全 文 ：第27卷第l期
2008年2月
生态
Ecological
科学
Science
27(1)：6-1I
Feb．2008
PhysiologicalresponseofmarinediatomPhaeodactylumtricornutumto
darknessstress
CAtZhuo．pin91，WEIWei2，DUANShun．shanl·
1．InstituteofHydrobiology,JinanUniversity,Guangzhou51063ZChina
2．GuangzhouInstituteofEnergyConversion,Guangzhou51064D．China
Abstract：Inthepresentstudy，Phaeodactylumtricorn tumwasgrowninlaboratoryunderdarknessconditionfor12daysin
ordertoinvestigateitsgrowthandsomechemicalompositionsinresponsetothedarknessenvironmentandtoevaluatethe
adaptationofthiseconomicmicroalgatodarknessstress．TheresultsindicatedthatPhaeodactylumtricornutumcouldstilIsurvive
inthedarknesscondition，whiledarkn ssstressimposedignificantlyinhibitoryinfluenceo thegrowthofPhaeodactylum
tricornutumd ringthecourseofthexperiment．AlgaleelldensityandbiomassinthedarknesstreatmentW rereducedto
2．93X10’cells·ml～and0．01lg·rnl-1onthedayofterminationofhexperiment，only8．0％and37．3％ofthecontr01．Moreover，
somechemicalcompositionsincludingchlorophylla，so ublesugarndproteinwerealsoapparentlylimitedinthedarkness
condition，thecont ntsofwhichwereapproximately89％，87％and85％lOWerthanthoseinthecontrolonthe12。day．Our
resultshaveprovidedsupportiveevidencethatPhaeodacCylumtricornutumtendedtoexhibitthecapacityofsustaininginthe
darknessstress，whichmig tbeduetoitspronouncedabilityinthebiosynthesisandmetabolismofprotein，solublesugarnd
chlorophylla．butitsgrowthperformancew sremarkablyrestrictedov rtime．Thefindingspresentedheresuggestedthatthe
adaptiveabilityofPhaeodactylumtricornutumtodarknessstresshouldbeconsideredintohecoI．ecollectionandscreening．as
wellascomprehensivexploitureofmicroalgalresources．
Keywords：growth；physiologicalresponse；d rknessstress；Phaeodactylumtri orn tum
CLCnumber：Q178．1Documentcode：A ArticleID：1008-8873(2008)01-06·06
三角褐指藻对黑暗胁迫的生理响应
蔡卓平1，危蔚2，段舜山p
1．暨南大学水生生物研究所，广州510632
2．中国科学院广州能源研究所，广州510640
【摘要】 为了研究黑暗条件对三角褐指藻生长及生化组成的影响，并探讨三角褐指藻对黑暗环境的生长适应能力，我
们对该藻进行12d的黑暗处理，着重测定了藻细胞密度、生物量、叶绿素a、可溶性糖和蛋白含量等指标。结果表明，
三角褐指藻对黑暗环境表现出一定的适应性忍耐能力，在12d的长期黑暗胁迫条件下依然可以存活，而藻细胞生化组成
对黑暗的响应程度很可能是该藻得以维持细胞低水平生长的主要原因。随着黑暗处理时间的延长，三角褐指藻生长状况
受抑制的程度增大，其生长及生化组成与对照组相比发生了极显著的变化。实验结束时，黑暗处理下的藻细胞密度和生
物最分别降低到2．93x105cells．ml以和0．Ollg m ～，仅为对照的8．0％和37．3％。同样地，黑暗环境也明显地抑制了三角褐
指藻体内生化物质的合成与积累，黑暗处理12d时藻细胞的叶绿素a、可溶性糖和蛋白含量分别比对照降低了约89％、
87％和85％。研究结果可为海洋微藻种质的筛选、种质资源库的构建及微藻生物资源的综合开发利用提供参考依据。
关键词：生长；生理响应；黑暗胁迫；三角褐指藻
收稿日期：2007．10—15收稿，2008．02．10接受
基金项目：国家自然科学基金项目(30370231，30770336)：广东省科技计划重点引导项目(2005833201001)：珠海市科技计划重点项目
(PC20061045)
作者简介：蔡卓平(1980一)，男，博士研究生，研究方向为微藻生态与生物技术。Email：zpeai@stall．edu．∞
·通讯作者
万方数据
l期 蔡卓平，等：三角褐指藻对黑暗胁迫的牛理响应 7
1Introduction
Microalgaeareamajornaturalsourcerichinavast
arraybioactivecompounds，suchas carotenoid
pigments，vitamins，proteins，fattyacids，sterolsand
polysaccharides【IJ，thereforetheycanbeappliedwi ely
in thehumanhealthyfood[21，animalfeeds[31，
pharmaceuticalandcosmeticsproducts[4】．biofuels【5】as
wellasbioremediationofpollutedwater』引．Inrecent
years，manystudieshavebeenconductedtoincrease
ourunderstandinginthephysiologicalresponsesof
microalgaetoth differente vironmentalco ditions
includingthevariationsinnutrient，temperature，salinity，
pHandlight，amongwhichlightisbelievedtoplaya
particularlycrucialro einthegrowthofunicellular
microalgae[7·91．Light．asthemainenergyresourcesfor
plants，maych ngethconformationofphytochrome
andultravioletreceptorsandthenalterthecellular
hormonelevelandgenexpressionmode，thus
regulatingthegrowthandevelopmentofplants[10】．
ThemarinemicroalgaPhaeodactylumtricornutum
isoneofthemostimportantmicroalgaespeciesthathas
beencommonlyusedinthelaboratoryresearchand
industrialproduction．Commercially，ithasbeservedas
animportantfoodsourceandfeedadditiventhe
successfulrearingofaquaticanimalslikemollusks，
crustaceansandfi hes[1l】．Earlierstudieshavepointed
outhatdifferentalgalspeciesmightappeartovaryin
theabilitytoresponsetothelightconditions，showing
differentgrowthcharacteristicsfromothers[12】．Infact，
inthenaturalecosystem，microalgaecanbeexposedto
environmentalstressfactors，suchasextremesof
temperature，light，salinityandpH[131．Fornstance，ice
coverinwinterinhi【ghlatitudesoreuphoticzoneinthe
watercolumnmaypotentiallyresultindarknessstress
forsomemicroalgaeinthemarineenvironment．Like
manyorganisms，microalgaecanbeab etor sponseto
thosevaryinge vironmentalfactorinsuchaswaythat
understandingtheser sponsesisveryessentialothe
studyofmicroalgalecologyandphysiology,whichwill
potentiallybeofgreatsignificanceinthecorecollection
andscreening，aswellascomprehensivexploitureof
microalgalresources．Therefore．inthecur ntstudywe
revealtheadaptiveabilityofthismicroalgainresponse
todarkness，andreportthegrowthandchemical
compositionsofPhaeodactylumtricornutums bjected
toalong—termdarknessconditionfor12days，soasto
gathermuchmorephysiologicalandecological
informationforthexploitureofthisconomicalga．
2Materialsandmethods
2．1Algalcultureconditions
ThemarinediatomPhaeodactylumtricornutumwas
obtainedfromtheInstituteofHydrobiology,Jinan
University,Guangzhou，China．Artificialseawaterwas
addedw thsterilef／2mediumfor icroalgalcultures
containingmacronutrients．trancemetalsandvitamins[14】．
S linityofheartificialse waterwas309／ooandtheinitial
pHoftheculturewas6．5—7．Thealgalculturesw re
inoculatedinliquidsterilizedme iumforaperiodof
daystoobtainexponentiallygrowingcultures，andthe
cellsinexponentialgrowthphasew reused．Therew
twotreatmentsinthexperiment：onewasthedarkness
reatmentandanotheronewasetasthecontr01．Forthe
control，algalcellsweregrownin1000mlErlenmeyer
flaskscontaining600mlsolutionandmaintainedina
emperature—programmablechamberat20oCwith
constantirradiance(5000Ix)andphotoperiodcycle(12h
light／12hdark)，whileforthdarknesstreatment，growth
conditionwasimilartothecontrolexceptthatflasksin
thechambercontainingalgalculturesw rewrappedwiⅡl
lighttightfilms，intheabsenceoflight．Allexperiments
werecarriedoutintriplicateandr peatedatleasttwice．
2．2CelIdensityandbiomassmeasurement
Algalcellsweremonitoredandcountedusinga
microscopewiththehelpofa hemocytometerev ry
otherdayuntilthecelldensityvariedlessthanlO％for
twodays．Algalbiomasswa alsoweighedbyan
electronicbalanceinthelabafteracertainvolumealgal
samplewasharvestedandcollectedev rytwodays．
2．3 Quantitativedeterminationofchlorophylla，
solublesugarndproteinco tent
Chemicalcompositionsofalgalcellswereassayed
万方数据
8 生态科学EcologicalScience 27卷
withtwodaysintervalduringthexperimentalpe—od
accordingtothemethods船describedbyLitl51．
Chlorophyllawasdeterminedspectrophotometricallyby
collectingacertainlgalcultureonglassfiltersand
extractingin90％acetone．Solublesugarwasm asured
usingthephenolmethodandperformedwitha
spectrophotometerat485nlTlwavelength．withsugar嬲
thestandardcalibration．Proteinwasassayedbyreading
theopticaldensitiesoftheextractsona
spectrophotometerat260nlnand280tml,afteralgal
cellsweredisruptedbyultrasonicationnallicebath．
3Results
3．1Changesinalgalcelldensityandbiomassduring
theincubation
nlealgalcellnumberandbiomass、Ⅳe糟estimated
everytwodaysduringthewholeexperimentalpcdod
(Fig．1andTab．1)．Obviously,Phaeodactylumtricornutum
inthecontrolexperiencedtheinitialgrowthstage，
exponentialgrowthstageandstablestage，amountingto
103x105cell·mL～afterincubationfor12days．Ⅵ嘶le
comparedtothecontrol，cellgrowthinthedarknesswa
restrictedseverelyovertime．Undertheconditionof
darknessstress，thehighestcelldensitywasdetectedon
the6也day,70．1％oftheinitialce ldensity．Thereafter,a
gradualdownwardtrendOCclllTedtotheendofthe
experiment,withthelowesteelldensityof2．93x10"
cell·mL’1onthe12mday,whichwas8．0％ofthatinthe
contr01．ItisalsoshowninTab．1thatalgalbiomassvaried
distinctlybetweenthecontrolandthedarknesstreatment
duringthecoursgofthexperiment．Thebiomassinthe
controlwasincreaseddramaticallyatthebeginning，and
reachedtothepeakof2．95mg·mL。1onthe8也day,after
whenitremainedsteadytothe ndoftheincubation．
However,cellbiomassinthedaFknesswaincreased
slowlyduringthefirst6days，reachingto1．82m ‘mL～，
36％higherthantheinitialbiomass．Aftertha ,italso
showeda egressivetendency,wiⅡlthemininlul／l
biomass(1．06mg·mL"1)occurringon龇10也day．
reflectthestatusofsynthesis，transformationand
me．tabolismofcarbohyrdrateinalgae[161．Proteinis
thoughttobeveryimportantforalgalcelldivision，and
chlorophyllaisthemainpigmenttransformingthel ght
nergyintochemicalenergy,whichdirectlyrepresents
thephotosyntheticabilityofalgae[171．Ther sults
obtainedfromurexperimentsindicatedthattherew re
significantdifferencesi thechlorophylla content
betweenthecontrolandarknesstreatmentthroughout
the xperiment．Asit isillustratedinFig．2，aftera
gradualincrease，thechlorophyllacontentinthecontrol
begantoraiseharplyfromthe8也day,andendedwith
about850lag·L～intheculture．Ontheotherhand，the
peakofchlorophyllacontentwasdetectedonthe6m
dayinthedarknesscondition，whichwas3．6times
high rwhencomparedwiththebeginningday,butit
wasonly18．6％ofthemaximuminthecontr01．
Whereafter,chlorophyllacontentwasreducedslowly
totheterminationofhexperiment．Manifestvariation
insolublesugarcontentwasalsofoundbetweenthe
controlandthedarknesstreatment．Incontrasttothe
gradualincreasingofsolublesugarcontenti the
control，thesolublesugarcontenti thedarkness
behavedinaverysimilarf shion弱thechlorophylla
content．Itwasobservedthathemaximumsoluble
sugarcontent(93．0lag·L_1)alsoappearedonthe6thday,
andtheminimumone(38．0lag·L‘1)wasfoundonme12恤
day,whichwasdecreasedtoapproximately13％ofthat
inthecontr01．Likewise，darknesstressh dsignificant
effectsontheproteinco tentascomparedwiththe
0 2 4 6 8 10 12
3．2changesinsomechemicaIcOmp。siH。nsacmss
culture‘ime(d)
thexperiment Fig．1GrowthcarvesofPhaeodactylumtricornutRmin he
Qualitatively,solublesugarcanbeemployedto darknesstreatment(@)andcontrol(O)acrosstheexperiment．
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万方数据
1期 蔡卓平，等：三角褐指藻对黑暗胁迫的生理响应 9
Table1 Cellbiomass(mg-mL"1)ofPhueodactylumtricornutumexposedtonormalfightconditionanddarknesstreatmentat very
otherdaysthroughouthexperiment．ThevaluesinthetablearethemeanswithstandardelTorsofthreer plications．Values
耐缅differentlettersinthesamecolumnaresignificantlydifferent(P0 2 4 6 8 10 12
O 2 4 6 8 10 12
0 2 4 6 8 10 12
Culturetime(d)
Fig．2ChangesinthechemicalcompositionsofPhaeodactylum
tricornutuminthedarknesstreatment(◆)andcontrol
(O)overtime．
contr01．Proteinc nte twaspeakedonthe4mdayinthe
darknesstreatment，andthenitdeclinedgraduallywith
thexperimentalprolongation．ItWas91pg·L-1atthe
timewhenthetrialWaSterminated．whichwas15％of
nlatinthecontr01．
4Discussion
MarinemicroalgaPhaeodactylumtricornutum
containsa veryhighcontentof hi．gh-value
polyunsaturatedfattyacids(PUFAs)，suchas
eicosapentaenoicacid(EPA)anddocosahexaenoicacid
(DHA)，whichhasseveralbiotechnologicaland
pharmaceuticalapp ications．Consequently，
physiologicalandgrowthresponsesofthiseconomic
microalgatotheadverseenvironmentalfac orshas
recentlyreceivedmuchmoreattention，whichwillshed
morelightonthescreeninga dcultivationof
microalgalspecies【18】．Ourdatarevealedthatcontrasted
tothecontrol，darknesswouldsignificantlyaffectthe
growthperformanceofPhaeodactylumtricornutum，
resultingindecreasedcelldensity,cellbiomass，B．8well
鹪contentsofsomechemicalcompositionsincluding
chlorophylla’so ublesugarndproteinduringthe
wholeexperimentalperiod,especiallyinthelast3days
beforetheterminationofthe xperiment．Ourresults
agreew llwiththoseobtainedbyZhangetal，who
examinedthgrowthofNannochloropsisculataunder
thecontinueddarknessstressfor12daysandfoundits
growthwasremarkablyrestrainedduringthecourseof
experiment，withthecelldensityonthedayofthe
terminationbeingreducedto67％ofthatinthe
beginningtl91．Itseemsreasonabletoassumethati Was
attributedtothefailuretocompleteth normal
physiologicalactivitiesbythealgaeinthedarkness
conditions，hencethecelldivisionmightberepressed
0
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万方数据
lO 生态科学EcologicalScience 27卷
andsloweddown，togetherwiththelowercapacityin
thebiosynthesisandaccumulationofsomechemical
compositions．Asitisknown，lightisoneofthemost
importantenvironmentalfactorstoregulatethegrowth
ofplants，whichactsasthemainandirectenergyfor
thephotosynthesisandplaysa vitalroleinallthe
developmentalphasesofplants[2们．Ithasalsobeen
reportedthatifferentlightintensitywouldalsohave
differenteffectsonthegrowthandbiochemical
compositionoftwomarinemicroalgae，Nitzschia
closterium￡minutissimaa dIsochrysisga，6anaParke
87011121．Whatismore．relatedstudieshaveb encarried
outontheimpactoflightonthegrowthrate，
chlorophylllevelandcellcycleinfouralgalspecies，
includingChlorella，lsochrysiszhangjiangersis，
PlatymonashelgolandicaDun liellaviridis。2“．
Overthelastfewdecades，agre tdealhasbeen
achievedintermsofmicroalgalphysiologyand
ecology[22·23I．Ithasbeenwidelyacceptedthatmany
algalspeciesmightphysiologicallya c imatein
responsetotheextremevariationofenvironmental
factorsbyadjustingtheirmetabolicstrategies，thus
increasingtheircapacitytoretaintheirg owth【矧．Algal
membranelipidcompositionhasbeenpiovedtobe
involvedin thedifferentresponsesto adverse
environmentalconditions，dependingonthealgal
speciesandthestrengthofenvironmentalstresses．For
instance，microalgaeouldenh nceth unsaturation
levalinmembranef ttyacidswhentheyweresubjected
tolowtemperature[251．Besides．earlierstu iesal o
pointedoutthatthedinoflageUateProrocentrum
minimumaintaineditsmetabolicintegritywithout
unrecoverablecellulard magesorcelldeathduring10
daysofdarkcultivationthroughalteringthestorage
lipidtriacylglycerideandgalactolipidsunderthe
prolongeddarkness[26]．Inthecurrentstudy,althoughthe
marinemicroalgawaskeptinthe12一day-longdarkness
condition，Phaeodactylumtricornutum，asexpected,
couldstillgrowslightlyinthebeginningofthe
experimentandbeabletomaintaininarelativelystable
leveltotheendoftheexperiment，implyingthat
Phaeodactylumtricornutummightavethecapability
toadapttosuchdarknessstressandsurviveinthe
adversecircumstances．Inconclusion，theresults
obtainedinourexperimentshaveprovidedstrong
evidencethathemarinediatomPhaeodact)7lure
tricornutummightpossesstheparticulartolerancetoth
darknessstress，whichmightbeduetoitspronounced
abilityinthebiosynthesisandmetabolismofprotein，
solublesugarandchlorophylla underdarkness
conditions．Nevertheless．furtherr s archhouldbes ill
neededinorderto evealthebiochemicalandmolecular
aspectsofPhaeodactylumtricornutuminvolvedinthe
adaptiveabilitytodarknessstress，andhencetotake
advantageofsuchadaptationforthecorecollectionand
screeningofmicroalgalresources．
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万方数据
三角褐指藻对黑暗胁迫的生理响应
作者： 蔡卓平， 危蔚， 段舜山， CAI Zhuo-ping， WEI Wei， DUAN Shun-shan
作者单位： 蔡卓平,段舜山,CAI Zhuo-ping,DUAN Shun-shan(暨南大学水生生物研究所,广州,510632)
， 危蔚,WEI Wei(中国科学院广州能源研究所,广州,510640)
刊名： 生态科学
英文刊名： ECOLOGICAL SCIENCE
年，卷(期)： 2008,27(1)
被引用次数： 1次

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2011(4)


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