a
StateKeyLaboratoryofBioreactorEngineering,EastChinaUniversityofScienceandTechnology,Shanghai200237,PRChina
b
JiaxingZeyuanBio-productsCo.,Ltd.,Jiaxing314007,PR
Shanghai
astaxanthin
system
hinunder
resistprotozoan
saving
Haematococcuspluvialiswasculturedforcommercialproductionof
(Guerinetal.,2003;Higuera-Ciaparaetal.,2006;Lorenzand
Cysewski,2000).Untilnow,thesyntheticastaxanthindominates
currentcommercialastaxanthinmarket,ofwhichthetotalvalue
ismorethan$200Mperyear(Lietal.,2011;Milledge,2011).
However,naturalastaxanthinismorefavorableinthemarketthan
besynthesized
Asastaxanthin
al.,2007;
Haematococ
pluvialisisrecognizedasthebestbiologicalsourcefornatural
xanthin,andfarsurpassesanyotherreportedsources(Lorenz
Cysewski,2000).
Althoughtheresearchanddevelopmentofastaxanthinproduc-
tionfromH.pluvialishavebeencommittedintheseyears,the
successfulstrategyforthecommercialproductionisestablished
baseonthetwo-stepculture(Aflaloetal.,2007;Fábregasetal.,
2001;Saradaetal.,2002).Thefirststage,greenvegetativegrowth
phase,isperformedtoobtainalargequantityofgreenvegetative
cellsunderthefavorablecultureconditionintubular,bubble
?
Correspondingauthor.Address:Mailbox301,MeilongRoad130,Shanghai
200237,PRChina.Tel./fax:+862164250964.
E-mailaddresses:wanminxi@gmail.com(M.Wan),ygli@ecust.edu.cn(Y.Li).
1
Theseauthorscontributedequallytothiswork.
BioresourceTechnology163(2014)26–32
Contentslistsavailable
BioresourceTec
Astaxanthin(3,3
0
-dihydroxy-b,b-carotene-4,4
0
-dione)isared
ketocarotenoidwithextraordinaryantioxidantcapability.Ithas
widespreadapplicationsinaquacultureanddietarysupplements
xanthinisnotbeingmet.Naturalastaxanthincan
bysomeplants,bacteria,fungi,andgreenalgae.
contentupto1–5%ofcelldryweight(Heet
andCysewski,2000),theunicellulargreenalgae
http://dx.doi.org/10.1016/j.biortech.2014.04.017
0960-8524/C2112014ElsevierLtd.Allrightsreserved.
Lorenz
cus
asta-
and
Received24February2014
Receivedinrevisedform4April2014
Accepted5April2014
Availableonline16April2014
Keywords:
Haematococcuspluvialis
Astaxanthin
Biomass
Attachedcultivation
Photoinduction
astaxanthinthroughtwocontinuousphases:cellgrowthandastaxanthininduction.Inthisstudy,the
efficiencyofanattachedsystemforproducingastaxanthinfromH.pluvialiswasinvestigatedandcom-
paredtothatofthesuspendedsystem(bubblecolumnbioreactor)undervariousconditions.Results
showedthatthisattachedsystemismoresuitableforphotoinductionofH.pluvialisthanthesuspended
bioreactor.Undertheoptimalconditions,theastaxanthinproductivityoftheattachedsystemwas
65.8mgm
C02
d
C01
and2.4-foldofthatinthesuspendedsystem.Thisattachedapproachalsooffersother
advantagesoversuspendedsystems,suchas,producingastaxanthinunderawiderangeoflightintensi-
tiesandtemperatures,savingwater,easetoharvestcells,resistingcontamination.Therefore,the
attachedapproachcanbeconsideredaneconomical,environmentallyfriendlyandhighly-efficienttech-
nologyforproducingastaxanthinfromH.pluvialis.
C2112014ElsevierLtd.Allrightsreserved.
1.Introductionsyntheticastaxanthin,whilethemarketdemandfornaturalasta-
Articlehistory:
Astheoptimalsourceofastaxanthin,
c
ShanghaiZeyuanMarineBio-productsCo.,Ltd.,
highlights
C15H.pluvialiscanaccumulateeffectively
C15Astaxanthinproductivityoftheattached
C15Attachedcellscanaccumulateastaxant
C15Thisattachedcultivationcanstrongly
C15Thisattachedcultivationissuperioron
articleinfo
China
200237,PRChina
withthisattachedcultivation.
is2.4-foldofthatsuspended.
lowlightandtemperatureupto35C176C.
contamination.
water,easetoharvest.
abstract
MinxiWan,DongmeiHou,YuanguangLi,JianhuaFan,JiankeHuang,SongtaoLiang,
WeiliangWang
a
,RonghuaPan
b
,JunWang
b
,ShulanLi
c
TheeffectivephotoinductionofHaematococcus
astaxanthinwithattachedcultivation
a,1a,1a,?
journalhomepage:www.elsevier
pluvialisforaccumulating
aaa
atScienceDirect
hnology
.com/locate/biortech
column,airliftphotobioreactors,ortheracewayponds.Andthe
secondstage,reddishinductiveproductionphase,istheinduction
ofcellstoaccumulateastaxanthinwiththetransitionofgreenveg-
etativecellstoreddishcystcellsinvariousstressconditions,such
asnitrogenlimitation,excessacetateaddition,stronglightinten-
sity,saltstress,phosphatedeficiency,ortheadditionofspecificcell
divisioninhibitors(Hataetal.,2001;Huetal.,2008;Lietal.,2010;
Saradaetal.,2002;Wangetal.,2003).Therefore,inductionsys-
temshaveadirectcorrelationwiththeastaxanthincontentofcells
andtotalproductivityofastaxanthin.Attachedsystemshavebeen
successfullyusedforculturingalgaetoremovenutrientsfrom
wastewater(Kebede-Westheadetal.,2006;WilkieandMulbry,
2002),andaredevelopingtogrowmicroalgaewiththepurpose
ofproducingbiofuelfeedstockduetolowconsumptionsofwater
2.2.1.Photobioreactor
M.Wanetal./BioresourceTechnology
Asimplebioreactorwasusedtoinvestigatetheinductionfeasi-
bilityofattachedalgaecells(Fig.1).Agauzesupportedbywire
meshwasverticallyplacedinamediumreservoir.Filteredwitha
0.2lmmicrofiltrationmembrane(thediameterof33±0.5cm
2
),
suspendedcellsformedanalgalfilmonthemembrane.Thenthe
membranewithalgalfilmwasplacedontothegauze.Inorderto
Light
SpargerAlgal“disk”
Inductionmedium
reservoir
Pump
Circulationpipe
Medium
flow
Algalfilm
Microfiltration
membrane
Wiremesh
support
Gauze
A
B
andenergyusingattachedsystems(JohnsonandWen,2010;Liu
etal.,2013;Ozkanetal.,2012).
Here,anattachedtechnologywasusedintheastaxanthinaccu-
mulationofH.pluvialisandcomparedwiththetraditionalsus-
pendedtechnology,andtheefficiencyofattachedtechnologyfor
astaxanthinproductivitywasevaluatedundervariousconditions.
2.Methods
2.1.Microalgalstrainandcultureconditions
H.pluvialisNIES-144wasobtainedfromtheNationalInstitutefor
EnvironmentalStudies,Tsukuba,Japan.ThemediumwasNIES-C
mediumwith10mMsodiumacetateastheorganiccarbonsource
(Hataetal.,2001).Thegreencellswereculturedundercontinuous
illuminationof25lmolm
C02
s
C01
at25C176C,andweresubsequently
inducedwithcontinuousilluminationof150lmolm
C02
s
C01
at
25C176Cunlessotherwisestated.Thepeak,dominant,centroidand
centralwavelengthsofilluminationfromthecoldfluorescence
lampwere445,504,437and437nm,respectively.Furthermore,
thecolortemperaturewas6199k.Otherphotoinductionconditions
foreachexperimentweredescribedinthesectionofexperimental
design.
2.2.Photobioreactorandexperimentaldesignfortheattached
induction
Fig.1.TheattachedbioreactorfortheinductionofH.pluvialis.(A)Theoverviewof
theattachedinduction.(B)Thedetailsofthealgae‘disk’.
fixthealgalfilm,thesmallmagnetswereputontheedgeofmem-
brane,andbroughtmagneticforcewithwiremeshsupport.During
theinduction,theinductionmediumfloweddownfromasparger
onthetopbrimofgauzesothatmediumpermeatedthegauzeand
microfiltrationmembranetowetalgalfilm.Theflowrateforeach
algaldiskintheattachedsystemis5ml/min,andcanbecontrolled
attherangesof0.05–190ml/mintochangemoistureofattached
cells.Fornormalexperiment,Thecontrolexperimentwasper-
formedin1Lcolumnbioreactor(height:26cm,radius:3.5cm,
theverticalcrosssectionwas182cm
2
)underthesamecondition.
Aerationandmixingweredonebyspargingairwiththerateof
0.5vvm.
2.2.2.Experimentaldesignfortheattachedinduction
Allexperimentswererepeatedthreetimes.Theinductionmed-
iumwasNIES-Nmedium(Kangetal.,2005)forallinductionexper-
imentsinthisstudy.Thismediumwithoutnitrogenledtocells
undernitrogendeficiency.Exceptspecialrequirement,H.pluvialis
wasinducedundercontinuousilluminationof150lmolm
C02
s
C01
at25C176C.Temperaturesandinitialcellamountweresetrespec-
tivelyto15,25,35C176Cand10,20,40gm
C02
toinvestigatetheir
effectsoninduction.Illuminationwasinthehorizontaldirection,
thustheverticalcrosssectionofcolumnbioreactorwasconsidered
light-receivingareainordertocompareperformancesbetween
bothattachedandsuspendedinductionsystems.Thebiomassof
suspendedcellswastransformedtothatofalgaldiskaccording
tothefollowingformula:
m
disk
?m
suspend
C3V=S
wherethem
disk
(g/m
2
)isthebiomassconcentrationofalgaldisk,
them
suspend
(gL
C01
)isthebiomassconcentrationinthecolumnbio-
reactor,V(L)isthevolumeofthecolumnbioreactor,S(m
2
)wasthe
light-receivingareaofcells.
Therefore,thestartingcelldensitiesperliterinthecolumnbio-
reactorwere0.18,0.36and0.65gL
C01
correspondingtothe10,20
and40gm
C02
intheattachedbioreactor.Theinductionwas
exposedtocontinuousilluminationof50,70,90,120,160,and
230lmolphotonsm
C02
s
C01
,respectively,tostudytheeffectoflight
densityoninduction.Furthermore,theflowrateoftheinduction
mediumwasadjustedinordertoinvestigatetheeffectofmoisture
oninduction.
2.3.Analyticalmethods
2.3.1.Dryweight
Forthecolumnbioreactor,thealgalcellswerecollectedbycen-
trifugingtheinductionfluidat4000gfor10min,washedwithdis-
tilledwateranddriedat80C176Cfor24h.Whilethecellofalgaldisk
waswasheddownandre-suspendedwithavolumeofdistilled
water,andthenthedryweightofalgaldiskwasmeasuredassame
asabove.Thusthebiomassofalgaldiskwascalculatedas:
M
t
?m
t
=S
wherethem
t
wasthedryweightofalgaldiskafterinduction,andS
wasthelight-receivingareaofcells.
Netbiomassproductivity:eM
t
C0M
0
T=t
whereM
t
wasdryweightafterinduced,andM
0
wastheinitialdry
weight.
2.3.2.Moistureofalgaldisk
Theflowrateofmediumwasadjustedtochangemoistureof
algaldiskwhichwascalculatedbytheratioofdrycellweightto
163(2014)26–3227
wetcellweight.Firstly,wetcellswerescrapedfromalgaldisk,
andthenweredividedintotwogroupsandweighedseparately.
Group1wasdirectlydriedat80C176Cfor24htomeasurethetotal
astaxanthincontentwasachievedattheinitialcellamountof
10gm
C02
.Butthegrowthratesatinitialcellamountof20and
40gm
C02
weresimilar,andwerehigherthanthatof10gm
C02
.
Theinductioneffectoftheattachedbioreactorwascomparedwith
thatofthecolumnbioreactor.Astheresults,thevariationtrendof
theastaxanthininthecolumnbioreactorwassimilartothatinthe
attachedinduction,namely,theastaxanthincontentsincreased
withreducinginitialcellamounts.Attheinitialcellamountof
10and20gm
C02
,theastaxanthincontentintheattachedinduction
washigherthanthatinthecolumnbioreactor.Aninterestingphe-
nomenoninthesuspendedinductionsystemhasbeenobserved
widelythatcellsareeasytoattachonthewallsofbioreactors,
andtheseattachedcellsreddenmuchfasterthanthosesuspended
inliquid.H.pluvialiswithhighastaxanthincontentisusuallyfound
024681012
0.2
0.4
0.6
Astaxanthin(%,w/w)
Time(d)
024681012
0
10
20
30
40
50
60
70
80
90
100
Attachedinductionwithinitialcellamountof10gm
-2
Attachedinductionwithinitialcellamountof20gm
-2
Attachedinductionwithinitialcellamountof40gm
-2
Suspendedinductionwithinitialcellamountof10gm
-2
Suspendedinductionwithinitialcellamountof20gm
-2
Suspendedinductionwithinitialcellamountof40gm
-2
Biomass(gm
-2
)
Time(d)
(b)
Fig.2.Theastaxanthincontents(a)andbiomassconcentrations(b)ofH.pluvialis
withthreedifferentinitialcellamountsintheattachedbioreactorandthecolumn
bioreactor.
Technology
dryweightofcellsandsaltsinsideinductionmedium.Group2was
washedwithdistilledwater,andthencellswerecollectedbycen-
trifugingthefluidat4000gfor10min,anddriedat80C176Cfor24h
tomeasuredrycellweight.Thusthemoistureofthealgaldiskwas
calculatedas:
Moisture?e1C0m
d1
=m
w1
T=e1C0m
d1
=m
w1
C0m
d2
=m
w2
T
wherem
d1
wasdryweightofGroup1(includingcellsandsalts),
andM
w1
wasthewetweightofGroup1(includingcells,water,
andsalts),m
d2
wasdryweightofGroup2(onlyincludingcells),
andM
w1
wasthewetweightofGroup2(includingcells,water,
andsalts).
2.3.3.Determinationofastaxanthin
Thealgalcellsintheattachedsystemwerewasheddownand
re-suspendedwithdistilledwaterfortheastaxanthindetermina-
tion.TheastaxanthincontentwasmeasuredwithmodifiedBouss-
ibamethod(BoussibaandVonshak,1991).Forastaxanthin
determination,5mLculturesamplewascentrifugedfor10min
at4000g,andthepelletwasfirstsaponifiedbyusingasolution
of5%KOHin30%(v/v)methanolat65C176Cfor15mintodestroy
thechlorophyll.Andthenthesupernatantwasdiscarded,andthe
pelletwasthenwashedthreetimeswithde-ionizedwaterto
removetheresiduallye.Theremainingpelletwasextractedwith
5mLDMSO(LabkimA.R.,99.5%)usinganultrasonicprocessor
for10mintorecovertheastaxanthin.Theextractionprocedure
wasrepeatedatleastthreetimesuntilthecelldebriswasalmost
colorless.Theabsorbanceofthecombinedextractswasmeasured
at490nm.
Perunitvolumeastaxanthinconcentration(c,mg/L)calculated
as:
c?4:5C2A490C2V
a
=V
b
wheretheV
a
wasthevolumeofextracts,theV
b
wasthevolumeof
culturesample(Fortheattachedculture,theV
b
wasthevolumeof
distilledwaterforthealgalcellswasheddownandre-suspended.),
andA490wastheabsorbanceofextractsat490nm.
Astaxanthincontente%T:c
t
=m
t
wherec
t
wastheastaxanthinconcentrationafterinducedandm
t
wasthedryweight(Fortheattachedculture,them
t
wasthedry
weightindistilledwaterforthealgalcellswasheddownandre-
suspended.)afterinduced.
AstaxanthinyieldeCT:C?cC3V=S
wherethecwastheastaxanthinconcentrationofalgaldiskafter
induction,Vwasthevolumeofdistilledwaterforthealgalcells
washeddownandre-suspended,andSwasthelight-receivingarea
ofcells.
Netastaxanthinproductivity:eC
t
C0C
0
T=t
whereC
t
wastheastaxanthinyieldafterinduced,andC
0
wasthe
initialastaxanthinyield.
3.Resultsanddiscussion
3.1.Theeffectofinitialcellamountonthecellgrowthandastaxanthin
content
Inordertoassesswhethertheattachedcultivationapproach
wouldbesuitableforH.pluvialisinduction,andhowmuchofthe
initialcellamountwouldbebetterfortheinduction,H.pluvialis
wasinducedwithdifferentinitialcellamountintheattached
C02C01
28M.Wanetal./Bioresource
bioreactorundercontinuousilluminationof150lmolmsat
25C176C.AsindicatedinFig.2,thelowertheinitialcellamount
was,thehighertheastaxanthincontentwas.Themaximal
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Attachedinductionwithinitialcellamountof10gm
-2
Attachedinductionwithinitialcellamountof20gm
-2
Attachedinductionwithinitialcellamountof40gm
-2
Suspendedinductionwithinitialcellamountof10gm
-2
Suspendedinductionwithinitialcellamountof20gm
-2
Suspendedinductionwithinitialcellamountof40gm
-2
(a)
163(2014)26–32
inthebottomofdriedoutrockpoolsandbirdbaths(Pocock,1960),
whichprovideasemiaridnicheandaresimilarwiththeattached
system,implyingthenicheintheattachedbioreactormaybeis
consistentwithnaturalenvironmentwherecellsaccumulateasta-
xanthin.Therefore,theattachedinductionstrategymaybemore
suitabletoastaxanthinaccumulationforH.pluvialisintheterm
ofbioecologycomparedtoanotherinductiontechnologywithsus-
pendedliquid.Intheearlyperiodofinduction,biosyntheticrateof
astaxanthinintheattachedbioreactorwassignificantlyhigher
thanthatinthesuspendedbioreactor,whichiswellmatchedwith
theabovephenomenon.
Thecellgrowthinthecolumnbioreactorwasslightlyhigher
gressonfeedstockproductionforbiofuel(Grossetal.,2013;
wasslowestat15C176C.Theastaxanthinproductivitywas32.3,
65.8,43.6mgm
C02
d
C01
andthebiomassproductivitywas1.1,3.7,
2.6gm
C02
d
C01
,respectively.Thus,thebesttemperatureforthe
attachedinductionwasabout25C176Candsimilartootherreports
(Dom?
′
nguez-Bocanegraetal.,2004;Kangetal.,2010,2006,
2005;Yooetal.,2012).
Inthisstudy,theinductioninthecolumnreactorwascarried
outtoverifytheperformanceofattachedinductionunderdifferent
temperature.Inthecolumnphotobioreactor,theastaxanthincon-
centrationwassimilarat15C176Cand25C176C,butbiomassat25C176Cwas
slightlyhigherthanthatat15C176C.But,theastaxanthinaccumula-
tionintheattachedphotobioreactorwasstillfasterthanthatin
thecolumnphotobioreactor.Thecellgrowthinthecolumnbiore-
actorwasslightlyhigherthantheattachedinductionbeforethe
6daysinduction,thenthegrowthrateinthecolumnbioreactor
sloweddown,butthoseintheattachedinductionstillincreased
rapidly.
At35C176C,theastaxanthinaccumulationwassimilarbetween
columnbioreactorandattachedbioreactorbeforethe6thday.
02468101214
0
10
20
30
40
50
60
70
80
Attachedinductionat15
o
C
Attachedinductionat25
o
C
Attachedinductionat35
o
C
Suspendedinductionat15
o
C
Suspendedinductionat25
o
C
Suspendedinductionat35
o
C
Biomass(gm
-2
)
Time(d)
(b)
Fig.3.Theastaxanthincontents(a)andbiomassconcentrations(b)ofH.pluvialisat
temperaturesof15C176C,25C176Cand33C176Cintheattachedbioreactorandthecolumn
bioreactor.
M.Wanetal./BioresourceTechnology
JohnsonandWen,2010;Liuetal.,2013;Ozkanetal.,2012),this
studyshowedattachedsystemhasagoodperformanceforasta-
xanthinproductionfromH.pluvialis.Furthermore,allstandard
deviationsofexperimentsinthisstudyarelessthan10%,indicat-
ingallresultswerestatisticallysignificant.
3.2.Theeffectoftemperatureonthecellgrowthandastaxanthin
content
Thelarge-scalecommercialproductionofastaxanthinfromH.
pluvialisunderoutdoorconditionwasaffectedbytheseason,
mainlyinthelightintensityandtemperature.Moststudieshave
beenreportedthesuitabletemperaturefortheastaxanthinaccu-
mulationofH.pluvialiswasbetweenthe20C176Cand28C176C(Kang
etal.,2010,2006,2005;Lababpouretal.,2005;Yooetal.,2012).
Furthermore,itwasreportedthatH.pluvialiscanaccumulateasta-
xanthinat35C176C(Tjahjonoetal.,1994).Thismaybebecauseofthe
differenceofH.pluvialisspeciesandinductioncondition.Inorder
toassessthescopeoftemperaturefortheattachedinduction,
theinductiontemperaturesweresetat15C176C,25C176Cand35C176C,
respectively.Accordingtoourresults(Fig3),theastaxanthincon-
tentat15C176Cwasthehighest,followedbythoseat25C176Cand35C176C
after12daysinduction,whilethebiomasswasachievedthe
maximumatthe25C176C,followedbythatat35C176C,andthegrowth
Table1
ThenetastaxanthinandbiomassproductivitiesofH.pluvialiswith
differentinitialcellamount.
Induction
bioreactor
Initialcell
amount
(gm
C02
)
Astaxanthin
productivity
(mgm
C02
d
C01
)
Biomass
productivity
(gm
C02
d
C01
)
Attached1044.1±1.72.1±0.2
2065.8±1.73.7±0.3
4058.7±2.33.3±0.3
thanthatintheattachedinductionbeforethe6daysinduction,
thenthecellgrowthinthecolumnbioreactorsloweddownbut
thatintheattachedinductionwasstillkeptatarapidrate.
Besides,boththeastaxanthinproductivityandbiomassproduc-
tivitywerereachedthemaximumattheinitialcellamountof
20gm
C02
(Table1).Theastaxanthinproductivityandthebiomass
productivityofattachedinductionwereabout2.4-foldand2.8-fold
ofthatinthesuspendedinduction,respectively.Attheinitialcell
amountof20and40gm
C02
,theastaxanthinproductivityandthe
biomassproductivityofattachedinductionwerehigherthanthose
ofthecolumnbioreactoraswell.
Forthesamealgaespeciesandinductionmedium,theastaxan-
thincontentwas1%underatemperatureof23C176Candlightinten-
sityof200lmolm
C02
s
C01
intheotherreport(Kangetal.,2005),and
waslowerthanthatinthisattachedinduction.Also,biomassand
astaxanthinproductivitiesinthisattachedinductionweresimilar
orhigherthanthoseinotherreports(Dom?′nguez-Bocanegra
etal.,2004;Hataetal.,2001;Kangetal.,2010,2007).Although
attachedcultivationsystemformicroalgaehavemadeagreatpro-
Suspended1022.4±0.80.9±0.2
2027.3±1.71.3±0.2
4033.9±3.11.1±0.2
02468101214
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Attachedinductionat15
o
C
Attachedinductionat25
o
C
Attachedinductionat35
o
C
Traditionalinductionat15
o
C
Traditionalinductionat25
o
C
Traditionalinductionat35
o
C
Astaxanthin(%,w/w)
Time(d)
(a)
163(2014)26–3229
Then,cellsincolumnbioreactorweredeath,butthoseinattached
photobioreactorweregrowing,suggestingastaxanthininduction
ofH.pluvialisintheattachedsystemcanbeconductedatahigher
temperaturethanthatinconventionalbioreactors.Therefore,the
attachedsystemofH.pluvialismaybeabletoreducethecostof
temperaturecontrol,comparedtotheconventionalapproach.
3.3.Therelationshipsoflightintensitywithcellgrowthand
astaxanthinaccumulationintheattachedbioreactor
Lightisakeyfactorforinducingrapidlytheastaxanthinbiosyn-
thesis.Differedwithsuspendedsystem,thickalgalfilminthe
attachedsystemmaycauseinsufficientilluminationfortheinner-
mostcells.Thusinitialcellamountwassettoahighlevelof
40gm
C02
tostudytherelationshipsoflightintensitywithcell
growthandastaxanthinaccumulation.Asthelightintensity
increaseduntil120lmolm
C02
s
C01
,theastaxanthincontentofcells
inattachedinductionincreased(Fig4).Andtheastaxanthin
contentreachedthemaximumunderlightintensityof
120lmolm
C02
s
C01
,anddecreasedslightlywiththeincreasinglight
keepingcellsliving.Thecurvesofbiomassandastaxanthincontent
duringtheinductionofH.pluvialiswithdifferentmoisturewere
showninFig5.Inthefirst4days,theaccumulationrateofgrowth
andastaxanthincontentunderthe95%moistureisfasterthanoth-
ers.Thenthegrowthrateswith85%and75%moistureincreased,
andtheastaxanthinaccumulationrateunderthreemoisturesslo-
weddown.Atthe12day,astaxanthincontentandbiomasswere
closeamongthreemoistures.Hence,thewetofthealgaldiskcould
notcausethegreatdifferencesinthegrowthandtheastaxanthin
accumulation,suggestingH.pluvialiscantogrowandsynthesize
astaxanthininlittlewater,andisverysuitabletotheinduction
inattachedbioreactors.
3.5.Thewatersavingpotentialandothermeritsforattachedinduction
approach
ForalargescaleproductionofH.pluvialis,1000–1500tonsof
freshwater(withoutwaterrecycling)willberequiredtoproduce
1tonofH.pluvialisdrymassinsuspendedinductionsystems
(Aflaloetal.,2007;García-Maleaetal.,2009;Lopezetal.,2006;
Torzilloetal.,2003;Zhangetal.,2009).Inparticular,drainage
withouttreatmentaftercellcollectionwillbringaseriousenviron-
ment.Suchhugeamountofwaterconsumptionandeffluenttreat-
mentaretwoofmainreasonscausedhighcostforproducing
astaxanthin.Fortheattachedinductionapproach,however,only
alittlewaterwiththenutrientswasrequiredtokeepthealgalcell
0.8
1.0
1.2
1.4
1.6
1.8
Moisture95%
Moisture85%
Moisture75%
(a)
30M.Wanetal./BioresourceTechnology163(2014)26–32
intensity.Thebiomassproductivitiesweresimilarunderlight
intensitiesbelow90lmolm
C02
s
C01
.Intherangeoflightintensities
from90to160lmolm
C02
s
C01
,thebiomassincreasedwithlight
intensity.However,theincreaserateofbiomasssloweddown
whenthelightintensityexceeded160lmolm
C02
s
C01
.Theseresults
indicatedthatthe160lmolm
C02
s
C01
oflightdensitycouldbecon-
sideredasthelightsaturationpoint(LSP)forthisattachedinduc-
tionsysteminourinvestigatedconditions.Thisresultwas
differentthanthoseinsuspendedinductionthattheastaxanthin
contentwasreachedthemaximumatthelightintensity
300lmolm
C02
s
C01
(Lietal.,2010;Liang,2009).Therefore,the
attachedinductionofH.pluvialis,evenatinitialcellamountof
40gm
C02
,canworkwellunderlowerlightintensitythansus-
pendedapproach.Furthermore,theattachedinductioniseasyto
avoidharmfulhighlightintensitybytiltingattachedbioreactor
towardthelightdirection,whichmeansmoreinductionbioreac-
torsinthesameilluminationareacanbeoperated.
3.4.Therelationshipsofmoisturewithcellgrowthandastaxanthin
accumulationintheattachedbioreactor
Intheattachedphotobioreactor,attachedcellsmustkeepa
certainmoisturetoavoiddeathcausedbylacksofwater.However,
highmoisturemeansmoreelectronicpowercostforenhancingthe
flowrateofinductionmedium.Inthisstudy,theeffectsof
moistureoncellgrowthandastaxanthinaccumulationofH.pluvi-
aliswereinvestigated.Duetowaterincells,thelowestmoistureof
H.pluvialiscellsintheattachedphotobioreactorwas75%when
406080100120140160180200220240
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Astaxanthin
Astaxanthin(%,w/w)
Lightintensity(μmolm
-2
s
-1
)
0
10
20
30
40
50
60
70
80
Biomass
Biomass
(
gm
-2
)
Fig.4.TheastaxanthincontentsandbiomassconcentrationsofH.pluvialisinthe
attachedbioreactorunderdifferentlightintensitiesfor12daysofphotoinduction.
02468101214
0.0
0.2
0.4
0.6
Astaxanthin(%,w/w)
Time(d)
024681012
10
20
30
40
50
60
70
Moisture95%
Moisture85%
Moisture75%
Biomass(gm
-2
)
Time(d)
(b)
Fig.5.Theastaxanthincontents(a)andbiomassconcentrations(b)ofH.pluvialis
withdifferentmoisturesintheattachedbioreactor.
Technology
wet.Asbeeninourexperiments,thewaterconsumptionofthe
attachedinducewaslessthan30%ofthatintheopenpond.There-
fore,theattachedinductionapproachcandramaticallyreducethe
waterrequirementandtheamountofwastewater.
Thecontaminationbyprotozoandoesgreatharmtomicroalgae
masscultivation(Tangetal.,2011).Thecontaminationcontrolfor
conventionalopenaqueous-suspendedcultivationwasquitediffi-
cultduetothehugewaterbody.AsshowninTable2,thecontam-
inationbyprotozoan,mainlybeparamecium,intheattached
systemwaslessthanintheopencolumnreactors.Furthermore,
smalldoseofpesticidesorantibioticswouldbeenoughtoreadily
controlthecontaminationintheattachedsystemduetothesmall
waterbody.Thus,theeffectofcontaminationoncellcultivation
couldbeminimized.
Othermeritsofattachedinductioninclude:(1)powercost-
effective.Mixtureofmediumisrequiredandenergy-intensivein
suspendedinductionsystems,butisnotneededanymoreinthe
attachedinduction.Furthermore,algalbiomasspastesarecol-
lectedeasyandcost-effectivelybyscrapingdownthebiomass
directlywithoutfurtherdewatering(Grossetal.,2013;Liuetal.,
2013;Ozkanetal.,2012).(2)wideinductioncondition.Cellscan
beinducedwiththeattachedapproachtosynthesizeastaxanthin
underhigherorlowertemperatureandlightintensityresulting
fromcloudydayortheseason,comparedtothesuspended
approach.Itisoneofnoteworthyfeaturesofattachedtechnology
forthecommercialapplication,becausecontrollingcellsundera
smallrangeoftemperaturesandlightintensitiesishigh-costfor
outdoorculture.(3)Easytoscale-up.Thescale-upofconventional
cultivationdevicesmayreducetheperformanceofbioreactordue
tothechangesofmixingcharacteristics.Fortheattachedinduction
systemwithoutmixing,theconstraintsinconventionalbioreactor
designandscale-upwerereleasedgreatly.Themediumcanbe
cycledwithspraysinsteadofpumpstowettocells,andthewire
meshsupportcanbereplacedbycheaper,thinnerandwaterreten-
tivematerials.
4.Conclusions
Inthepresentstudy,anattachedcultivationapproachwassuc-
cessfullyappliedintheinductionofHaematococcuspluvialisfor
astaxanthinproduction.Undertheoptimalcondition,biomass
andastaxanthinproductivitiesintheattachedcultivationwere
2.8-fold(3.7gm
C02
d
C01
)and2.4-fold(65.8mgm
C02
d
C01
)ofthose
inthesuspendedbioreactor,respectively.Furthermore,the
attachedcultivationapproachissuperiortosuspendedinduction
Table2
Theamountofparameciumintheattachedbioreactorandthecolumnbioreactor.
InductionbioreactorThetotalamountofparamecium(cellsperdryalgal
cellweight,C210
7
cells/g)
3days12days
Attachedinduction7±135±2
Suspendedinduction51±5103±12
M.Wanetal./Bioresource
approachinotheraspects,suchas,lowerwaterconsumptionand
smallerriskofcontamination,indicatingthisapproachprovidesa
promisingwaytoboosteconomicbenefitandconsiderablyreduce
productioncostofastaxanthinfromH.pluvialis.
Acknowledgements
ThisresearchwasfundedbyNationalBasicResearchProgram
China(973Program:2011CB200903&2011CB200904),National
KeyTechnologiesR&DProgram(2011BAD23B04),China
PostdoctoralScienceFoundation(2013M530183).
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