IAMjavmoo.info

Peietal.
BiotechnolBiofuels(2017)10:76DOI10.
1186/s13068-017-0759-3RESEARCHTowardfacilitatingmicroalgaecopewitheffluentfromanaerobicdigestionofkitchenwaste:theartofagriculturalphytohormonesHaiyanPei1,2*,LiqunJiang1,QingjieHou1andZeYu1AbstractBackground:Althoughnumerousstudieshaveusedwastewaterassubstitutestocultivatemicroalgae,mostofthemobtainedweakeralgalviabilitythanstandardmedia.
Somestudiesdemonstratedapromotionofphytohor-monesonalgalgrowthinstandardmedia.
Forexploitingastrategytoimprovealgalbiomassaccumulationineffluentfromanaerobicdigestionofkitchenwaste(ADE-KW),theagriculturalphytohormonesgibberellin,indole-3-aceticacid,andbrassinolide(GIB)wereappliedtoChlorellaSDEC-11andScenedesmusSDEC-13atdifferentstagesofalgalgrowth.
Previousstudieshavedemonstratedapromotionofphytohormonesonalgalgrowthinstandardmedia,butattemptshavebeenscarce,focusingonwastewatercultivationsystem.
Inaddition,theeffectsofwastewateronalgalmorphologyandultrastructurehavenotbeenrevealedsofar,muchlessonthemechanismoftheroleofphytohor-monesonalgae.
Results:ADE-KWdisruptedthemembranesofnuclearandchloroplastinultrastructuralcellofSDEC-11,andreducedtheroombetweenchloroplastandcellmembraneandincreasedthestarchsizeofSDEC-13.
Thisreducedalgalgrowthandbiocompoundaccumulation,butSDEC-13hadgreateradaptationtoADE-KWthanSDEC-11.
Moreover,inoculationwithanalgalseedpretreatedwithGIBaidedtheadaptabilityandviabilityofalgaeinADE-KW,whichforSDEC-13wasevenpromotedtothelevelinBG11.
GIBmitigatedtheinhibitionofADE-KWonalgalcelldivisionandphotosyntheticpigmentsandapparatus,andincreasedlipiddroplets,whichmightresultfromthechangeinthesynthesisandthefateofnicotinamideadeninedinucleotidephosphate.
GIBadditionsignificantlypromotedlipidproductivityofthetwoalgalspecies,following13mgL1d1ofSDEC-11inB+ADE-KWandespecially13mgL1d1ofSDEC-13achievedduringtheprimingofalgalseedwiththehormones,whichis139%higherthan5mgL1d1achievedinADE-KWcontrol.
Conclusions:AgriculturalphytohormonescouldbeappliedasastrategyforpromotingbiomassandbiocompoundaccumulationofalgaeinADE-KW,inwhichpretreatmentofthealgalinoculumwithhormonesisauniquewaytohelpalgaesurviveunderstress.
Consideringourresultsandtreatmenttechnologyforkitchenwaste,amorefeasibleandeconomicplantcanbebuiltincorporatinganaerobicdigestion,algaecultivationwithADE-KWassistedwithphytohormones,andbiodieselproduction.
Keywords:Agriculturalphytohormones,Anaerobicdigestion,Kitchenwaste,AlgaebiomassTheAuthor(s)2017.
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0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
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OpenAccessBiotechnologyforBiofuels*Correspondence:haiyanhup@126.
com1SchoolofEnvironmentalScienceandEngineering,ShandongUniversity,No.
27ShandaNanRoad,Jinan250100,ChinaFulllistofauthorinformationisavailableattheendofthearticlePage2of18Peietal.
BiotechnolBiofuels(2017)10:76BackgroundTomakemicroalgae-basedbiofuelpracticallyviable,combiningwastewaterandalgaebiomasscultivationhasbeenregardedasaneconomicandenvironmentallysustainableapproachbymanyresearchers.
Thecombi-nationcouldeasethebottleneckofhighcostinnutrientdemandandvastwaterinputrequiredforsimultaneousalgaegrowthandwastewatertreatment[1–4].
Forsev-eralkindsofwastewaterthathavebeenreportedtobefeasiblealternativesforalgaecultivation,thebiomassesorlipidproductivitiesdecreasedcomparedtothelev-elsachievedinstandardmedium.
Therefore,somestepsmustbetakentofacilitatealgaetoperforminwastewa-teraswellas,orifpossiblebetterthan,theydointheirfavorableartificialmedia,priortolarge-scaleproduction[5].
Astrategymustbeadopted,whichsupportsmicro-algaetomaximizetheirpotentialtosurviveinnonidealenvironment.
Asregardsthewastewaterinthisstudy,wechosetheeffluentfromanaerobicdigestionofkitchenwasteforthefollowingreasons.
Kitchenwasteisgenerateddailyallovertheworldandaccountsforalargepartofmunici-palwaste[6]:forexample,approximately60billionkil-ogramsoffoodsupplyarewastedintheUnitedStates[7];itcontributesupto60%ofthetotalmunicipalsolidwasteinEgypt[8];morethan60billionkgofkitchenwasteareproducedinChinaeveryyear.
Anaerobicdiges-tionisaneffectiveandcommonwaytodealwithkitchenwaste,asitcanreducebiodegradablewasteandproduceH2orbiogas,andhence,therenewableenergytoo[9].
However,intheprocessofanaerobicdigestion,thevastamountofresidualeffluentproducedbecomesanotherproblemduetothepresenceofluxuriouslyrichnutri-entsinit.
Duetothegraduallymaturingandadvancedtechnologyofanaerobicdigestionforkitchenwaste,thelargeamountofeffluentsgeneratedinthisprocesscouldbechosenforcultivatingmicroalgaetoproducebiomassalongwithnutrientremovalfromwastewater.
Moreover,infrastructureforbiodieselproductionusuallyexistsasacomponentofkitchenwastetreatment,takingadvan-tageofoilseparatedfromthewaste,whichmightbeemployedtoproducealgae-basedbiofuel.
Meanwhile,theexistingprocessesforanaerobicdigestionarealsoimportantformicroalgae-basedbiodieselsustainability,whichcansolvetheissuesofalgalresidueafteroilextrac-tionandenergybalance[10].
Therehavebeenafewapplicationsofusingeffluentfromanaerobicdigestionprocesses,withthedigesterfeedstocksincludinglivestockwaste[11],municipalwastewater[2],andfoodwastewater[12].
However,sofar,limitedresearchhasbeenreportedonusingeffluentfromanaerobicdigestionofkitchenwaste(ADE-KW)tocultivatealgae.
Therearedifferencesbetweentheeffluentsfromthedigestionofkitchenwasteandthosefromlivestockwaste,municipal,andfoodwastewaters,especiallyintermsofdarkcolor,highorganicmatterlevels,andlimitedphosphorusconcentration,asshowninAdditionalfile1:TableS1[2,11–15].
Theimbalanceinnutrientratiosandthedarkcolorposechallengestoalgalgrowth.
ItisnecessarytoconfirmwhetheritisfeasibletouseADE-KWtocultivatealgaeoremployalgaetodevelopaprocessfornutrientremovalfromthewastewater.
Intermsofincreasingbiomassandlipidproductiv-ity,phytohormonesaretakenintoconsiderationduetotheirroleinplantgrowthregulation[16,17].
Theirusehasbeenextendedtothefieldofalgaeproduction[18–22].
Planthormonesaregrowthregulators,andatanappropriateconcentration,theyappeartoplayaregula-toryroleinmicroalgaecelldevelopmentincludingcelldivisionorelongation,andinchlorophyllandproteinmetabolisms[21–23].
Besidespromotinggrowth,phyto-hormonesalsohavebeenreportedtoenhancetolerancetostress,suchasheavymetals[20,24],oxidativecondi-tions[25],andosmoticandsaltstresses[26].
Neverthe-less,thehormonesappliedheretoforeinalgalcultivationwerepurechemicals,whichaccountedforthehighcostofproductionandadditionrequiredafterstartingbatchcultivation.
Thehormonescommonlyappliedinagriculture,i.
e.
,agriculturalphytohormones,arecharacterizedbymixedmaterialcontainingmorethanonekindofphytohor-mone,andamatureproductionlinewithlowinvestmentforlarge-scaleproduction.
However,theapplicationofagriculturalphytohormonestomicroalgaehadnotbeenstudied.
Furthermore,thepreviousstudiesdidnotmen-tiontheimpactofphytohormonesonalgalactivityinresponsetoacomplexstressenvironmentsuchasrealwastewater,exceptinapplyinghormonestohelpalgaedealwithasimpleunfavorablefactor[20,24].
Therefore,themainaimofthisstudywastodemonstratewhethertheagriculturalphytohormonescouldenhancethealgae'sabilitytotoleratetheadverseeffectofwastewaterandachieveaconsiderableamountofbiomassandaccu-mulationofbiocompounds.
Inspiredbytheapproachofapplyingphytohormonetohigherplants,wetriedapplyingitinseedprimingandduringinoculumpreparation,astheformerenhancestheactivityofhigherplantsunderstress[27],whilethelat-terpretreatsthealgalseedbeforebatchcultivation.
Thisstudyenablesustounderstandtheinfluenceofeffluentfromanaerobicdigestionofkitchenwaste(ADE-KW)onalgaegrowthandbiocompoundsaccumulation,andfur-therverifythepotentialofusingphytohormonestohelpalgaeovercometheadverseeffectsfromthewastewaterPage3of18Peietal.
BiotechnolBiofuels(2017)10:76feedstockandprovideaneconomicalwaytoaccumulatealgaebiomassbasedonADE-KWasmedia.
MethodsMaterialsTheeffluentfromanaerobicdigestionofkitchenwaste(ADE-KW)wascollectedfromShandongShifangEnvi-ronmentalProtection&Bio-EnergyCompany(Jinan,PRChina).
Thedigestionprocessoperatescontinuouslyinthecompanyandultimatelyproducesconsistentefflu-ent,witharelativedeviationinwatercharacteristicsoflessthan10%betweendifferentbatches.
FollowingourprevioussurveyontheeffectofADE-KWloadingonalgalgrowth(datanotyetpublished),ADE-KWdilutedbyafactorof15withtapwaterwasputintouseasthegrowthmediumforthetestedalgae,withoutemploy-inganyotherpretreatmentssuchasfiltering,auto-claving,orpHadjustment.
Thechemicalcompositionofthe15-folddilutedADE-KWwasasfollows:TN138.
22±0.
72mgL1,NH3-N105.
41±0.
73mgL1,TP1.
75±0.
07mgL1,CODCr390.
16±2.
32mgL1,protein33.
52±2.
23mgL1,carbohydrates11.
09±2.
78mgL1,pH8.
54±0.
05.
ThetestedmicroalgaeChlorellaellipsoidea(SDEC-11)andScenedesmusquadricauda(SDEC-13)weresimilartothoseusedbyJiangetal.
[22],whichwereisolatedandevaluatedasthegoodcandidatesforbiofuelproductionbyourlaboratory.
ChlorellaandScenedesmushavebeendemonstratedasidealcandidatesforbiofuelindustri-alizationduetotheirstrongadaptationtowastewater,inhibitionofbacterialgrowth,andrelativelyhighlipidproduction[28,29].
AlgaeinoculumswerepreparedfromexponentiallygrownseedculturesinBG11mediumwhichcontains1.
5gL1NaNO3,40mgL1K2HPO4,75mgL1MgSO47H2O,36mgL1CaCl2·2H2O,6mgL1citricacid,6mgL1ferricammoniumcitrate,1mgL1EDTA-Na2,20mgL1Na2CO3,and1mLL1A5.
A5isatracemetalsolutioncontaining2.
86gL1H3BO3,1.
86gL1MnCl2·4H2O,0.
22gL1ZnSO4·7H2O,0.
39gL1Na2MoO4·2H2O,0.
08gL1CuSO4·5H2O,and0.
05gL1Co(NO3)2·6H2O.
Ifthereisnospecialstate-ment,thechemicalsareproducedbySinopharm.
Thephytohormones(VitaCat,Germany)werecom-monlyappliedinagriculture,andconsistedof0.
135%(w/w)gibberellin(GA3),0.
00052%(w/w)indole-3-aceticacid(IAA),0.
00031%(w/w)brassinolide(BL),andanauxiliaryingredientthathelpstheformerthreephyto-hormonestobecomepowderthatcaneasilydissolveinwater,calledGIBforshort.
ExperimentalsetupGIBphytohormoneswereincorporatedintothemediumat3daysbeforeinoculationduringthepreparationphaseofthealgaeinoculumseed(S+)andatthebeginningofthebatchcultivationphaseofthealgae(B+).
TheprimingseedforS+groupwaspreparedwiththeseprocedures:(1)cultivatingtestedalgaetoreachingthemiddleexpo-nentialphase;(2)addingthephytohormonestoculturementionedatlaststep;(3)3dayslater,recoveringthealgaebycentrifugationandwashedthreetimeswithdeionizedwater;(4)inoculatingthepelletfromthethirdstepintothewastewaterforS+ADE-KWrun.
TheseedforB+ADE-KWgroupwaspreparedaccordingtoabove-mentionedprocessinabsenceofthesecondprocedure.
CultivationinBG11andADE-KWwithnoGIBadditionwasconductedsimultaneouslyascontrolsforallexperi-ments.
Theoverallexperimentalset-upispresentedinTable1andrunnamessuggestthephaseatwhichhor-moneswereaddedandtheculturemedium(e.
g.
,B+ADE-KWindicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase).
GA3isthemainhormoneinthephytohormones,sotheGIBdosewascalculatedaccordingtothepreviousresearchbyTatkowskaandBuczek[30]andTateetal.
Table1Experimentalset-up,maximumbiomassconcentration,andaveragebiomassproductivitiesofChlorellaSDEC-11andScenedesmusSDEC-13eRunnamesindicatethephaseatwhichhormoneswereaddedandtheculturemedium,e.
g.
,B+ADE-KWindicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase.
–Phytohormonenotadded.
Dataarethemeansofthreeindependentexperiments±SD*DatainthesamecolumnfollowedbydifferentlettersaresignificantlydifferentbyDuncan'stestatp2daughtercells2daughtercells612182430Morphologicalcomposition(%)03691215four-andeight-celltwo-cellcTheratioofAandBsemi-axes(m)MeanAMeanBMeanA/BBG11ADE-KWS+ADE-KWB+ADE-KWaCellweightCelldryweight(pg.
cell-1)BG11ADE-KWS+ADE-KWB+ADE-KW6080100120CulturesystemScenedesmusSDEC-13ChlorellaSDEC-11bdCulturesystemFig.
2Theeffectofphytohormonesoncelldivision(a,b)andmorphology(c,d)ofChlorellaSDEC-11andScenedesmusSDEC-13.
Incandd,A:semi-majoraxis;B:semi-minoraxis;A/B:theratioofAandBaxes.
Therunnamesontheabscissaindicatethephaseatwhichhormoneswereaddedandtheculturemedium;e.
g.
,B+ADE-KWindicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphasePage7of18Peietal.
BiotechnolBiofuels(2017)10:76qualityreachingthealgalcells.
Increasedratiosinchl-a/chl-bhavebeenconsideredtodecreaselightcollectioninrelationtotherateofphotosystemII(PSII)photochem-istry,whileincrementsincarotenoids/chl-a+b(caro/chl-a+b)indicatedthedifficultyinlightharvestingcom-plexandPSIIactivity[37].
Forthetwoalgae,theratioofchl-a/chl-bandcaro/chl-a+bincreaseddramaticallyinADE-KWmediacomparedwithBG11culture(p<0.
05),whichindicatedabnormalchloroplastphotosyntheticphosphorylationactivityandacclimationofchlorophyllwhensubjectedtotheharshcondition.
TheADE-KWwastewaterdeceleratedthealgalgrowthincludingbiomassaccumulationandcelldensity,whichmightoccurthroughslowingdownoftherateofcelldivisionandchlorophyllactivity.
Intermsofthedegreeofdeclineintheabovemetrics,ScenedesmusSDEC-13hasastrongeradaptabilitytoADE-KWthanChlorellaSDEC-11.
RoleofphytohormonesinstimulatingalgaevitalityinADEKWTheeffectoftheGIBphytohormonesonthegrowthofalgaecultivatedinADE-KW,expressedasbiomasscon-centrationandcellnumber,ispresentedinFig.
1.
Hor-moneadditionduringtheseedpreparationphaseorthebatchcultivationphaseincreasedbiomassaccumulationslightlyforChlorellaSDEC-11,butwithastatisticaldif-ferenceforScenedesmusSDEC-13(p<0.
05),comparedwithnontreatedADE-KWcontrol(Table1).
Oziokoetal.
alsofoundnosignificanteffectsofGA3oncellgrowthofChlorellasp.
IAM-C212,asexpressedbydrycellconcen-tration[38].
However,thispretreatmentoftheseedwithphytohormonescausedthetwoalgaestrainstohavenolagphasewhentheyencounteredADE-KW,andespe-ciallygaveSDEC-13strongerbioactivitytosurviveinADE-KW,attaining0.
40gL1biomass,nearlydoublethatof0.
22gL1obtainedintheADE-KWcontrol.
Conformancebetweentheempiricalresultsandsimu-lationfromtheVerhulstmodelwasobserved(Table1).
Nosignificantdifferenceappearedforbiomassproduc-tionofthetwoalgaestrainsundertheconditionofGIBhormones.
Norwasthereanydifferenceinthecellmor-phologiesofthetwoalgaestrains(Fig.
3b–d,f–h).
How-ever,incomparisonwiththenon-GIB-treatedcontrol,theobviousnuclearandchloroplastforSDEC-11andclearmitochondrionofSDEC-13occurredwiththeaddi-tionofphytohormone,whichallexpressedstrongercellviability.
IncorporationofGIBwithADE-KWtocultureSDEC-11isbetterdoneinthebatchcultivationphase,whichyieldedahighspecificgrowthrate(0.
18d1).
ForScenedesmusSDEC-13,thetwomethodsofphytohor-moneadditionhadpositiveeffectsontheculturesinFig.
3DetailsofultrastructureofChlorellaSDEC-11andScenedesmusSDEC-13treatedwithADE-KWandphytohormones.
aSDEC-11inBG11;bSDEC-11inADE-KW;cSDEC-11inS+ADE-KW;dSDEC-11inB+ADE-KW;eSDEC-13inBG11;fSDEC-13inADE-KW;gSDEC-13inS+ADE-KW;hSDEC-13inB+ADE-KW.
Cchlorophyll,Sstarch,Llipid,Wcellwall,Ncellnucleus,Mmitochondrion,Vvacuole.
Theaboverunnamesindicatethephaseatwhichhormoneswereadded,andtheculturemedium;e.
g.
,B+ADE-KW,indicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase.
Scalebar1μmPage8of18Peietal.
BiotechnolBiofuels(2017)10:76termsofbiomassaccumulationandgrowthrate,inwhichadditionintheseedpreparationphaseperformedbetterwithaspecificgrowthrateof0.
27d1,comparedto0.
18d1.
Fromthevariationsofthecellnumber(Fig.
1),seedpreparedwiththehormonessignificantlyincreasedthecellgrowthsofthetwoalgaestrains,especiallythemaxi-mumcelldensityachievedonthe10thday(p<0.
05).
Moreover,itisnotablethattheGIBphytohormonesexcitedthegrowthactivityofScenedesmusSDEC-13inADE-KWtothecorrespondinglevelinBG11.
ThechangeincellnumbersconfirmedthesamestimulationbyGIBofalgaegrowthasdemonstratedforbiomassconcentration.
Thereductioninalgaebiomassisnormalduetothelowadaptationofalgaeinwastewaterwithcomplex-ity[39].
However,comparedwithGIB-freeADE-KW,ahigheractivityofthetwoalgaespecieswasobservedinADE-KWinoculatedwiththealgalseedpretreatedwithphytohormones,wherenolagphasesoccurred.
Thatistosay,primingthealgalseedwiththephytohormonescouldmitigatetheinfluenceofadifferentenvironment,theADE-KWmedium,onalgalgrowth.
Moreover,theimprovementismoreapparentintreatedgroupsofScenedesmusSDEC-13thanChlorellaSDEC-11.
ResponseofpigmentcontentstothephytohormonesPhotosynthesisinplantandalgaecellsoftenundergoeschangesinresponsetoenvironmentandphytohor-mones,andsothechangesintheproductionofpigmentsinvolvedinphotosynthesisweretested(Table2).
Chlo-rellaSDEC-11cultivatedinS+BG11groupsattainedincreasesinpigmentcontentsof19%inchl-aand25%inchl-b.
TheS+ADE-KWgroupbroughtenhancementof33%inchl-aand26%inchl-bincomparisonwiththeADE-KWrun.
TheincreaseinchlorophyllagreedwiththeappearanceofchloroplastinGIB-treatedgroupscomparedwiththylakoid-likenetsinADE-KWcontrol(Fig.
3c,d).
IntermsofScenedesmusSDEC-13grownwithADE-KW,theadditionofphytohormonesintheseed-preparationphasecontributedtosignificantincreasesofabout51%inchl-aand22%inchl-baccumu-lationinrelationtothecontrol.
LargerspaceoccupiedbychloroplastandmoreclearthylakoidinitconfirmedtheincreaseofGIBonchlorophyllofSDEC-13.
TheresultsobtainedsuggestedthatstimulationbytheGIBphyto-hormonesofphotosynthesisandphotosyntheticpigmentcontentinalgaecellsbypretreatingthealgaeseedwithGIBallowedtheadverseeffectsoftheADE-KWenviron-menttobeendured,whilepreventingchlorophylldegra-dationandabsorbinglimitativelightintervenedbydarkcolorinwastewater.
TatkowskaandBuczekstudiedtheeffectsofIAAandGA3uponchlorophylllevelsofS.
quadricaudaandfoundthat,whenaddedseparately,allofthesephytohormonesstimulatedchlorophyllcontentinalgaecellssignificantlycomparedwiththecontrol[30].
OfthefivehormonestestedbyParketal.
,IAAandGA3exhibitedthehigh-estincrementsinconcentrationofchl-a+b,namely81and68%,respectively[40].
BajguzandPiotrowska-Nic-zyporukreportedthepositiveeffectofBLchlorophyllinChlorellavulgaris[12].
Piotrowska-Niczyporuketal.
foundthatexogenouslyappliedGA3couldmodifythephytotoxicityofheavymetalsonC.
vulgarisandincreasephotosyntheticpigmentaccumulation[15].
ObviouslyGA3,IAA,andBL,eitherindividuallyorthroughthecombinationofthesesubstances,hadabeneficialeffectonphotosyntheticapparatusinmicroalgaecultivatedingeneralmediumorsomemediawithstress.
Moreo-ver,carotenoidscouldhaveanantioxidativeroleinthescreeningandtrappingofexcessivelight,whichisoth-erwiseabsorbedbythechloroplast[41].
Thecarote-noidcontentsofthetwoalgaestrainstreatedwithGIBwereincreasedinrelationtothecorrespondingcontrol.
Table2Photosyntheticpigmentcompositions(mgL1)ofChlorellaSDEC-11andScenedesmusSDEC-13inADE-KWwithphytohormonesaRunnamesindicatethephaseatwhichhormoneswereaddedandtheculturemedium;e.
g.
,B+ADE-KW,indicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase1Chlorophyll-a2Chlorophyll-b3CarotenoidsRunaChl-a1Chl-b2Caro3Chl-a+bChl-a/Chl-bCaro/Chl-a+bSDEC-11SDEC-13SDEC-11SDEC-13SDEC-11SDEC-13SDEC-11SDEC-13SDEC-11SDEC-13SDEC-11SDEC-13BG114.
5±0.
23.
6±0.
12.
4±0.
12.
7±0.
80.
9±0.
00.
7±0.
07.
0±0.
36.
3±0.
51.
9±0.
01.
4±0.
10.
1±0.
00.
1±0.
0ADE-KW1.
2±0.
02.
1±0.
20.
7±0.
1.
4±0.
10.
6±0.
00.
8±0.
11.
8±0.
03.
4±0.
12.
2±0.
11.
5±0.
00.
3±0.
00.
2±0.
0S+ADE-KW1.
6±0.
03.
1±0.
10.
8±0.
01.
7±0.
40.
6±0.
20.
8±0.
22.
5±0.
14.
8±0.
32.
0±0.
11.
9±0.
20.
3±0.
10.
2±0.
0B+ADE-KW1.
4±0.
12.
7±0.
20.
9±0.
01.
5±0.
10.
6±0.
40.
9±0.
02.
3±0.
14.
3±0.
21.
5±0.
01.
8±0.
20.
3±0.
10.
2±0.
0Page9of18Peietal.
BiotechnolBiofuels(2017)10:76Hence,theapplicationofthephytohormonesinlarge-scalecultivationofmicroalgaeshouldplayapositiveroleinhelpingalgaetomitigatethephotoinhibitioncausedbysunlight.
CellmorphologyinresponsetothephytohormonesAttheearlystationarystageofgrowth,thecombinationofGA3,IAA,andBLsignificantlyaffectedthecelldivi-sionexpressedasthepercentageofcellsundergoingcelldivisionandthenumberofdaughtercellsretainedinasinglecell(Fig.
2).
ForChlorellaSDEC-11,thereleaseoftwodaughtercellswasthemainmodeofdivision.
TheB+ADE-KWgroupofSDEC-11showedsuperiorityintheamountofcellsindivisionandinthecellsthataredividedintotwodaughtercells.
AlthoughinS+ADE-KWtherewasnosig-nificantincrementinthepercentageofcellsindivision,theproportionofcellscontainingmorethantwodaugh-tercellsincreased,whichwasalsoareasonforthehighercelldensity.
Theseresultsareconsistentwiththevaria-tionsofcelldensityasshowninFig.
1.
DivisionintomorethantwodaughtercellswasusualinScenedesmusSDEC-13culture.
TheGIBhormonessignificantlypromotedcelldivisionviaincreasingthepercentageofcellsindivision(18.
46%inS+ADE-KW;22.
38%inB+ADE-KW)comparedwiththeADE-KWcontrol(8.
05%)(p<0.
05).
Thestimulationofmicroal-gaebyphytohormoneswasalsoreportedbyParketal.
,whereintroducingbothGA3andIAAatasingleaddi-tiontimeincreasedthepercentageofcellsindivisionandthecellscontainingfourdaughtercells[40].
Thus,thepresentstudyisthefirsttoreportthatphytohormonesaffectedthedivisionofmicroalgaeinamannerrelatedtothestageofthehormonedose.
Incontrasttocelldivision,thephytohormonesdidnotgeneratesignificanteffectsonthecellsizeofChlorellaSDEC-11.
Thedryweightofacellwascalculatedaccord-ingtothecorrespondingbiomassconcentration,andcelldensitywasinfluencedtoagreatdegreebythehor-mones.
ForSDEC-11,allthegroupsunderGIBtreatmenthadlightercellsthantheothergroups,whichmaybecausedbythefasterspeedofcelldivision.
EspeciallyfortheS+BG11group,theintervalfordaughtercellgrowthbeforebecomingamothercellmightbeshortenedbytheactionofGIBhormones,leadingtolessmaterialconcen-trationinthecellandlowerdryweightcomparedwiththecontrol.
OnthebasisoftheratiooftheAandBaxesfromSDEC-13,phytohormonetreatmentresultedinamarkedchangeinmorphology.
ComparedwiththeADE-KWcontrol,GIBtreatmentreducedthevalueofA/B(p<0.
05),whichshowedsphericalizationofcellsunderthemicroscope.
Thisphenomenonalsooccurredinourpreviousresearchintotheeffectofdiethylaminoethylhexanoate(DA-6)onSDEC-13[13].
Sphericalizationcouldgeneratecellswithlargerspecificsurfaceareastoabsorbnutrientsforgrowth,whichisespeciallyneces-saryforwastewatertreatment.
Thefinalconcentrationsinthewater(Table3)demonstratedtheimportanceofthecell'scharacter,withlowerfinalnutrientconcentra-tionintheS+ADE-KWgroupofSDEC-13.
Moreover,cellsculturedinADE-KWfromtheseedpretreatedwithGIBweighed122.
55pgcell1,whichismuchheavierthanthecellsgrowninADE-KWwithoutanytreatment(103.
06pgcell1)(p<0.
05)andevenslightlyheavierthanthecellscultivatedinBG11.
Thechangeofcellmasscouldbesensitivetogrowthmediumorphytohormone.
Intermsofgrowthmedium,Polishchuketal.
usedaneffluentfromanaerobicdigestion(DE)ofexcess-acti-vatedsludgeandartificialseawatermedium(ASW)togrowNannochloropsisoculata,findingthatcellsgrownintheDEwerealmosttwiceasheavyasthecellsgrownintheASWmedium[42].
Scenedesmusisapleomorphicstrainwhichchangesitsmorphologytoproduceunicellsandcoenobiaundervariousenvironmentalconditions(lengthofphotoper-iod,pH,nutrients)andinresponsetopredators[43].
ComparedwithBG11,algaeinADE-KWhadasmallerproportionoffour-andeight-cellcoenobiaandmoretwo-cellcoenobiaandunicells.
Panchaetal.
observedtheecomorphicchangesofScenedesmussp.
CCNM1077,whichformedmoretwo-andfour-cellcoenobiawithlimitednitrateandsequentialnitrateremoval[33].
Nev-ertheless,SiverandTrainorreportedaunicelldominanceinanammonia-richenvironment[44].
ThismeantthatunicellswereproducedbyScenedesmussp.
withsuffi-cientnitrate.
GIBaddedintheseedpreparationorbatchcultivationphasestriggeredmorphologicalchangestofour-andeight-cellcoenobiaanddecreasedthenumberTable3ThefinalwatercharacteristicsofADE-KWcultivat-ingChlorellaSDEC-11andScenedesmusSDEC-13aRunnamesindicatethephaseatwhichhormoneswereadded,andtheculturemedium;e.
g.
,B+ADE-KW,indicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphaseRunaTN(mgL1)NH3-N(mgL1)TP(mgL1)CODCr(mgL1)ChlorellaSDEC-11ADE-KW120.
7±0.
166.
9±1.
30.
34±0.
01216.
8±19.
1S+ADE-KW113.
3±1.
754.
8±0.
70.
32±0.
01219.
7±25.
5B+ADE-KW111.
2±1.
357.
0±0.
60.
24±0.
01208.
2±12.
8ScenedesmusSDEC-13ADE-KW94.
3±1.
761.
7±4.
20.
34±0.
03211.
2±13.
1S+ADE-KW66.
4±3.
054.
3±1.
10.
42±0.
01203.
3±17.
3B+ADE-KW89.
4±1.
757.
0±0.
60.
29±0.
03201.
6±12.
3Page10of18Peietal.
BiotechnolBiofuels(2017)10:76oftwo-cellcoenobiainScenedesmusSDEC-13(Fig.
2b).
ThiswasconsistentwiththefindingofPrasadthatGA3andIAApromotedtheformationoffour-celledcoenobiainS.
obliquusratherthantwo-celledcolonies[45].
TheseresultssuggestedthatapplicationofGIBcouldincreasetheformationoffour-andeight-cellcoenobia,andtheammoniaandorganiccarboninADE-KWcouldweakentheecomorphicexpressionofthecoenobiainScenedes-musSDEC-13.
TheGIBphytohormonesmitigatedtheinhibitionofADE-KWonalgalcelldivision,whichexplainedtheincrementofbiomassconcentrationandcelldensityintheGIB-treatedgroups.
Themorphologyincludingcellsizeandthenumberofcellincoenobiaalsofluctuatedunderthehormonetreatment.
GA3,togetherwithIAAfromGIB,isusuallyinvolvedinde-repressionofplant-counteringstress[17],andorganismcouldrecallsomeeventsdependingonthetypeofprimarystimulusandconstructadiversesetofdefensemechanismsafterbeingprimed[27].
ThealgalactivityinB+ADE-KWgroupindicatesthepositivefunctionofGIBonalgaeembracingthewastewater,possiblybymeansofadjustinginternalphytohormonesysteminalgalcellandmakingchloroplastandnucleusresistanttoanunusualenvironmentincludingimbalanceinnutrients,bacterialinfection,anddarkcolor.
Thepromotionsonchlorophyll,division,andcellmorphologyofalgaeinS+ADE-KWsuggestedthattheseedpreparedwiththephytohor-monesrecallsthestimulationofGIB,followedbyreadilycopingwithADE-KW.
NolagphaseinS+ADE-KWalsoexhibitedthesysteminalgalcellbeinginreadystateforconfrontingsomestressordifferentcondition,aftertheprimingprocess.
BiomasscompositionsinresponsetothephytohormonesLipidinalgaeisthemostpromisingcomponentforbio-fuelproduction;however,tomaximizeeconomicandenvironmentalbenefitswhileminimizingwasteandpol-lution,exploitationofdiversityandsyntheticbiologyfortheproductionofalgalbiofuelsisneeded[46].
Usuallyproteinandcarbohydratespecies,thenonlipidfractions,havethepotentialtoproduceanimalfeed,biogasthroughanaerobicdigestion,andsoon[47].
Thus,thecharac-teristicsofcarbohydrate,protein,andlipidinChlorellaSDEC-11andScenedesmusSDEC-13areevaluatedandshowninFigs.
4and5fordifferentculturemediaandphytohormones.
ProteinandcarbohydrateIngeneral,proteincontentsarewellcorrelatedwiththemetabolicactivityinthecells[48].
Inotherwords,theaccumulationofproteinsoccurswhenthemicroalgaecellsdisplayhighmetabolicandmitoticactivities.
ThedecreasedproteincontentcomparedtothatinBG11(p<0.
05)indicatedthattheADE-KWmediumwasanonidealenvironment,aswasalreadyindicatedbybio-massandchlorophyllobservations.
Thisenvironmentledtomoreenergysourceaccumulationforalgaeself-protection,asSinghetal.
concludedthatproteincontentinplantswasanimportantindicatorofreversibleandirreversiblechangesinmetabolism,aknownresponsetoawidevarietyofstressors[49].
Anotherobviousfea-tureoftheseresultswasthemassratioofcarbohydratetoprotein,whichinADE-KWwashigherthanthatinBG11forthetwoalgae.
TheapparentdifferenceinmassratiosofSDEC-11betweenADE-KWandBG11alsosug-gestedaweakeradaptationofChorellaSDEC-11thanthatofScenedesmusSDEC-13,withaslightlyincreasedmassratioinADE-KW.
Nevertheless,GIBplayedaprevi-ouslyunconsideredroleinimprovingtheproteinactivityinthetwoalgaespeciestested.
Theproteincontentsofallgroupstreatedwiththephytohormonesattainedlevels010203040cbbca*abbCarbohydrateProteinaab0.
00.
30.
60.
9Massratio010203040Massratio)%(tnetnocnietorproetardyhobraCBG11S+ADE-KWADE-KWB+ADE-KWbaab0.
00.
30.
60.
9CulturesystembcabbcFig.
4Carbohydrateandproteinaccumulationsof(a)ChlorellaSDEC-11and(b)ScenedesmusSDEC-13cultivatedunderphytohor-mones.
Massratioistheratioofcarbohydrateandprotein.
Therunnamesontheabscissasindicatethephaseatwhichhormoneswereaddedandtheculturemedium;e.
g.
,B+ADE-KW,indicatesthatphy-tohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase.
*DatainthesameparametermarkedwithdifferentlettersaresignificantlydifferentbyDuncan'stestatp<0.
05Page11of18Peietal.
BiotechnolBiofuels(2017)10:76thatwerealittlehigherthanthoseofthecorrespond-ingcontrolwithouttreatment.
ThissamephenomenonoccurredinthecaseofthetwoalgalstrainswhentheyweretreatedbyDA-6.
NoappreciabledifferencebetweenproteincontentsinSDEC-11andSDEC-13wasobservedasresponsetophytohormones,whichisdifferentfrom6121824303642Lipidcontent(%)Lipidcontentaa*bacLipidproductivityaLipidproductivity(mg.
L-1d-1)babc3691215020406080100CulturesystemCulturesystemcFattyacidcomposition(%)C18:3C18:2C18:1C18:0C16:1C16:0C15:0BG11ADES+ADEB+ADEBG11ADES+ADEB+ADEScenedesmusSDEC-13dChlorellaSDEC-11Fig.
5Lipidaccumulation(a,b)andfattyacidprofile(c,d)ofChlorellaSDEC-11andScenedesmusSDEC-13cultivatedinADE-KWunderphytohor-mones.
Therunnamesontheabscissaindicatethephaseatwhichhormoneswereaddedandtheculturemedium;e.
g.
,B+ADE-KWindicatesthatphytohormoneswereaddedtoalgaeculturedwithADE-KWatthebatchcultivationphase.
*DatainthesameparametermarkedwithdifferentlettersaresignificantlydifferentbyDuncan'stestatp<0.
05Table4BiomasscompositionsofmicroalgaeunderdifferentphytohormonetreatmentsaThedatainthisformationpresentthattheformercamefromS+ADE-KWgroupandthelatterfromB+ADE-KWgroupbThedatacomefromtheADE-KWcontrolAlgalspeciesPhytohormoneProteincontentCarbohydratecontentReferenceTreatedControlTreatedControlC.
vulgarisGA312.
4*108mgcell19.
5*108mgcell14.
8*108mgcell13.
2*108mgcell1[11]C.
vulgarisBLandkinetin390fgcell1100fgcell1350fgcell160fgcell1[12]C.
vulgarisCytokinins38*108gcell118*108gcell13.
8*108gcell11.
1*108gcell1[15]SDEC-11DA-639%39%17%14%[13]SDEC-13DA-637%39%15%13%[13]C.
reinhardtii1-triacontanol44%abovethecontrolNodifference[39]C.
vulgarisIndomethacin43*108gcell130*108gcell15.
2*108gcell12.
7*108gcell1[49]SDEC-11GIB22/25%a24%b17/22%a23%bThisstudySDEC-13GIB30/34%a32%b16/20%a21%bThisstudyPage12of18Peietal.
BiotechnolBiofuels(2017)10:76thenoticeablepromotiononotheralgaetypesreportedintheliterature[11–13,15,39,49](Table4).
Unlikeprotein,theaccumulationofcarbohydrateinalgaewasdisturbedslightlybyADE-KWandthecar-bohydratecontentdecreasedtosomedegreeingroupstreatedwiththephytohormones.
Thereductionwasoppositetothephenomenonofhighercarbohydratecon-tentunderthetreatmentofphytohormonesobservedbyFalkowskaetal.
[11],BajguzandPiotrowska-Niczy-poruk[12],Piotrowskaetal.
[50],andPiotrowska-Niczy-poruketal.
[15](Table4),whichmightresultfromtheimprovedphotosynthesisactivity.
However,Yuetal.
alsofoundthatgibberellintreatmentreducedthecontentofglucoseandpromotedtheutilizationofglucose(carbonsource)forconversionintoothermaterials[51].
Com-paredwithcellsinADE-KW,moremitochondriathatappearedincellsofSDEC-13treatedbyGIBalsosug-gestedapossiblehigherconsumptionofprimaryenergymaterials,suchasglucoseandstarch.
Theremightbeanotherreasonthatthephytohormonecouldchangethemetabolicpathwaysofcarbon.
Accordingtothebiosyn-theticroutineofbiochemicalcomponentsinmicroal-gaeproposedbyLvetal.
,diminishmentincarbohydratemightbeattributedtothetransformationofmostglycer-aldehyde-3-phosphates(G3P),theintermediateproductfromphotosynthesis,intolipid[52].
Nolinearcorrelationappearedbetweencarbohydratecontentandcellsizeundertheimpactofhormones,asshowninthepreviousresearchontheeffectsofDA-6onSDEC-11[13].
ThismightbecausedbytheeffectofGIBontheintracellularcarbohydratesynthesispathwayofthestrains.
LipidandfattyacidprofilesTheeffectsoftheGIBphytohormonesonlipidaccumu-lationinSDEC-11andSDEC-13cultivatedinADE-KWareevaluatedandillustratedinFig.
5.
Consideringtheeffectsofgrowthmediumonlipidaccumulation,areduc-tionwasobservedforSDEC-11inADE-KW,andtherewasnodifferencebetweenthetwomediaforSDEC-13.
However,significantdifferences(p<0.
05)werefoundforlipidaccumulationinthetwoalgalstrainsbetweengroupswithGIBtreatmentandthecorrespondingcon-trolgroups.
ForlipidcontentofSDEC-13,allthetreat-mentswiththephytohormonesexhibitedabout130%increaseoverthecontrol.
ItisworthnotingthatthelipidcontentofSDEC-11inB+ADE-KWreachedupto38%,enhancedby1.
52timesthevalueof25%fortheGIB-freecontrol,whichwasvisuallyexhibitedbybiglipiddropletsinultrastructuralcellsofB+ADE-KWgroup(Fig.
3a,d).
Theincreaseinlipidcontentbalancedthedecreaseincarbohydratecontent,duetothecompetitionforG3P.
Inpreviousstudies,theauxinsappearednottostimulatesignificantchangesinlipidcontent[53,54],butgibber-ellinspromotedastatisticaldifferenceinlipidaccumu-lation[50].
ItmaybeGA3intheGIBhormonesthatworkedonlipidcontentinthetwotestedalgaestrains.
Lipidproductivity,whichcombineslipidcontentandbiomassproductivity,wasdecreasedbythewastewaterthroughloweringofbiomassorlipidaccumulation,butallthegroupstreatedwiththephytohormonesshowedsuperiorlipidproductivity(p<0.
05).
Becauseofthehighlipidcontent,algaeSDEC-11inB+ADE-KWgroupyielded12.
73mgL1d1inlipidproductivitywithrela-tivelylowbiomassconcentration(0.
33gL1),whichwas70%higherthan7.
48mgL1d1achievedinADE-KWcontrolandalmostequalto13.
67mgL1d1inBG11.
ThesuperiorityofGIBtreatmentwithSDEC-13wasmoreobviousduetothestimulationofbothalgalgrowthandlipidbiosynthesis.
EspeciallyforSDEC-13intheS+ADE-KWgroupwith12.
67mgL1d1inlipidproductivity,thetreatmentincreasedthelipidproductivityby139%comparedto5.
30mgL1d1inADE-KWandwasevenhigherthanthatinBG11(9.
10mgL1d1).
Thefattyacid(FA)profilesofmicroalgaecultivatedinBG11andADE-KWmediaareshowninFig.
5.
Thesatu-ratedfattyacidswerecomposedofpentadecanoicacid(C15:0),palmiticacid(C16:0),andstearicacid(C18:0).
Palmitoleicacid(C16:1)isoneofthemembersofmon-ounsaturatedFAs(MUFA),withC18:1dominatingMUFA.
Linoleicacid(C18:2)andlinolenicacid(C18:3)arepolyunsaturatedFAs(PUFA).
TheobviousdifferencebetweenFAcompositionsinBG11andADE-KWwasfoundinC15:0andC18:2.
C15:0waspresentbuttherewasnoC18:2inthetwoalgaestrainsgrowninADE-KWmedia,quitetheoppositeoftheresultinBG11media.
TreatmentsofSDEC-11inS+ADE-KWandSDEC-13inB+ADE-KWrecorded21and22%increasesinsaturatedfattyacid(SFA)content,respectively,relativetothecor-respondingvaluesinADE-KWcontrol;introducingGIBtoSDEC-11duringthebatchcultivationandtoSDEC-13duringtheseedpreparationdecreasedtheSFApropor-tion.
Theinfluenceofthephytohormonesonfattyacidcompositionexhibitedacomplexdifferencedependingonthedosingphaseandalgalspecies.
AsregardsthecomplicatedimpactsofplanthormonesonFA,Jusohetal.
alsofoundthattheoperatingtimeofIAAonC.
vul-garis(UMT-M1)significantlyaffectedtheoilaccumula-tion,fattyacidcompositions,andgeneexpression[55].
DifferenceswereexhibitedintheinfluenceofGIBonFAcompositionbetweenthegroupswithdifferentdos-ingphasesandalgalspecies,whichcouldbefurtherstud-iedinsubsequentresearch.
ItisencouragingthattheGIBphytohormonescouldmakeabeneficialchangeinthelipidbiosynthesispathway.
Page13of18Peietal.
BiotechnolBiofuels(2017)10:76Thepossiblemechanismofphytohormone'sinfluenceonalgalactivityGA3,IAA,andBLintheGIBphytohormonesareimpor-tantplantgrowthregulatorsinmultipledevelopmentalprocesses,includingorchestrationofcellularhomeosta-sistocopewithvariableenvironmentalfactors,suchasethylenebiosynthesis;celldivisionandelongation;DNA,RNA,andproteinsynthesis;photosynthesis;andsoon[7].
Thepositiveeffectsofthethreephytohormonesonthegrowthorenvironmentalstressresponseofmicroal-gaehavealsobeenexploredbymanyresearchers,andareattendantwithpromotionofcelldivisionandexpansion,DNAandRNAlevels,shorteningthecelldevelopmen-talcycle,orstimulationofantioxidantenzymeactivities,restoringalgaegrowth,andprimarymetabolitelevels[9,12,15,30,40].
TheGIBhormonesusedinourstudystimulatedthealgaetodealwiththeunfavorablecondi-tionsinADE-KW,includinghighammonia(105mgL1),limitedphosphorus(1.
75mgL1),extremeimbalanceintheratioofnitrogenandphosphorus(78.
95),andevendarkcolorandsuspendedsolidsasanobstacletolighttransmission.
TheseconditionsinADE-KWdecreasedchlorophyllbiosynthesisandlightqualityreachingthealgae,whichthenresultedinthereducedpercentageofcellsundergoingdivisionandevenreducedlipidcontent.
ThisstimulationofalgaebyGIBinADE-KWmightbesupportedbytheregulationofphotosyntheticappa-ratusbythephytohormones.
Theprimaryenergysourceinamicroalgaecellisformedinchloroplastscontainingchlorophyll,asshowninFig.
6.
Nicotinamideadeninedinucleotidephosphate(NADPH)derivedfromchlo-rophyllmainlyservesproliferationandcellgrowth,butlipidbiosynthesisisaffectedwhenanexcessofNADPHappears.
SignificantimprovementofchlorophylllevelsbyGIBoccurredincellsofthetwoalgaestrains(increaseof25–40%intotalchlorophyllcontentasshowninTable2andimprovementofthylakoidorchloroplastasvisuallyrevealedinFig.
3),whichcouldabsorbmoresolarradia-tionandthenmoreNADPH.
Thismeantmoreenergywasconvertedintocellproliferation,whichultimatelypromotedcelldivision,leadingtoanincreasedpropor-tionofSDEC-11cellscontainingmorethantwodaughterFig.
6AsimplifiedschemeshowingcelldivisionandlipidbiosynthesisinmicroalgaecultivatedinaBG11,bADE-KW,andcADE-KWwithphy-tohormonesaddition.
Thewidthofthearrowsrepresentstheenergyflowinginto,ortheeffecton,thematerialpointedto.
NADPHnicotinamideadeninedinucleotidephosphate;ADE-KWeffluentfromanaerobicdigestionofkitchenwaste;GIBagriculturalphytohormonescontaininggibberel-lin,indole-3-aceticacid,andbrassinolide;S+ADE-KWculturingalgaeinADE-KWwithinoculumpretreatedwithGIB;B+ADE-KWintroducingGIBintoalgaeinADE-KWatbatchcultivationstagePage14of18Peietal.
BiotechnolBiofuels(2017)10:76cellsandincreasedthepercentageofSDEC-13cellsundergoingdivision(Fig.
2).
EvenlipidaccumulationwasenhancedwhenmicroalgaewereconfrontedwithanexcessofNADPHfollowingtheincrementinphotosyn-theticpigments.
Thereisapositivecorrelationbetweenthetotalchloro-phyllcontentandlipidcontentforSDEC-11(R2=0.
45)and,SDEC-13(R2=0.
92).
Howeversucharelationshipwasnotobservedwhenconsideringdataofthetwoalgaespecies,whichmightindicateadifferentenergyparti-tioningbetweenChorellaSDEC-11andScenedesmusSDEC-13.
Indeed,theproductionofNADPHisalsorelatedtotheconditionofthylakoid,inwhichafunc-tionalmembranestackessentiallyensuresthatthepho-tosystemoperatesefficiently[35,56].
ApparentlyforSDEC-11,clearthylakoidsinchloroplastwereobservedafterphytohormonestreatment,whichcouldbeanotherreasonforthehigherlipidcontentinGIB-treatedgroups.
Moreover,TanandLee[57]summarizedthatthegenera-tionofNADPHcouldmainlyderivefromthemetabolismofstarch.
ComparedwithultrastructuralcellsinADE-KWcontrol(Fig.
3b),thecellsofSDEC-11(Fig.
3d)inB+ADE-KWexhibitedfewerstarchgranulesandhigherlipidcontent(Fig.
5).
AsimilarphenomenonbetweencellsofSDEC-13incontrol(Fig.
3f)andS+ADE-KW(Fig.
3g)wasobserved.
Carotenoidsconstitutethemainprotectionagainstexcesslightenergyandpossiblytransfertheabsorbedradiation[56].
CarotenoidsynthesisinalgaealsoincreasedunderthehormonetreatmentasshowninTable2,whichsuggestedsuperfluouslighttransferfornormalalgalgrowthandthepossibleexcessofNADPH.
Over-reductionwilloccurinthephotosyntheticelec-trontransportchainduetorichnessofNADPH,whichcouldleadtosomereactionswithhighlyreducedprod-ucts,suchaslipidsynthesis[58].
Inadditiontocarbonsupply,denovoFAsynthesisalsorequiresacontinu-ousprovisionofreducingpowerintheformofNADPH[57].
ItmaybetheefficientstimulationbyGIBofchloro-phyllinalgaethatultimatelyleadstoanincreaseinlipidaccumulation.
Themainingredientsinmembranelipid(ML)involveC16:0,C18:0,cis-9-C18:1,cis-9,12-C18:2,andcis-6,9,12-C18:3,andFAinothertypesaremainlyfoundinstoragelipid(SL)[35].
BasedonthestructuralformulaeobtainedaccordingtotheGC-MSretentiontime,thereisnofattyacidincis-structurepresentinChorellaSDEC-11,andonlycis-9-C18:1andcis-9,12-C18:2arepresentinScenedesmusSDEC-13culturedwithBG11.
FattyacidslikeC16:0andC18:0alsoconstitutethemainfattyacidsintriacylglycerol(TAG),thestoragelipid[59],andso,whilethevalueofMLinAdditionalfile1:TableS2mightnotexpressthemembraneactivity,theproportionofSLcouldsuggestthepercentageofstoragelipid.
InrelationtoBG11;thehigherSLlevelsinalgaegrowninADE-KWindicatedanimprovementinstoragelipidpathway,whichwasinagreementwithstimulationunderaharshenvironmentofTAGsynthesisthroughproductionofanexcessofNADPH,asvisualizedinFig.
6.
TheGIBphy-tohormonesdecreasedthecontentofSL,whichmightindicatethatthecelldivisionoccurringinGIB-treatedgroups,manifestingasincreasedcelldensity,consumedNADPH,thefeedstockofTAG.
Hence,comparedwiththeADE-KWcontrol,thehigherlipidcontentinGIB-treatedgroupsconsistedofmorestructurallipidorstor-agelipidformedwithsaturatedfattyacids.
ThebenefitaccruedbytheGIBphytohormonestothealgaewasaffectedbythebiosynthesisandflowdirectionofNADPH,i.
e.
,thecompetitionofcellproliferationandlipidconversion.
NADPHisproducedbychloroplastsandsourcedfromlightabsorbedbychlorophyll,sotheimpactofthephytohormonesonphotosynthesiswillbethekeytopromotesimultaneousbiomassandlipidaccu-mulationinmicroalgae.
CouplingalgaebasedbiofuelproductionwithanaerobicdigestionofkitchenwasteTheaboveresultsindicatedanimprovementbytheGIBphytohormonesintheadaptabilityandlipidaccumula-tionofmicroalgaeculturedineffluentfromanaerobicdigestionofkitchenwaste.
Table5liststhecostofalgaebiomassproductionfromthreeculturepatterns,BG11,B+ADE-KW,andS+ADE-KW,withoutconsideringthefreshwaterneededbybothdilutionofADE-KWandpreparationofBG11.
TheADE-KWwasusedasazerocostcontroltoestimatetheadditionalinvestmentfornutrientsorphytohormones,andthecalculationofGIBamountneededbyS+ADE-KWgroupwasbasedonthefactthat1Lseedculturewassufficientfor5Lbatchcul-tivationaccordingtothebiomassconcentrationsattheTable5AcomparisonofcostsofalgaebiomassproductionfromBG11,B+ADE-KW,andS+ADE-KW,respectivelyaThecostofBG11rootsinchemicalsneededbythemediumAlgaespeciesBG11a(CNYkg1biomass)S+ADE-KW(CNYkg1biomass)B+ADE-KW(CNYkg1biomass)ChlorellaSDEC-11140.
3±1.
917.
7±0.
776.
3±2.
8ScenedesmusSDEC-13182.
0±15.
413.
5±0.
575.
1±2.
6Page15of18Peietal.
BiotechnolBiofuels(2017)10:76beginningandendofgrowthperiod.
Apparently,addi-tionoftheGIBphytohormonesinADE-KWforalgalcultivationismoreeconomicthandosingnutrientsinBG11medium,especiallyinS+ADE-KWculturalpatternwheretheinvestmentonlyaccountedforahundredthofthatinBG11.
Thecomparisonlaidafoundationtoacon-ceptplantcouplingalgae-basedbiofuelproductionwithADE-KW,asshowninFig.
7.
IntheplantwheretheADE-KWwascollected,therearemature,advancedtechnologicaloperationsinclud-ingbiodieselproductionfromtheoilseparatedfromkitchenwasteandanaerobicdigestionofkitchenwaste(Fig.
7).
Thisinfrastructurecouldbenefittheconversionofthe'microalgaebiomasstobiodieselorbiogas,'Basedontheresultsinthelaboratory,thestageforintroduc-tionofGIBtoalgaewassetbetweenalgaeprimingandthebeginningofalgaecultivation,asshowninFig.
7.
Microalgaearethengrownineffluentfromanaerobicdigestionuntilharvest.
Theharvestedbiomassmaybesentintwodirections,assessedbasedonlipidcontent,withacriticalpointof40%,accordingtothereportofSialveetal.
[21].
Biomasscontainingmorethan40%lipidentersthetransesterificationprocessafteroilextraction,andtheresidueswouldbeanaerobicallydigested.
Chistidiscussedthetreatmentofthemicroalgaeresiduesafterbiodieselproduction,highlightingitspotentialtorecoverbiogasandthenoffsetapartoftheenergydemandsoftheseprocesses[60].
Basedonenergybalanceandener-geticrecoveryofcellbiomass,itisbettertotransferthebiomasswithlessthan40%lipidcontentdirectlyintotheanaerobicdigestionprocesswiththekitchenwaste,whichcouldalsoimprovethemethaneyieldbyareduc-tionofthecarbon-to-nitrogenratiointhedigester.
Theenhancementinthemethaneyieldisnormalinco-diges-tionofalgaewithotherdigesterfeedstocks,becausethehighnitrogencontentinalgaecouldimprovethedigest-ibilityofcarbon-richsubstrate[61],likekitchenwastewithhighratioofcarbontonitrogen[62].
TheconceptplantwehaveproposedandshowninFig.
7combinesalgae-basedbiofuelproductionwithADE-KWtreatmentandformsacyclewherealmostnonitrogenorphosphorusislost.
Ajeejetal.
proposedthatcouplingwastewatertreatmentwiththemicroal-gaeculturewasapromisingavenuetowardtheproduc-tionofrenewablebiodieselorbiogas[61].
Moreover,theconceptplantbasedonakitchenwastetreatmentplanttakesfulladvantageoftheexistingtransesterificationandanaerobicdigestioninfrastructure,whichcouldmitigatesomeenvironmentalandeconomicburdens.
Thiswillbeanenvironment-friendlyproject.
Fig.
7Aconceptplantforcouplingalgae-basedbiofuelproductionandanaerobicdigestionofkitchenwaste.
ADE-KW:effluentfromanaerobicdigestionofkitchenwaste;GIBagriculturalphytohormonescontaininggibberellin,indole-3-acetic,acidandbrassinolide;S+ADE-KWculturingalgaeinADE-KWwithinoculumpretreatedwithGIB;B+ADE-KWintroducingGIBintoalgaeinADE-KWatbatchcultivationstagePage16of18Peietal.
BiotechnolBiofuels(2017)10:76ConclusionsThestudytriedtostimulateaccumulationofbiomassandbiocompoundsinalgaegrowninADE-KWwithagriculturalphytohormones(GIB)addedatdifferentalgalgrowthstages.
RegardingcellularultrastructureunderADE-KWculture,thedamagewasmostobvi-ouslyobservedincellsofSDEC-11includingdisinte-gratedmembranesystemandformationofvacuoles,whiletheinterferenceincellsofSDEC-13justexhib-itedadecreaseinthespacebetweenchloroplastandtheincreaseinthesizeofstarchgranule.
Improve-mentsintermsofbiomassconcentration,celldensity,andphotosyntheticpigmentsoftwoalgaestrainscouldbefoundinallgroupstreatedbyGIB.
Thesecharacter-isticswerealsoexhibitedwithultrastructureinclud-ingstrongerchloroplastandcellnucleus.
Theresultswereaccompaniedbyvariationsofcellmorphologyandcellweight,whichwereallrelatedtothestagesofGIBaddition,thegrowthmedium,andthealgaespe-cies.
Theaccumulationsofcarbohydrateandlipiddif-feredwithregardtodifferentGIBdosingstagesandculturemedia,andtherewasapositiveresponsetoGIBadditionintwoalgaestrainsculturedwithADE-KW.
Theseeffectsmighthaveresultedfromthesyn-thesisandconsumptionofG3PandNADPHbeinginfluencedbythehormones.
ItisnotablethatlipidproductivityofSDEC-13inS+ADE-KWincreasedto12.
67±0.
54mgL1d1from5.
30±0.
11L1d1undertheADE-KWcontrol.
Primingofalgalseedwithhormonesprovidesanoptionforimprovingalgalactivityinanonidealenvironment.
Additionofagri-culturalphytohormonescouldbeabeneficialstrategyforpromotingtheaccumulationsofbiomassandbio-compoundsinalgaeculturedwithwastewater.
Basedonthesefindings,aconceptplantforcouplingalgae-basedbiofuelproductionandanaerobicdigestionofkitchenwastecanbeestablished.
AbbreviationsADE-KW:effluentfromanaerobicdigestionofkitchenwaste;GA3:gibberellin;IAA:indole-3-aceticacid;BL:brassinolide;DA-6:diethylaminoethylhexanoate;GIB:agriculturalphytohormonescontaininggibberellin,indole-3-aceticacid,andbrassinolide;S+ADE-KW:culturingalgaeinADE-KWwithinoculumpre-treatedwithGIB;B+ADE-KW:introducingGIBintoalgaeinADE-KWatbatchcultivationstage;FA:fattyacid;MUFA:monounsaturatedfattyacid;PUFA:polyunsaturatedfattyacid;ML:mainfattyacidsinmembranelipid;SL:otherfattyacidsmainlyinstoragelipid;SFA:saturatedfattyacid;G3P:glyceralde-hyde-3-phosphate;NADPH:nicotinamideadeninedinucleotidephosphate;TAG:triacylglycerol.
AdditionalfileAdditionalfile1:TableS1.
Comparisonofeffluentfromanaerobicdigestionofdifferentdigesterfeedstocks.
TableS2.
Fattyacidlevels(%oftotalFAME)inChlorellaSDEC-11andScenedesmusSDEC-13.
Authors'contributionsHPdesignedtheexperimentsandeditedthemanuscript.
LJcarriedoutthewholeexperimentsanddraftedthemanuscript.
QHandZYparticipatedinbuildingashakertablewithillumination.
Allauthorsreadandapprovedthefinalmanuscript.
Authordetails1SchoolofEnvironmentalScienceandEngineering,ShandongUniversity,No.
27ShandaNanRoad,Jinan250100,China.
2ShandongProvincialEngineeringCentreonEnvironmentalScienceandTechnology,No.
17923JingshiRoad,Jinan250061,China.
AcknowledgementsNotapplicable.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
AvailabilityofdataandmaterialsDatasharingisnotapplicabletothisarticleasnodatasetsweregeneratedoranalyzedduringthecurrentstudy,andalldatageneratedoranalyzedduringthisstudyareincludedinthispublishedarticle.
FundingThisresearchwasfundedby:NationalScienceFundforExcellentYoungSchol-ars(51322811);ScienceandTechnologyDevelopmentPlanningofShandongProvince(2012GGE27027);theProgramforNewCenturyExcellentTalentsintheUniversityofMinistryofEducationofChina(GrantNo.
NCET-12-0341).
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