyaZDS210illuminometerandcontrolledbyvaryingthenumberoffluorescentlamps.
tractwasinjectedintothecolumn.
1.2 GrowthofC.Vulgaris
Thecellmassconcentrationofalgaeinculturalflu2idswasdeterminedbythemeasuredabsorbancewithanUC22102spectrophotometerat540nm.ThedrycellweightofC.vulgariswasdeterminedbyweighing.
2 ExperimertalResults
2.1 GrowthofC.VulgarisinDifferent
CulturalConditions
Table1andFig.1showcharacteristicsandgrowthofindifferentisthespecificgrowthvulgarisunderHCandMCareunderAC,themaximumbiomassesarerespectively4.65and5.93timesthoseunderAC,andthespecificgrowthratesarerespectively2.13and3timesthoseunderAC.Furthermore,thelogarithmicphasesofHCandMCextendandriseup.
Table1 GrowthcharacteristiesofC.vulgarisindifferentcultural
conditions
CulturalconditionACHCMC
Specificgrowthrate/d-
10.150.320.45
Doublinggeneration
time/d4.622.161.54
Maximumbiomass/(g・L-1)0.864.05.1
1.3 SpecificGrowthRateandDoubl2ing
GenerationTime
)wasdeterminedbySpecificgrowthrate(μ
tingthenaturallogarithmofdrycellthatis,μ=(lnX2-lnXt2arere2spectivelythecellweightsinthetimeperiod.timeDT=ln2/μ.
[2]
1.4 PreparationofAlgaePowder
Culturalsolutionwascollectedandcentrifugatedtoobtainsediment.Algaepowderwasthenobtainedbyvac2uumdryingandgrinding.
1.5 DeteriminationoftheBiochemical
ComponentsinC.Vulgaris
ChlorophyllcontentwasmeasuredaccordingtoArnonπsmethod[3].SolubleproteinwasquantifiedusingtheFolin2phenolreagent[4].Thecontentsofsolublesug2arsandcrudefatwererespectivelydeterminedbyusingsulfuric2phenolreagent[4]andSoxhletmethod.
1.6 DeterminationoftheFattyAcidsin
C.Vulgaris
Themethylationoffattyacidswasperformedbyus2ingtheborontrifluoride(BF3)2methanolmethodasde2scribedbyYangetal[5].
ThecompositionoffattyacidsinC.vulgariswasdeterminedbyaHP4890DGasChromatography.ThechromatographwasfittedwithHP2INNOwax(withthelengthof30mandtheinnerdiameterof0.32mm).Thetemperaturewasprogrammedfrom190℃to220℃withanincreasingrateof2℃/minandmaintainedatthefinaltemperaturefor13min.Thenitrogencarriergaswassetwithaflowrateof2mL/min.Thetemperatureofthein2jectionportwasmaintainedat250℃andtheflameionozationdetectorat270℃.1μLofthederivatizedex2
Fig.1 GrowthcurvesofC.vulgarisindifferentculturalcondi2
tions
Theabsorbancewasmeasuredatthewavelengthof540nm
2.2 EffectofIlluminenceonMixotrophic
Cultivation
Fig.2andTable2showtheeffectofilluminenceonmixotrophicgrowth.Itcanbeseenthatthegrowthisaffectedbyilluminenceinmixotrophicculture.Withtheincreasingofilluminence,thespecificgrowthrateandmaximumbiomassincrease,andthattheeffectsoftheilluminenceat2.5and4.0klxarenotremarkably
No.4
WangHai2yingetal:GrowthandBiochemicalComponentsofChlorellaVulgarisUnderAutotrophic,
tions
CulturalconditonAC(4.0klx)HC(0.0klx)MC(1.0klx)MC((4.)
Massfractionofsolubleprotein/%
Massfractionofsolublesugar/%
Massfractionofcrudefat/%
Contentofchlorophyllindrypowder/(mg・g-1)
49
different.Thecellmassconcentrationinstationaryphaseat4.0klxisalmostthesameasthatat2.5
klx.
Table3 BiocomponentsofC.vulgarisindifferentculturalcondi2
24.3212.7614.5519.15.14.0911.90..12.35
17.6830.23.8921.5621.25
35.148.4018.2924.3131.28
2.4 FattyAcidCompositionsofC.Vul2
Fig.2 EffectofonofC.vul2
garisunderTheabsorbancewasmeasuredatthewavelengthof540nm
garisinDifferentCulturalCondi2tions
ThefattyacidcompositionsofC.vulgarisunderdif2
ferentcultivationsareshowninTable4,fromwhichitcanbeseenthat,forAC,themajorfattyacidsare16∶0,181∶,182∶and183∶,andthemassfractionsoflinoleicacidandlinolenicacidarerespectively26.24%and17.49%.Therearesignificantcontentsof16∶0and18∶3un2derHCandMC.Thecontentoflinolenicacidreaches27.65%inheterotrophicalgae.ThemassfractionoflinolenicacidunderMCislessthanthatunderHC,andthemassfractionoflinoleicacidunderMCincreaseswiththeilluminenceincreasing.
Table4 FattyacidcompositionsofC.vulgarisindifferentcultural
conditions
Fattyacidcomposition
160∶161∶162∶163∶170∶180∶181∶182∶183∶
Massfractionsindifferentculturalconditions/%
ACHCMCMCMC()()()()()17.5629.4125.4526.3126.52.056.674.781.092.1421.9826.2417.49
2.0814.273.661.682.2810.098.8827.65
2.289.475.175.744.5113.8911.8221.67
5.727.084.694.834.6911.3613.7421.58
5.248.532.834.216.5312.2414.5419.38
Table2 EffectofilluminenceonthegrowthcharacteristicsofC.
vulgarisunderMC
Illuminence/klx1.02.54.0
Specificgrowthrate/d-10.350.430.45
Doublinggeneration
time/d1.981.611.54
Maximumbiomass/(g・L-1)4.34.95.1
2.3 BiocomponentsofC.
Vulgarisin
DifferentCulturalConditions
ListedinTable3arebiocomponentsofC.vulgarisindifferentculturalconditions.AsshowninTable3,thebiocomponentsofC.vulgarisvarywiththeculturalcon2ditions.Thecontentsofsolubleproteinandchlorophyllinautotrophicalgaearerespectively1.91timesand4.18timesthoseinheterotrophicalgae,whilethecontentofcrudefatinheterotrophicalgaeis1.71timesthatinau2totrophicalgae.Thecontentofsolublesugarsinau2totrophicalgaeisslightlyhigherthanthatinheterotrophicandmixotrophicalgae.MCcanimprovethecontentsofsolubleproteinandchlorophyllcomparedwithHC.UnderMC,thebiocomponentsofC.vulgaris,especiallythecontentofchlorophyll,isaffectedbytheilluminence.
3 Discussion
ThegrowthofC.vulgarisissignificantlyaffectedbythesupplyofcarbonsource.Thefinalcellmasscon2centrationachievedunderACisverylow.Glucosecaus2esagreatchangeinthemetabolismsystem,leadingto
50JournalofSouthChinaUniversityofTechnology(NaturalScienceEdition)Vol.32
highcellmassconcentrationunderHC.ThespecificgrowthrateofC.vulgarisunderMCislessthanthesumofthoseunderACandHC.
Themixotrophic
growthofC.vulgarisisaffectedbytheilluminence.Attheilluminencesof2.5klxand4.0klx,thegrowthofC.vulgarisisalmostthesame,witharelativelysta2blespecificgrowthrateandmaximumbiomass,espe2ciallyinthestationaryphase.
Microalgaeareusuallyphotoautotrophs,withlightasanenergysourceandcarbondioxideasasource.Thecontentsofprotein,carbo2hydrateachievedunderACderHCandMC.is,2scaleat2tentivemethodoforganiccarbonsubstancesassoleandenergysources.Intheex2periments,thecontentofcrudefatunderHCismuchgreaterthanthatintheothertwoculturalconditions,andthepercentageoflinolenicacidisveryhigh.ByusingHC,therequirementforlightiseliminated.Therefore,thecellmassconcentrationofmicroalgaewillconsider2ablyincrease,
andthe
commercialproduction
of
linolenicacidbecomespossible.Thelackoflightmakestheaccumulationofproteinandchlorophylldecrease.Mixtrophismusuallyrepresentsthesimultaneoususeoflightandglucoseasenergysources.
Mixotrophic
metabolismresultsinahighenergyefficiencywithmini2malamountofenergydissipated.ComparedwiththoseunderHC,thecontentsofproteinandchlorophyllin2creasesignificantlyunderMC.Thecomponentofcellisaffectedbyilluminance,whichsuggeststhatlightisoneofkeyfactorsforchlorellagrowth.
MCisanefficientwaytoprepareproductsofhigh
celldensityandgreatvaluesimultaneously.Moreover,sincethepenetratingdistanceoflightisinverselypropor2tionaltothecellmassconcentration,cellgrowthwillbelimitedbytheavailabilityoflight.Thismaybetherea2sonforthedecreaseofcellmassconcentrationat4.0klxinstationaryphase.Toavoidthetrouble,Ogbonnapro2posedthesequentialcultivation,mini2jarfer2phaseandainautotrophicphase.Productsofandhighbiomasscanbeobtainedbyusingthissystem
[6]
.
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jing:ChinaLightIndustryPress,1999.61.(inChinese)[2] Weidong,ZhangXuechen.Effectofcellgrowthphaseontotal
lipidcontentandfattyacidcompositionoftwomarinemicroal2gae[J].JournalofOceanUniversityofQingdao,2000(3):503-509.(inChinese)
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[5] YangBo,YaoRuhua,Panli,etal.EffectsofpHcontrolinγ2
linolenicacidfermentation[J].ChinaOilsandFats,2001(3):55-57.(inChinese)
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autotrophiccultivation—AnefficientmethodofproducingChlorellabiomassforhealthfoodandanimalfeed[J].Appl
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(下转第55页)
第5期徐 丽等:菌落PCR方法的建立及其与常规PCR方法的比较55
Detectionoftotalandhemolysin2producingVibriopara2haemolyticusinshellfishusingmultiplexPCRamplificationof
tlh,tdhandtrh[J].JofMicrobiolMethods,1999,36:215-225.
EstablishmentofColonyPCRMethodandItsComparisonwith
ConventionalPCRMethod
XuLi Cai(CollegeofFoodandBiologicalEngineering,SouthChina,,Guangdong,China)
Abstract:Toestablishaquick,fordirectlydetectingtlhandadhgenes2carryingVibrioparahaemolyticus,acolonyPCRmethodwasdevelopedanditsresultswerecompared
withthoseof,thusestablishingacolonyPCRmethod.ByusingtheVibrioparahaemolytic2
usstrainsas,marinebacteriastrainspreservedinthelabwereamplifiedthroughthecolonyPCR
methodandtheconventionalPCRmethod,bywhichthereliabilityofcolonyPCRmethodforthecorrespondinggenede2tectionwasproved.ThecomparisonshowsthattheresultsobtainedbycolonyPCRmethodareingreatagreementwiththoseobtainedbytheconventionalPCRmethod,andthatthetlhandtdhgenesinpositivecontrolscanbeamplifiedbythesetwoPCRmethods.
Keywords:colonyPCR;conventionalPCR;tdhgene;tlhgene
(上接第50页)
自养、异养和混养下小球藻的生长及生化成分3
王海英 郭祀远 郑必胜 李存芝
(华南理工大学轻化工研究所,广东广州510640)
摘 要:对小球藻在自养、异养和混养条件下的生长状况及细胞的生化成分(如可溶性蛋白、可溶性糖、叶绿素及脂肪酸组成)进行了研究.结果表明:异养和混养培养的小球藻的生物量远大于自养时的生物量;异养和混养培养的小球藻的比生长速率分别是自养时的2.13倍和3倍;光照对混养条件下的细胞生长有影响,但光照强度为2.5klx和4.0klx时细胞生长的差别并不明显,尤其在稳定生长期;与自养生长相比,异养过程中小球藻的脂肪含量明显增加,可溶性蛋白和可溶性糖的含量则有不同程度的降低,叶绿素含量大大减少;异养有利于亚麻酸的积累;在混养条件下,光照使可溶性蛋白、可溶性糖和叶绿素的含量增加,其强度对细胞的生化成分有影响.
关键词:小球藻;生化成分;生长;自养;异养;混养中图分类号:Q815 文献标识码:A文章编号:1000-565X(2004)05-0047-04
责任编辑:傅晓琴
收稿日期:2003-10-29
3基金项目:广东省科技攻关项目(99M01409G)
作者简介:王海英(1974-),女,博士生,主要从事微藻生物技术研究.E2mail:[email protected]