全 文 ：OptimizationoftheConditionsforUsingSewagetoCulti-vateNaviculatenera
WUXia-yuan, LIHuan＊ , WEIPing
ColegeofLifeScienceandPharmaceuticalEngineering, NanjingUniversityofTechnology, Nanjing210009
Abstract　[Objective] ThisstudywastoimprovethebiomassofNaviculatenera, andthustoprovidereferenceforachievingtheindustrialpro-
ductionofNaviculatenera.[ Method] ThefeasibilityofusingsewagetocultivateNaviculatenerawaspreliminarilyinvestigatedbasedonthecon-
siderationofregionalcharacteristics;andaseriesofcultureconditionsincludingthenutrientsourceofnitrogen(N), phosphorus(P), iron(Fe),
silicon(Si)andthesalinityinmediumforculturingNaviculatenera, wereoptimizedbysinglefactortestandorthogonaldesign.[ Result] Theop-
timizedconditionsforcultivatingNaviculatenerausingsewageareasfolows:thewaterfromXiaoerlouArtificialLakeofNanjingUniversityof
Technologyasbasicsolvent;360mg/Lurea;150mg/LN2HPO4· 12H2O;50mg/Lferriccitrate;2 000mg/LNa2SiO3· 9H2O;2.0mol/Lsa-linity.Naviculatenerawasstronglyadaptivetosewageandcouldweluptakethenutrientsourcesinthesewage.Undertheoptimizedcondi-
tions, theculturecostdecreased, andmeanwhilethebiomassofNaviculatenerareached4.766g/Lwhichis3.57multiplesoveroriginalmedi-umand1.9multiplesoveroptimizedmediumNo.1.[ Conclusion] ThisstudylaidafoundationforthecombinationofculturingNaviculatenerain
largescaleandsewagetreatment.
Keywords　Naviculatenera;Sewage;Culturecondition;Nutrientsource;Orthogonaldesign
Received:December19, 2008　　Accepted:March5, 2009
SupportedbyNationalBasicResearchProgramofChina(973 Pro-
gram)(2003CB71600).
＊Correspondingauthor.E-mail:lihuan45678@163.com
　　Oceanbenthicdiatomfloraareimportantfoodsforrare
andpreciousaquaticanimalssuchasabaloneandtrepang[ 1] .
Ofthem, Naviculateneraisatypeofunicelularalgaecom-
monlyusedasseashelforculture.Naviculatenerabelongsto
NaviculaofNaviculaceaeofNaviculales, Bacilariophyceae
anditisabundantinfatcontentandfatspecies.Fromthere-
sultsofgaschromatography-massspectrometry(GC-MS)a-
nalysis, Naviculateneraisrichinvariousunsaturatedfat
acids[ 2] ;furthermore, itcansecretesulfatedpolysaccharide
thatisendowedwithvariousbioactivitiesintheprocessof
growth.Therefore, Naviculateneraisnotonlyanoptimal
weaningfoodforaquaticanimals, butalsoakindofmarine
organismwithpotentialmedicinalvalue.Inearly1980s, dia-
tomflorahasbeenhighlyconcernedbyscientistsfromrelated
researchfieldowingtoitshighfatcontent, andwasthoughtto
beoneofthealgaespeciesthatcanmostlikelyachievein-
dustrialproductionofgrease[ 3] .Astheincreaseofglobalpop-
ulation, therigidincreaseoffossilfuelresultedfromtheag-
gregationofindustrialprocessandtheincreasingdeterioration
ofenvironmentalpolutionincludinggreenhouseeffect, Navic-
ulateneraasapotentialmicro-algaerichinoilisworthyoffur-
therresearchandexploitation.
Highculturedensityisthepremisefortheintegratedex-
ploitationandutilizationofNaviculatenera.Uptothepresent,
veryfewreportsinvolveNaviculateneraathomeandabroad,
andthecultureconditionsreportedarenotcomprehensive.
ThisstatusdirectlycontributestothelowerbiomassofNavicu-
latenera, hugelyrestrictingitsexploitation.Inviewofthis, we
firstlyatemptedtocombinetheculturingNaviculatenerain
largescaleandsewagetreatment, inordertoimproveitsbio-
mass, decreaseitsculturecost, andprovidebasisforinte-
gratedindustrialexploitationofNaviculateneraassoonas
possibleaswel.
MaterialsandMethods
Experimentalalgaandmedia
NaviculatenerasuferedfromspaceflightbyShenzhou
5wasgiftlyprovidedbyProf.ZHENGWei-fafromXuzhou
NormalUniversity.Theinitialculturemediawereoptimized
fromtheformulaelistedinreference[ 4] .
Mainreagentsandinstruments
CO(NH2 )2 , Na2HPO4 · 12H2O, Na3SiO3 · 9H2O and
fericcitratewerealatanalyticpuregradeandpurchased
fromSinopharmChemicalReagentCo., Ltd.;seawatercrys-
talwaspurchasedfromQingdaoGoeHaidaSeaSaltCo.,
Ltd.DU650spectrophotometer(BECKMAN, US).
Algaculture
AlgaatlogarithmicgrowthphasewiththeinitialOD680 be-tween0.2 and0.5 wasinoculatedina500 mlflaskforsha-
kingculture(toexcludetheeffectoforiginalculturesolution,
thealgawascentrifugedbeforeinoculation).Culturecondi-
tionswereasfolows:temperature(24±1)℃, ilumination18
h/d, lightintensity3 000 lx, aerobicincubation.Eachtest
containsthreeparaleltreatments.Theculturesolutionwas
sampledeverytwodayatafixedtimetomeasuretheOD680
andbasedonthattogeneratethegrowthcurveofNavicula
teneracels.
Determinationofrelativegrowthrate
Theformulaforcalculatingtherelativegrowthratewasas
folows:
K=(lgOD-lgOD0)/T
WhereODrepresentsOD680 ofalgasolutionafterculture, OD0
representsOD680 ofalgasolutionbeforeculture, Trepresentscultureduration(d).
EffectsofdifferentwaterqualitiesonthegrowthofNavic-
ulatenera
ThewatersamplesfromXuanwuLakeinNanjing, Jin-
chuanRiverinQingshiVilage, XiaoerlouArtificialLakeof
NanjingUniversityofTechnologyandtapwaterwererespec-
tivelyusedasbasicsolventtoprepareartificialseawaterme-
dium, whichwasusedtocultureNaviculatenera.Thebasic
solventoffolowingtestswastheoptimizedwatersample
here.
AgriculturalBiotechnologyAgriculturalScience＆Technology, 2009, 10(1):68-73Copyright 2009, InformationInstituteofHAAS.Alrightsreserved.
DOI :10.16175/j.cnki.1009-4229.2009.01.039
SinglefactortestsforoptimizingthefactorsincludingN,
P, Fe, Siandsalinity
① OptimizationofN.Originalmediumasbasis, appen-
dedwithvariousNsourcesofKNO3 , NH4 Clandurea, was
usedtocultureNaviculateneraattheoriginalNconcentration
of3 mmol/L;anddifferentureaconcentrationsof45, 90,
180, 360 and720mg/Lweremeanwhilesetup.
② OptimizationofP.DiferentNa2 HPO4 · 12H2 Ocon-
centrationsof30, 60, 120, 240 and480 mg/Lweresetupfor
optimizingPfactor.
③ OptimizationofFe.Fe-EDTAandferriccitratewere
respectivelyusedtocultureNaviculateneraattheoriginalFe
concentrationof0.15 mmol/L;anddiferentureaconcentra-
tionsof0, 25, 50, 100, 150 and200 mg/Lweredesignedto
optimizeFefactor.
④ OptimizationofSi.DifferentNa3SiO3 · 9H2Oconcen-
trationsof100, 400, 700, 1 000, 1 200 and1 500mg/Lwere
designedtooptimizeSifactor.
⑤Optimizationofgradientsaltfactor.Diferentureacon-
centrationsof0.5, 1.0, 1.5, 2.0 and2.5 mg/Lwere
designedtocultureNaviculatenera.
OrthogonaldesignforoptimizingfivefactorsofN, P, Fe,
Siandsalinity
Basedontheresultsofthesinglefactoroptimizationtests
mentionedabove, theorthogonaltestforalthefactorswere
carriedout.Orthogonaldesign[ L16(45)] wasreferredtoref-
erence[ 5 ] , thedetailedlevelsofeachfactorwereas
folows:
Urea(A):45, 90, 360 and720 mg/L;Na3SiO3 · 9H2 O(B):
700, 1 000, 1 500and2 000mg/L;Na2HPO4· 12H2O(C):60,
150, 300 and600mg/L;fericcitrate(D):50, 100, 200and400
mg/L;mediumsalinity(E):0.5, 1.0, 2.0and2.5mol/L.
ResultsandAnalysis
Effectsofdifferentwaterqualitiesonmedium
Recentyears, thearbitrarydischargeofdomesticsew-
ageandindustrialwastewaterhasmadetheeutrophicationof
lakewaterandriverwateraggregated.Aimingatachieving
thedualtargetsofutilizingsewageanddecreasingtheculture
costofNaviculatenera, wefirstatemptedtocultureNavicula
tenerawiththewatersamplesfromdiferentsourcesinNan-
jingregion.Usingdistiledwatertopreparemediumforcultu-
ringmicro-algae, micrometalelementsandvitaminsarere-
quired.Interestingly, waterfromlakeandriverisrichinmicro
metalelementsandvitamins, thuswhetheritisessentialto
addingtheseelementstomediumstilneedstobeproved.On
thisbasis, wedesignedfolowingtestgroups:normaldistiled
watergroup(CK), tapwatergroup, tapwater+micrometal
elementsandvitaminsgroup(abbreviatedasmicro);Jin-
chuanRiverwatergroup, JinchuanRiverwater+ micro
group, XuanwuLakewatergroup, XuanwuLakewater+mi-
crogroup, XiaoerlouArtificialLakewatergroup, XiaoerlouAr-
tificialLakewater+microgroup.Ofthesewaterorigins, Xu-
anwuLakewasmainlypolutedbyurbandomesticsewage
andsecondlyindustrialwastewater, JinchuanRiverwasbyur-
bandomesticsewage, XiaoerlouArtificialLakebydomestic
sewageofNanjingUniversityofTechnologyandexperimental
wastewater.
AsshowninFig.1, NaviculatenerainXiaoerlouArtificial
Lakewatergroupgrewbeterthanothertreatmentstested, its
growthrateonthecontrarydecreasedintheXiaoerlouArtifi-
cialLakewater+microgroup, whichmayresultfrom the
abundantmicrometalelementsandvitaminsthathasbeen
containedinXiaoerlouArtificialLakewater, thusextraaddition
wilinsteadgoagainstthegrowthofNaviculatenera.Navicula
teneraintapwatergroupgrewpoorestinalthetreatments,
thisisduetothedeficiencyofmicrometalelementsandvita-
minsortheuncleanedchlorineintapewater.Inthisstudy,
wefoundthatNaviculatenerawasstronglyadaptivetoenvi-
ronment, itcouldgrowandpropagateinvarioussewages
withoutthepolutionofotheralgaeorbacteria.Thisonone
handcoulddecreasethecostofculturingNaviculatenera, on
theotherhandcouldcontributetosewagetreatment, laying
goodfoundationforfurtherculturingNaviculatenerainlarge
scaleanditscomprehensiveutilization.Hence, XiaoerlouAr-
tificialLakewaterwaschosenasbasicsolvent(mediumpre-
paredwithXiaoerlouArtificialLakewaterwasmarkedasopti-
mizedmediumNo.1)tooptimizethecultureconditionsof
Naviculatenera.
Fig.1　EfectsofdiferentwaterqualitiesonthegrowthofNa-
viculatenera
Resultsofsinglefactortestsforoptimizingthefactorsin-
cludingN, P, Fe, Siandsalinity
Nitrogen　Althethreekindsofnitrogensourceswereavaila-
bleforthegrowthandpropagationofNaviculatenera, of
whichureaperformedbest(Fig.2).Concerningtheurea
concentration, 360 mg/LisoptimalforculturingNaviculaten-
era;45 -360 mg/Lcouldremarkablypromotethegrowthof
Naviculatenera;exceeding360 mg/L, thegrowthrateofNa-
viculateneradecreasedsharply, itsgrowthwasobviously
restrained.
Fig.2　EfectsofdiferentnitrogensourcesonthegrowthofNa-
viculatenera
Phosphorus　Phosphorusisanessentialelementforinvitro
synthesisofATP, GTPandphospholipid[ 6] .Theeffectofdif-
ferentconcentrationsofNa2 HPO4 · 12H2Oonthegrowthof
Naviculatenerawasinvestigated.AsilustratedinFig.4, 30-
240 mg/LNa2HPO4 · 12H2Ocouldremarkablypromotethe
69WUXia-yuanetal.OptimizationoftheConditionsforUsingSewagetoCultivateNaviculatenera
growthofNaviculatenera;toohighconcentrationobviously
restrainedthegrowthratesofNaviculatenera.
Iron　Ironisatraceelementindispensabletothegrowthof
Naviculateneraandnitrogenfixation, andthesynthesisof
chlorophylaswel, deficiencyofwhichcouldinhibitcel
growthinmetabolism.Forculturingbenthicdiatomflora, re-
searchersusedtoaddferriccitrateintoseawater, butlittleis
knownabouttheoptimalironformandoptimalconcentration.
Thus, Fe-EDTAandfericcitratewerechosentocultureNa-
viculatenera.Theresultsshowedthatferriccitrateperformed
beterthanFe-EDTAinpromotingthegrowthofNaviculaten-
era(Fig.5).Withregardtothedetailedferriccitrateconcen-
tration, within0-50 mg/L, thegrowthofNaviculatenerawas
remarkablypromotedwiththeincreaseofferriccitrateconcen-
tration, whilethatinthemediumwithoutferriccitrategrew
slowly;onceexceeding50 mg/L, Naviculateneraaggregated
andprecipitatedinthebottomofflask, algafrondturnedlight
andgrowthratedecreasedsharply, algacelsenteredintode-
clinephasesoon, indicatingthathighconcentrationofferic
citrateisharmfulforalgacels.
Silicon　Apartfromcomposingcelwalstructure, siliconas
anessentialnutrientelementparticipatesinvariousmetabo-
lismsandgrowthprocessesincludingthesynthesisofphoto-
syntheticpigment, protein, DNAandceldivision[ 7] .Ourre-
sultsshowedthatarelativelyhighersiliconconcentrationwas
requiredforthegrowthofNaviculatenera;intheexperimental
concentrationsofsilicon, theNaviculateneragrewdependent-
lywiththedoseofsilicon, andthepeakbiomassappearedat
thehighestconcentration(1 500 mg/L).
Salinity　Fivesalinityconcentrationswerechosentooptimize
thegrowthofNaviculatenera.Theresultsshowed, withinthe
concentrationsof0.5-2.0 mol/L, thattheaccumulationof
algabiomassincreasedgradualywiththeriseofNaClcon-
centrationinmedium, suggestingthehighsalttoleranceof
Naviculatenera(Fig.8).Intheinitialyinoculatedperiod, the
growthrateofNaviculatenerainthemediumwith0.5 mol/L
NaClwashighest, whilethatwith1.0 -2.5 mol/LNaClwas
relativelylower, possiblybecauseNaviculatenerahadnota-
dapttothehighsalthabitatatthattime, itsgrowthwastem-
porarilyinhibited.Aboutfourdayslater, algacelsgradualy
becameadaptivetohighsalthabitat(1.0-2.5 mol/LNaCl),
growthrateincreasedhugelyandespecialythatat2.0 mg/L
NaClconcentration;whilethegrowthofNaviculatenerainthe
mediumthatcontainedNaClexceeding2.0 mg/Lwasobvi-
ouslyinhibited, harmfulforthecultureofNaviculatenera.
Resultsoforthogonaldesignforoptimizingfivefactorsof
N, P, Fe, Siandsalinity
Theinfluenceorderofthefivefactorsandoptimizedcom-
binationforculturingNaviculatenera　Itcouldbeseen
fromTable1 thattheinfluenceorderofthefivefactorstothe
70 AgriculturalScience＆TechnologyVol.10, No.1, 2009
Fig.8　EfectsofdiferentsalinitiesonthegrowthofNavicu-
latenera
growthofNaviculatenerawasC>A>D>E>B, i.e., forthe
growthofNaviculatenera, factorD(fericcitrate)andfactor
A(urea)arethemajorinfluencingfactors, otherscomesec-
ondly.Theoptimizedformulawereasfolows:360 mg/Lure-
a, 150 mg/LN2HPO4· 12H2O, 50 mg/Lferriccitrate, 2 000mg/LNa2 SiO3 · 9H2O, 2.0 mol/Lsalinity, markedwithopti-
mizedmediumNo.2.
Table1　Resultsoftheorthogonaldesignfornutrientsandsalinityfor
culturingNaviculateneraanddirectanalyses
Test
No.
Factorandlevel
A
mg/L
B
mg/L
C
mg/L
D
mg/L
E
mg/L
Relative
growth
rate
1 45 700 60 50 0.5 0.021 431
2 45 1 000 150 100 1.0 0.028 582
3 45 1 500 300 200 2.0 0.032 682
4 45 2 000 600 400 2.5 0.024 057
5 90 700 150 200 2.5 0.021 732
6 90 1 000 60 400 2.0 0.011 101
7 90 1 500 600 50 1.0 0.028 274
8 90 2 000 300 100 0.5 0.028 162
9 360 700 300 400 1.0 0.013 916
10 360 1 000 600 200 0.5 0.028 459
11 360 1 500 60 100 2.5 0.042 009
12 360 2 000 150 50 2.0 0.046 665
13 720 700 600 100 2.0 0.037 602
14 720 1 000 300 50 2.5 0.039 153
15 720 1 500 150 400 0.5 0.023 887
16 720 2 000 60 200 1.0 0.031 984
R 0.011 0.009 0.003 0.016 0.007
ThegrowthofNaviculatenerainoptimizedmedium　
Comparedwiththeoriginalmedium andoptimizedmedium
No.1, optimizedmediumNo.2 performedbeteringrowth
ratesandbiomassofNaviculatenera, wherethepeakOD680
reached4.25.Byconversion, theregressionequationfordry
weightofNaviculatenerawasdeterminedtobeY=25 ×
(0.047X-0.009 1)(P<0.001, R2 =0.994 9), fromwhich
thepeakbiomassesinthetheoriginalmedium, optimizedme-
diumNo.1 andoptimizedmediumNo.2 werecalculatedas
1.334, 2.510 and4.766 g/L, respectively.Theresultex-
plainsthatoptimizedmediumNo.2couldwelsuittherequire-
mentsofNaviculateneragrowth, anditshouldbetheopti-
mizedmediumformula(Fig.9).
Conclusion
Atpresent, traditionalF/2 mediumisoftenemployedto
cultureNaviculatenerainlaboratoryandfield;andtheculture
Fig.9　ThegrowthofNaviculateneraintheoptimizedme-
diumformula
conditionshaveneverbeenwelinvestigated, resultinginthe
lowdensityofNaviculatenerainculture.WUSu-zhenetal[ 4]
carriedoutaoptimizationtestforthecultureconditionsofNa-
viculatenera, butfailedtoimproveitsbiomasssignificantly.
Lowculturedensityhasbecomethebottleneckforeficiently
exploitingandutlizingNaviculatenera.Togetherwiththede-
tailedenvironmentalcondition, wefirstlyoptimizedaseriesof
conditionsforculturingNaviculatenera, theresultslaidasolid
foundationforthecomprehensiveexploitationandutilizationof
Naviculatenera.Usingsewageaswatersource, Navicula
tenera, itdecreasedtheculturecostofNaviculatenera, and
meanwhilelaidabasisforcombiningtheoutdoorcultureof
Naviculatenerainlargescaleandsewagetreatment.Theop-
timizedconditionsforcultivatingNaviculatenerausingsewage
areasfolows:thewaterfromXiaoerlouArtificialLakeofNan-
jingUniversityofTechnologyasbasicsolvent;360mg/Lure-
a;150 mg/LN2HPO4 · 12H2O;50mg/Lferriccitrate;2 000mg/LNa2SiO3· 9H2O;2.0 mol/Lsalinity.Culturedwiththis
formula, thepeakdrybiomassofNaviculatenerareached
4.766 g/L, whichis3.57 multiplesoverthatfromtheoriginal
medium andhigherthantheheterotrophicpeakvalue(2.8
g/L)reportedbyWUSu-zhenetal[ 4] .ThegrowthofNavicu-
lateneraiscomprehensivelyinfluencedbyvariousnutrients
andenvironmentalfactors.Inthisstudy, justseveralmajorin-
fluencingfactorshavebeenoptimized, otherfactors, theinflu-
encingmechanismsofeachfactor, waterqualityanalysisand
utilizationmechanism arealstilunknownandneedtobe
studiedinfurtherstudy.
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Responsibleeditor:CHENJuan　　　Responsibletranslator:DUANYong-bo　　　Responsibleproofreader:WUXiao-yan
舟形藻的污水培养及条件优化
吴夏芫 , 李 环＊ , 韦萍　(南京工业大学制药与生命科学学院 ,江苏南京 210009)
1　材料与方法
1.1　藻种及培养基　经 “神舟 5号 ”航天搭载后的舟形藻由徐
州师范大学郑维发教授惠赠 。试验所用原始培养基由参考文献
[ 4]的配方优化而成 。
1.2　试剂及仪器　CO(NH2)2 、Na2HPO4·12H2O、 Na2SiO3·9H2O、柠
檬酸铁均为分析纯 ,国药集团化学试剂有限公司;海水晶 ,青岛
通用海大海水素有限公司;分光光度计 , DU650型 ,美国 BECK-
MAN公司生产 。
1.3　藻类的培养　在 500ml摇瓶中培养 ,取处于对数生长期的
舟形藻接种 ,接种初始 OD680保证在 0.2 ～ 0.5(接种前离心以排
除原培养液的影响)。培养条件:温度(24±1)℃、光照时间 18
h/d、光强 3 000lx,通气培养 ,每组试验设 3个平行对照 。隔天固
定时间取样于分光光度计下测 OD680 ,绘制藻细胞生长曲线。
1.4　相对生长速率的测定　相对生长速率按公式 K=(lgOD-
lgOD0)/T计算 。式中 , OD:经过 T时间培养后的藻液 OD680值 ,OD0:T培养时间开始时的藻液 OD680值 , T:培养时间(d)。
1.5　不同水质对舟形藻生长的影响　分别以南京市玄武湖水、
青石村金川河水、南京工业大学小二楼人工湖水 、自来水代替蒸
馏水作为培养基基础溶剂配制人工海水培养基 ,培养舟形藻。
以下试验培养基的基础溶剂为该试验确立的最佳培养用水 。
1.6　N、P、Fe、Si、盐度各因素单因子优化试验　①N因子优化
试验 。以原始培养基为基础 ,改变氮源种类 ,分别以 KNO3 、
NH4 Cl、尿素为氮源 ,初始 N浓度均为 3 mmol/L,对舟形藻进行
培养;分别设置不同尿素浓度:45、90、180、360、720mg/L培养舟
形藻 。 ②P因子优化试验 。分别设置不同 Na2HPO4·12H2O浓度:30、60、120、240、480 mg/L培养舟形藻 。③Fe因子优化试验。
分别以 Fe-EDTA、柠檬酸铁作为铁盐对舟形藻进行培养 ,初始 Fe
浓度均为 0.15mmol/L;分别设置不同柠檬酸铁浓度:0、25、50、
100、150、200mg/L培养舟形藻。 ④Si因子优化试验。分别设置
不同 Na2SiO3·9H2O浓度:100、400、700、1 000、1 200、1 500mg/L
培养舟形藻 。 ⑤盐度因子优化试验 。分别设置 5个盐度梯度:
0.5、1、1.5、2、2.5mol/L培养舟形藻 。
1.7　N、P、Fe、Si、盐度 5因素正交优化试验　根据上述各单因
子对舟形藻生长影响的考察结果 ,进行 5因素正交优化试验。
正交表的设计参照文献 [5]进行 ,选用 L16(45 )设计 ,正交试验中
各因素的水平设计分别为尿素 (A)45、90、360和 720 mg/L,
Na2SiO3·9H2 O(B)700、1 000、1 500和 2 000 mg/L, Na2 HPO4·
12H2O(C)60、150、300和 600 mg/L,柠檬酸铁(D)50、100、200
和 400 mg/L,培养基盐度(E)0.5、1.0、2.0和 2.5 mol/L。
2　结果与分析
2.1　不同水质配制培养基的影响 　蒸馏水配制的微藻培养基
需加微量金属元素和维生素 ,而湖河水一般已含有丰富的微量
金属元素和营养元素 ,是否需要另加微量金属元素和维生素要
进行考察。据此笔者设计了如下试验组:正常蒸馏水组 (CK)、
自来水组、自来水加微量金属元素和维生素(以下简称微)组 、金
川河水组、金川河水加微组 、玄武湖水组 、玄武湖水加微组 、小二
楼水组 、小二楼水加微组 。据调查得知:玄武湖水中主要有城市
生活污水及部分工业废水的排放 ,金川河水主要为居民生活污
水的排放 ,小二楼水则主要为南京工业大学部分生活污水及实
验废水的排放 。
图 1(图见第 69页 Fig.1:不同水质对舟形藻生长的影响)
表明 ,最适合舟形藻生长的是小二楼水组 ,而小二楼水加微组生
长速率反而下降 ,可能是因为小二楼水中已有足够的微量金属
元素和维生素 ,如若再额外添加反而不利于舟形藻的生长 。舟
形藻在自来水组中生长最差 ,这可能是因为自来水中微量金属
元素和维生素的缺乏或氯气未除尽造成的 。试验发现 ,舟形藻
的生长适应能力很强 ,可以在多种污水中生长繁殖且不易被杂
藻或杂菌污染 ,这一方面可使舟形藻的培养成本得到降低 ,另一
方面也可使污水得到治理 ,为将来舟形藻的室外大规模培养以
及综合开发打下良好的基础。因此以下试验选定以小二楼水作
为基础溶剂(以小二楼水配制的培养基记为优化培养基 1号),
对该藻的培养条件进行优化。
2.2　N、P、Fe、Si、盐度单因子优化试验
2.2.1　氮盐的影响。舟形藻能很好地利用这 3种形式的氮源
生长繁殖 ,最适合舟形藻生长的氮源为尿素(图见第 69页 Fig.
2:不同形式 N源对舟形藻生长的影响)。以尿素为 N源考察不
同浓度尿素对舟形藻生长的影响 ,结果如图 3(图见第 70页 Fig.
3:不同浓度尿素对舟形藻生长的影响)所示 ,尿素浓度为 360
mg/L时生长最好;尿素浓度超过 360 mg/L,舟形藻生长速率迅
速下降 ,生长受到明显抑制 。
2.2.2　磷盐的影响 。 Na2 HPO4·12H2O在 30 ～ 240 mg/L浓度范围内可以明显促进舟形藻的生长;当其浓度大于 240 mg/L时 ,
舟形藻的生长速率呈明显下降趋势(图见第 70页 Fig.4:不同浓
度 Na2 HPO4·12H2O对舟形藻生长的影响)。
2.2.3　铁盐的影响 。在培养底栖硅藻时 ,多在自然海水中补充
柠檬酸铁 ,但并未对铁盐的最适形式和最适浓度作详尽探讨 。
该试验分别选择 Fe-EDTA、柠檬酸铁作为铁盐培养舟形藻 ,结果
(图见第 70页 Fig.5:不同形式铁盐对舟形藻生长的影响)发现
柠檬酸铁对舟形藻生长的促进作用明显优于 Fe-EDTA。不同浓
度柠檬酸铁对舟形藻生长的影响(图见第 70页 Fig.6)表明 ,在
0 ～ 50 mg/L范围内 ,随着柠檬酸铁浓度的升高 ,舟形藻的生长受
到明显促进 ,培养基不含柠檬酸铁时藻体生长缓慢;但当柠檬酸
铁浓度超过 50 mg/L时 ,舟形藻聚集成团沉入摇瓶底部 ,藻体颜
色变淡 ,生长速率明显下降 ,藻细胞很快进入衰亡期 ,说明高浓
度的柠檬酸铁对藻细胞有毒害作用 ,不利于该藻的生长 。
2.2.4　硅盐的影响 。舟形藻对硅盐的需求相对较多 ,在硅盐浓
72 AgriculturalScience＆TechnologyVol.10, No.1, 2009
度小于1 500mg/L时 ,其生长与硅盐浓度呈现一定的剂量依赖
关系 ,并在试验所设定的最高硅盐浓度 1500 mg/L下生长速率
最高 ,生物量达到最大(图见第 70页 Fig.7)。
2.2.5　盐度的影响。不同盐度对舟形藻生长的影响(图见第 71
页 Fig.8)表明 ,该藻的耐高盐能力很强 。接种初期 , 0.5mol/L
NaCl浓度下舟形藻生长繁殖最迅速 ,而 1.0 ～ 2.5mol/LNaCl浓
度下舟形藻生长繁殖速率较慢 ,可能因为藻细胞还未能适应高
盐环境 ,藻体生长受到暂时抑制;培养 4 d左右 ,藻细胞逐渐适
应 1.0 ～ 2.5 mol/LNaCl的高盐环境 ,生长繁殖速率大幅度提
高 ,且在 2.0 mol/LNaCl浓度下藻细胞生长最佳 ,而盐度超出
2.0 mol/L时舟形藻的生长明显受到抑制。
2.3　N、P、Fe、Si、盐度 5因素正交优化试验
2.3.1　5因素对舟形藻生长影响的顺序和最优水平组合。 5因
素对舟形藻生长的影响主次顺序为 C>A>D>E>B,即对于舟
形藻而言 ,因素柠檬酸铁和尿素为主要影响因素(表 1)。取所
有因素的最佳水平 ,得到有利于舟形藻生长的 5因素最佳水平
组合为:尿素 360 mg/L、Na2SiO3·9H2O2 000 mg/L、Na2 HPO4·
12H2O150mg/L、柠檬酸铁 50 mg/L、盐度 2.0 mol/L,记为优化培养基 2号 。
2.3.2　舟形藻在优化培养基中的生长状况 。与原始培养基和
优化培养基 1号相比 ,优化培养基 2号中舟形藻明显生长速率
和生物量均得到很大的提高 ,最高 OD680值达到 4.25 ,通过换算
得到的 OD680值对应藻体干重回归线形方程:Y=25×(0.047x-
0.009 1)(P<0.001 , R2 =0.994 9),可以分别得到原始培养基、
优化培养基 1号与优化培养基 2号最高生物量干重为 1.334、
2.51和 4.766g/L。这充分表明经过优化后的培养基 2号更适
合舟形藻生长的需要(图见第 71页 Fig.9:舟形藻在优化培养基
中的生长情况)。
3　结论
该试验首次结合当地环境条件对舟形藻的培养进行了一系
列考察 ,试验所得该藻的最优培养条件为:以南京工业大学小二
楼人工湖水为培养基基础溶剂 ,尿素 360mg/L、Na2 HPO4·12H2 O
150mg/L,柠檬酸铁 50 mg/L、Na2SiO3·9H2 O2 000 mg/L、盐度
2.0mol/L。通过培养基的优化 ,舟形藻最高生物量干重达 4.766
g/L,是原始培养基的 3.57倍 。舟形藻的生长是各种营养物质
和环境因素综合作用的结果 ,该研究仅对其中几个主要的影响
因子进行探讨 ,对于其他因素 、各因素影响机理 ,各种污水水质
分析及舟形藻利用机制等问题都有待于进一步研究 。
表 1　营养盐和盐度在舟形藻培养中的作用正交试验结果及直观分析
试验号
因子与水平
Amg/L Bmg/L Cmg/L Dmg/L Emg/L
相对生长率
1 45 700 60 50 0.5 0.021 431
2 45 1 000 150 100 1.0 0.028 582
3 45 1 500 300 200 2.0 0.032 682
4 45 2 000 600 400 2.5 0.024 057
5 90 700 150 200 2.5 0.021 732
6 90 1 000 60 400 2.0 0.011 101
7 90 1 500 600 50 1.0 0.028 274
8 90 2 000 300 100 0.5 0.028 162
9 360 700 300 400 1.0 0.013 916
10 360 1 000 600 200 0.5 0.028 459
11 360 1 500 60 100 2.5 0.042 009
12 360 2 000 150 50 2.0 0.046 665
13 720 700 600 100 2.0 0.037 602
14 720 1 000 300 50 2.5 0.039 153
15 720 1 500 150 400 0.5 0.023 887
16 720 2 000 60 200 1.0 0.031 984
极差 0.011 0.009 0.003 0.016 0.007
基金项目　国家 973项目资助(2003CB71600)。
作者简介　吴夏芫(1984-),女,江西宜春人, 硕士研究生 ,研究方向:
微藻生物柴油。 ＊通讯作者。
收稿日期　2008-12-19　　修回日期　2009-03-05
(上接第 14页)
T1处理叶面积变化叶面积变化规律较为一致 ,呈明显的二次
曲线趋势 , T2处理叶面积变化较为平滑 ,多数时间内 ,单叶叶
面积随着 UV-B辐射的增加而减小 (图见第 13页 Fig.4:玉米
单叶叶面积变化)。各处理单叶叶面积差异的显著性检验(表
见第 13页 Table4)表明 , 6月 29日 、7月 14日 、7月 19日 、7月
29日和 8月 24日测定的叶面积差异显著 ,其他时间测定的叶
面积没有明显差异 , T1和 T2之间所有时间测定的叶面积均没
有显著差异 。
2.3　UV-B辐射增强对玉米绿叶数的影响　总体上 , UV-B辐
射增强对玉米单株绿叶数影响较小 , 3个处理的绿叶数仅在 6
月 24日 ～ 7月 4日间有明显差异 ,其他时间的差异均不显著
(表见第 13页 Table5:UV-B辐射增强对玉米绿叶数的影响)。
2.4　UV-B辐射增强对玉米产量构成的影响　UV-B辐射增
强从多方面影响产量构成:其中 T1处理的穗位高 、果穗长 、果
穗粗及百粒重与 CK处理接近 ,其他产量构成因素 (秃尖长 、穗
粒数和穗粒重)都是 CK处理最优 、T1处理次之 、T2处理最差;
此外 , T2处理的穗位高 、果穗长 、果穗粗及百粒重均比 CK、T1
处理低或者差 。也即随着 UV-B辐射增强 ,玉米各产量构成因
素均不同程度变差 。由于产量构成因素受较大不利影响 ,因此
玉米最终理论产量随着 UV-B辐射增强而减小 , T1处理的理论
产量为 CK的 83.5%,而 T2处理的理论产量仅为 CK的 61.8%
(表 6)。
　　表 6 UV-B增强对玉米产量构成的影响
处理 穗位高∥cm 果穗长∥cm 果穗粗∥cm 秃尖长∥cm 穗粒数∥个 穗粒重∥g 百粒重∥g 理论产量∥kg/hm2
CK 131.0 24.1(100) 5.6(100) 3.9(100) 569.7(100) 198.3(100) 34.9(100) 17 761.5(100)
T1 131.7 23.9(101) 5.4(99.2) 4.8(123) 512.0(89.8) 174.1(87.8) 34.1(97.7) 14 876.5(83.5)T2 122.0 20.4(93.1) 4.9(84.6) 5.1(131) 375.7(65.9) 116.0(58.5) 31.0(88.8) 10 968.0(61.8)
注:圆括号内数字表示与 CK的比例(%)。
3　结论
该研究表明 ,在中高海拔的干旱地区 , UV-B辐射增强降低
了玉米的株高 ,但增加低剂量和高剂量 UV-B辐射强度的处理
间没有显著差异 。玉米叶片长和叶宽均随 UV-B辐射增强而
变短或变窄 ,因而导致玉米叶片叶面积减小 。玉米绿叶数受
UV-B辐射增强的不利影响较小 ,绿叶数仅在拔节期至抽雄期
随着 UV-B辐射增强而明显减少。 UV-B辐射增强对玉米产量
构成产生了较大不利影响 ,最终导致玉米理论产量随着 UV-B
辐射强度增加而大幅下降 。
基金项目　宁夏自然科学基金项目(A1012)。
作者简介　张磊(1974-), 男 ,宁夏永宁人 ,硕士 , 工程师 , 主要从事
农业气象 、生态学等方面的研究。 ＊通讯作者。
收稿日期　 2009-01-06　　修回日期　 2009-02-10
73WUXia-yuanetal.OptimizationoftheConditionsforUsingSewagetoCultivateNaviculatenera