Understanding the salinity effect on cationic polymers in inducing flocculation of the microalga Neochloris oleoabundans

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ContentslistsavailableatScienceDirect

Journal of Biotechnology

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j b i o t e c

Understanding the salinity effect on cationic polymers in inducing ﬂocculation of the microalga Neochloris oleoabundans

G.P. ‘t Lam

^a,∗

, J.B. Giraldo

^a

, M.H. Vermuë

^a

, G. Olivieri

^a,b

, M.H.M. Eppink

^a

, R.H. Wijffels

^a,c

aBioprocessEngineering,AlgaePARC,WageningenUniversity,P.O.Box16,6700AAWageningen,theNetherlands

bDipartimentodiIngegneriaChimica,deiMaterialiedellaProduzioneIndustriale,UniversitàdegliStudidiNapoliFedericoII,PiazzaleVincenzoTecchio, 80,80125Napoli,Italy

cUniversityofNordland,FacultyofBiosciencesandAquaculture,N-8049Bodø,Norway

a r t i c l e i n f o

Articlehistory:

Received28October2015

Receivedinrevisedform24February2016 Accepted3March2016

Availableonline18March2016

Keywords:

Marinemicroalgae Harvesting Flocculation Mechanism Cationicpolymers Cationiccharge

a b s t r a c t

Amechanisticstudywasperformedtoevaluatetheeffectofsalinityoncationicpolymericflocculants, thatareusedfortheharvestingofmicroalgae.ThepolyacrylamideSynthofloc5080Handthepolysac- charideChitosanwereemployedfortheflocculationofNeochlorisoleoabundans.Inseawaterconditions, amaximumbiomassrecoveryof66%wasobtainedwithadosageof90mg/LChitosan.Thisrecovery wasapproximately25%lowercomparedtoSynthofloc5080Hreachingrecoveriesgreaterthan90%with dosagesof30mg/L.Althoughdifferentrecoverieswereobtainedwithbothflocculants,thepolymers exhibitasimilarapparentpolymerlength,aswasevaluatedfromviscositymeasurements.Whileboth flocculantsexhibitsimilarpolymerlengthsinincreasingsalinity,thezetapotentialdiffers.Thisindi- catesthatpolymericchargedominatesflocculation.Withincreasedsalinity,theeffectivityofcationic polymericflocculantsdecreasesduetoareductionincationiccharge.Thismechanismwasconfirmed throughaSEManalysisandadditionalexperimentsusingflocculantswithvariouschargedensities.

1. Introduction

Thelowenergyrequirementsforflocculationestablishesitasa promisingtechniqueforconcentratingmicroalgae(Udumanetal., 2010;Vandammeetal.,2013).Flocculationofseawatercultivated microalgae,however,isstillverychallenging.Insea-water,ionic hindranceoccurswhichinhibitstheinteractionoftheflocculant moleculeswiththe microalgae(Bilanovic etal., 1988; Uduman etal.,2010;Vandammeetal.,2010,2013).Unfortunately,onlya smallnumberoftechniquesarereportedtobesuccessfulforfloc- culationofmarinespecies:i.e.pH-increase,inorganicflocculation, andpolymericflocculation (Wu et al.,2012;Chatsungnoen and Chisti,2016;‘tLametal.,2014).ApH-increaseinducesthepre- cipitationofsalts.Thoseprecipitateswillsettleand,meanwhile, willsweepthebiomass(Wuetal.,2012).Intheirstudy,several microalgaehavebeensuccesfullyflocculatedbyincreasingthepH, resultingin a precipitationof thedivalention magnesium.The useof inorganicflocculantsinseawatersalinities hasalsobeen reported(ChatsungnoenandChisti,2016).However,asmentioned byUdumanetal.(2010),theuseofinorganicflocculantsinseawa-

∗Correspondingauthor.

E-mailaddress:gerard.tlam@wur.nl(G.P.‘tLam).

tersalinitiescommonlyrequireshighdosagesthatareabout5–10 timeshighercomparedtopolymericﬂocculants.Withpolymeric ﬂocculation,polymericbridgesbetweenindividualcellsareformed and,subsequently,aggregatesofbiomassevolve(Vandammeetal., 2013;‘tLametal.,2014).

Amongpolymericflocculants,cationicpolymersareregarded assuccessful,thoughnotallareequallyefficientininducingfloc- culationofmarinemicroalgae.Currently,onlypolyacrylamidesare reportedtobesuccessful(‘tLametal.,2014;Königetal.,2014;

Roseletetal.,2015).

Despite the success of cationic polyacrylamides in harvesting marine microalgae, ‘t Lam et al. (2015) reported that, when commercially available cationic polymers are applied as flocculants, the required flocculant dosage is quite high (40–100mg_flocculant/g_biomass),resultinginalowereconomicfeasi- bility.Additionally,theuseofpolyacrylamidesisforbiddenforfood andfeedapplicationsasseveraloftheseflocculantsarereportedto betoxicandnon-foodgradepetroleumprocessingtechniquesare commonlyusedtomanufacturethem(Leeetal.,2014).Toover- cometheselimitations,otherflocculantsthatpreferablyhavean equalofevenbetterperformanceandthatareallowedinthefood andfeedindustryshouldbeselectedordesigned.Toallowtheratio- nalselectionordesignofnovelflocculants,themechanismthatis

http://dx.doi.org/10.1016/j.jbiotec.2016.03.009

0/).

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However,recentstudiesofRoseletetal.(2015)showedthatthe cationicchargeofthepolymericflocculantshadapositiveeffect onthebiomassrecoverywherethepolymerlengthwasofminor importanceandthatisnotinaccordancewiththepreviouslyspec- ifiedexplanationofpolymericcoiling.Itis,therefore,stilldifficult toexplainwhycertaincationicpolymersaresuccessfulininducing flocculationinseawatersalinitieswhileothersarenot.

The goal of this study was to provide further information tobetterunderstandcationicpolymericﬂocculationinseawater salinitiesandpossiblyrevealwhycationicpolyacrylamidesremain functionalinhighsalinitieswhileothercationicpolymersdonot.

Thisgainedinsightalsoprovidedinformationthatcanbeapplied inoptimizingthedesignofﬂocculants.

Inthisstudy,Synthofloc5080HandChitosanwereexploitedas flocculants.Synthofloc5080Hisacationicpolyacrylamidethatis reportedtobesuccessfulinflocculatingmarinemicroalgae(‘tLam etal.,2014).Chitosanisanaturalpolysaccharidewhichisrecog- nizedasbeingsuccessfulininducingflocculationunderfreshwater conditionsbutbecomeslesssuccessfulinseawatersalinitiesandin neutralpH(Bilanovicetal.,1988).Theapparentpolymerlengthand nettcationicchargeofbothflocculantswerecomparedwitheach otherasafunctionofsalinity.

TheusedmicroalgainthisstudywasNeochlorisoleoabundans whichisabletogrowinbothfreshandsaltwaterconditions.It.has beenreportedtocontainahighproteincontentand,understressed conditions,a highlipidcontent.ThismakesN.oleoabundansan interestingspeciesforseveralapplications(Popovichetal.,2012;

Breuer et al., 2012). In addition, N.oleoabundans is a spherical Chlorophyta,hence,itsshapeeliminatespossibleside-effectsof thecellshapeduringﬂocculation.

2. Materialandmethods 2.1. Biomasscultivation

The microalgal strain N. oleoabundans UTEX1185 was culti- vatedinartiﬁcialseawatermediumwithvarioussalinities:NaCl:

15g/L(brackish),25g/L(seawater),35g/L(saline);KNO₃:1.7g/L;

Na₂SO₄: 0.5g/L; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): 23.83g/L; MgSO4·7H2O: 0.73g/L; CaCl2·2H2O:

0.36g/L;K₂HPO₄:0.43g/L;Na₂EDTA·2H₂O:0.03g/L;MnCl₂·4H₂O:

0.004g/L; ZnSO₄·7H₂O: 0.0012g/L; CoCl₂·6H₂O: 0.0003g/L;

CuSO4·5H2O:0.0003g/L;Na2MoO4·2H2O:0.00003g/L;NaFeEDTA:

0.01g/L.

Biomasswascultivatedin100mLshakeﬂasksinanInforsMul- titronincubator(InforsAG,Bottmingen,Switzerland).Thecultures werecontinuouslyilluminatedat120␮molm⁻²s⁻¹inatmospheric airenrichedwith2.5%CO₂atatemperatureof25^◦C.Theﬂaskswere orbitallyshakenat90rpm.

Partoftheculturedbiomasswasharvestedusingpipettingtwo daysafterinoculation.Ontheseventhday,newcultureswereinoc- ulatedforfurthercultivation.Byre-inoculatinganewﬂaskevery

provided by Sachtleben Wasserchemie GmbH, Germany. All ﬂocculants are large polyacrylamides with various cationic chargesandarecommonlyusedinwastewaterapplications.

Chitosan(purchasedfromSigma-Aldrich,productnr.:448869- 50G)wasdissolvedovernightin0.1%(v/v)aceticacidafterwhich thepHwasadjustedtopH7±0.2.Flocculantswerestoredat4^◦Cin adarkenvironmentandwereneverstoredlongerthansevendays.

2.3. Biomassrecovery

After harvesting the biomass, the initial optical density at 750nmwasestablishedat0.8±0.01usingculturemedium(cor- respondswitha dryweightof0.24±0.07g/L).Aftersetting the OD₇₅₀,10mLofthesamplewastransferredtoabeakerglassand stirredat500rpm.Fromastocksolution,ﬂocculantwasaddeduntil thedesireddosewasachieved(rangingbetween0and90ppm).

Afterﬁveminutesofmixingat500rpmfollowedbyatenminute period ofmixing at100rpm,samples weretransferredto4mL polystyrene cuvettes. The mixing protocol that was used ﬁrst involvedaseveremixingfollowedbyagentlemixingtimeandis inaccordancewithprotocolsreportedinotherstudies(Bilanovic etal.,1988).Usingthephotometricmethodof(Salimetal.,2012), thegradualbiomassrecoverywasfollowed inaBeckman Coul- ter DU730 photometer. After two hours of sedimentation, the biomassrecoveriesweredeterminedandcalculatedaccordingto (Salimetal.,2012).Allexperimentswereperformedinduplicate:

Recovery(%)=OD_750(t0)−OD750(tsupernatant)

OD_750(t0) ×100

2.4. Viscosity

The viscosity of a polymeric solution is correlated with the apparentpolymer length.Tostudy theeffectof thesalinity on theapparentpolymerlengthof theflocculants,theviscosityof theflocculant solutionsin varioussalinities wasmeasured.The flocculant concentrations ranged between 0 and 100ppm. The viscosity was measured using a Physica MCR 301 Rheometer.

Polymericsolutionsweremadewithvarioussalinitiesbyvarying theNaCl-concentration(0–10g/LNaCl).Aftertheadditionofthe ﬂocculant solution in the rotational cylinder, theviscosity was measuredatshearratesrangingfrom1to100s⁻¹.

2.5. ␨-Potential

␨-Potential measurementswere performedtodeterminethe effectofsalinityonthenetcationicchargeoftheﬂocculant.Several ﬂocculantsolutionswithdifferentNaClconcentrationswerepre- pared.Flocculantdosagesrangedbetween30 and200ppm.The salinityrangedbetween0and4g/LofNaCl.Thechargewasmea- suredusingaMalvernZetasizerNano.

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Fig.1. Biomassrecoveryasafunctionoftheflocculantdosageatsalinitiesof25g/L( ),35g/L(䊏)and45g/L().RecoveriesobtainedwithSynthofloc5080HinfigureA, ChitosaninFigureB.Allsamplesrepresentbiologicalduplicates.

Fig.2.ViscosityofSynthofloc5080Hmeasuredatasharerateof100s⁻¹.Everyclusterofbarsrepresentsaflocculantdosage.Withineverycluster,thesalinitywasincreased, correspondingwiththelegendattherightsiteofthefigure.

Table1

ComparisonofobtainedbiomassrecoverieswithChitosanatneutralpHinvariousstudies.

Species Cx(g/L) pH dosage(mg/L) fresh/marine recovery Reference

C.sorokiniana 0.27±0.07 7 5 fresh >90% Xuetal.(2013)

C.vulgaris 1 7 120 fresh 92%±0.4 Rashidetal.(2013)

N.oleoabundans 0.5 7.2 100 fresh 95% Beachetal.(2012)

S.obliquus 0.54 7 80 fresh 95% Chengetal.(2011)

N.salina – 8 8 marine >90% Garzon-Sanabriaetal.(2013)

N.oleoabundans 0.24±0.07 7 90 marine 66% thisstudy

2.6. SEMimaging

The scanning electron microscopy objects were prepared accordingtotheprotocoldescribedinSalimetal.(2014).Inthis protocol,aliquotsofthemicroalgaeweremixedwiththefloccu- lantfor.,fiveminutesofseveremixing(500rpm)followedbyten minutesofgentlemixing(100rpm).Immediatelyafterthemixing, adropofsuspendedflocswastransferredtoapoly-L-lysinecoated microscopycoverslip.Afteronehour,thecoverslipwasrinsed, andtheremainingcells onthecoverslipwerefixatedina3%a glutaraldehydesolutioninaPBS-bufferforonehour.Thecellswere post-fixatedina1%OsO₄ solutionforanotherhour.Afterwards, thefixatedcellswererinsedanddehydratedusingethanol.They weresubsequently, dried using critical point CO2 drying. After drying,thecover slips werecoatedwitha 10nmIridiumlayer usingsputter-coating.

3. Resultsanddiscussion 3.1. Flocculation

Thebiomassrecoveriesweremeasuredatvariousdosagesof Synthoﬂoc5080H(Fig.1A)andChitosan(Fig.1B)atthreedifferent salinities:25,35,and45g/LofNaCl.

WithSynthoﬂoc5080H,thebiomassrecoveryisalwayshigher than90%regardlessofthesalinity.Alowerbiomassrecoveryis recordedwhenChitosanisappliedasacationicpolymericﬂoccu- lantusingasimilardosage.

Atelevateddosages,thebiomassrecoveryinallthreesalinities decreaseswith7%recoverywhenusingSynthofloc5080Hasafloc- culant.Thisisinagreementwiththemodelpresentedinprevious work(‘tLametal.,2015)inwhichthereisanoptimumflocculant-

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Fig.3.ViscosityofChitosanmeasuredatasharerateof100s⁻¹.Everyclusterofbarsrepresentaﬂocculantdosage.Withineverycluster,thesalinitywasincreased, correspondingthelegendattherightsiteoftheﬁgure.

biomassratio.Whenthisratioisexceeded,ﬂocculationbecomes inhibitedduetorestabilization.

ThesuccessfuluseofChitosaninfreshwaterconditionshaspre- viouslybeenreported(Table1),andtheobtainedresultsofFig.1B werecomparedwiththesestudies.Inallofthestudiesmentionedin Table1,thebiomasswascultivatedinnutrientrepleteconditions.

Possiblebiological effectssuchastheformation ofextracellular polymericsubstancesduetonutrientstress(Salimetal.,2013), werethuseliminated.

Thecomparisonbetweenthebiomassrecoveriesobtainedwith Chitosaninthisstudyandotherstudiesdemonstratedthat,insea- watersalinities,aconsiderablylowerbiomassrecoveryisobtained usingmerelychitosan(Table1).AlthoughGarzon-Sanabriaetal.

(2013)didinciteelevatedbiomassrecoveriesbyusingChitosanin seawatersalinities,itisnotknowniftherewasapossiblepHeffect involvedasthepHafterflocculantadditionwasadjustedto8in theirstudy.Inadditiontothelowerbiomassrecovery,otherstud- iesinTable1usedsubstantiallowerflocculantdosages.Theuseof lowerflocculantdosageswithhigherbiomassrecoveriesimplies that,inotherstudiesinfreshwaterconditions,Chitosanwasamore efficientflocculant.

The differences in polymeric properties that were observed betweenSynthoﬂoc5080HandChitosan inincreasingsalinities havebeenattributedtothedegreeofpolymericcoiling(Bilanovic etal.,1988).Theyconcludedthat,asafunctionofthesalinity,a polymershrinksuntilitreachesitsmallestdimensions.

3.2. Viscositymeasurements

Toverifyifpolymericcoilingprovidesanexplanationforthe lower biomass recovery observed with Chitosan compared to Synthoﬂoc5080H,viscositymeasurementsofbothﬂocculantsdis- solvedinwaterwithdifferentsalinitieswereperformed.

The viscosity of a polymeric solution is proportional tothe apparentlengthofthepolymers(Yamakawa,1971;Tricot,1984;

Bilanovicetal.,1988).

InFigs.2and3,thetwobardiagramsillustratetheviscosityas afunctionoftheﬂocculantdosageandasafunctionofthesalinity.

In Fig.2, thedecrease in viscosityobtainedwithSynthoﬂoc 5080HisinagreementwiththetrenddescribedbyBilanovicetal.

(1988).Intheirstudy,alsoadecreasein viscosityasafunction ofthemedium salinitywasobserved.Butdespitetheobserved substantialviscositydecreaseoftheSynthofloc5080Hsuspension inhighsalinities,itstillinducesflocculation(Fig.1).Moreover,the viscosityofSynthofloc5080Hdropsdramaticallytovaluescloseto theviscosityofwateralreadyinmediumwithsaltconcentrations

Fig.4.␰-potentialasafunctionof[NaCl](g/L).Synthoﬂoc5080H,potentialsmeasuredat:100mg/L( )and200mg/L( ).Chitosan,potentialsmeasuredat:30mg/L (),60mg/L()and90mg/L(䊏).Errorbarsareduplicates.

lowerthan 1g/L ofNaCl.Thisillustrates thatSynthoﬂoc5080H polymerisverysensitivetosurroundingionicforcesandbecomes coiled.

With Chitosan (Fig. 2), the viscosity remains similar to the viscosityofwaterregardlessoftheflocculantdosageandsalinity thatisapplied.Theseresultsdemonstratethatnocoilingoccurred toexplainthelowerbiomassrecoveriesobtainedinFig.1with ChitosancomparedtoSynthofloc5080H.Inaddition,bothfloccu- lantshadaviscositysimilartowaterinsalinitiesof10g/LNaCland aflocculantdosagelowerthan100ppm.Thisresultillustratesthat bothflocculantshadasimilarapparentpolymerlengthinthese conditions.

Althoughpolymericcoilingobviouslyoccursinelevatedsalinity, itdoesnotexplainthesuccessofSynthofloc5080Hinhighsalinity andthedecreasingfunctionalityofChitosanwithincreasingsalin- ityasthesalinityofseawaterisapproximately35g/L.Theseresults illustratethatanothercharacteristicoftheflocculantsshouldbe responsibleforthedegreeofsuccessofflocculantsinhighsalinities.

3.3. ␨-Potential

Inadditiontotheapparentlengthofthepolymericchain,the chargeof cationicpolymersmaybeanimportant feature.With increasingsalinity, thenett cationic charge ofpolymers should

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Fig.5. SEMimaging,A:controlat25g/Lsalinity.B:controlat45g/Lsalinity.C:FlocwithSynthoflocat25g/L.D:flocwithSynthoflocat45g/L.E:zoominonthebridges withSynthoflocat25g/L.F:zoominonthebridgeswithSynthoflocat45g/L.Usedflocculantconcentrationwas60mg/L.

decrease due to the surrounding of anions. ␨-Potential mea- surementswereperformedtomeasuretheimpactofincreasing salinityonthenettchargeofthecationicpolymers(Fig.4).Forboth ﬂocculants,thepolymericpotentialwasmeasuredasafunctionof salinity.ThesalinitywasincreasedbyanadditionofNaCl.These measurementswereperformedwithvariousdosages(Fig.4).

Withbothflocculants,the␨-potentialdecreasesasafunctionof thesalinity.Whenthe␨-potentialasafunctionofsalinityofSyn- thofloc5080Hiscomparedwiththe␨-potentialofChitosan(Fig.4), itappearsthat the␨-potentialofSynthofloc5080His generally morethantwiceashighregardlessofthesalinity.Bothflocculants demonstrateaninitialsharpdecreasein␨-potentialwithsalinity, butSynthofloc5080Halwayshasatleasta20mVorhighercharge thanChitosan.

Thecombinationoftheobserveddifferenceincationiccharge forbothflocculantswiththeobservedsimilaritiesinviscositywith salinitysuggeststhatthecationicchargeisapredominantparame- terinfluencingtheflocculationefficiencyofN.oleoabundansunder salineconditions.

3.4. SEMimaging

Inadditiontoviscosity-and␨-potentialmeasurements,Scan- ning Electron Microscopy (SEM) was performed to verify if a differencebetweenthetwoﬂocculantsandanyeffectofsalinity onthestructureoftheﬂocculatedmicroalgaecouldbeobserved.

Theintentionwastovisualizeiftheﬂocculantisindeedadsorbedto thecellwall.Inaddition,thepicturescanalsorevealhowindividual cellsareattachedtoeachother:bridging,patching,acombination, oranotherpossibility.

InFig.5,thecellsandformedaggregatesaredepictedatbrackish salinity(25g/L,Fig.5A,CandE)andathighsalinity(45g/L,Fig.5B, DandF)afteradding60mg/LofSynthoﬂoc5080H.

Fig.5Aillustratesthecellswithoutflocculantinbrackishsalin- ity.Accordingtothefigure,thecellsareclusteredwhichmaybe caused fromthedehydration of thesamplesduring theprepa- ration. However, despite this clustering, thecells have smooth surfacesandarenotboundtoeachotherbyafibrousnetworkof flocculants.Afteradditionoftheflocculantinbrackishconditions, Synthofloc5080Hwasstronglyinteractingwiththesinglecells

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Fig.6. SEMimagingA:controlat25g/L.B:controlat45g/L.C:FlocwithChitosanat25g/L.D:ﬂocwithChitosanat45g/L.E:zoominonthebridgeswithChitosanat25g/L.

F:zoominonthebridgeswithChitosanat45g/L.Usedﬂocculantconcentrationwas60mg/L.

(Fig.5CandE).Thepolymersadsorbtothesurfacesandforma fibrousnetworkbetweenthesinglecells.Asaresult,largeaggre- gatesofflocsareformed.Inaddition,alloftheflocculantsappear tobe adsorbedtothe cells as nonon-absorbedflocculants are observed.

Fig.5Bshowsthatthesinglecellsalsohaveasmoothsurface inverysalineconditions.AccordingtoFig.5DandF,largeagglom- eratesareformedjustasthoseinbrackishconditions.However,in thishighsalinity,Synthofloc5080Happearstoexperienceaweaker interactionwiththecellsasthelargepolymericfibrousnetworks werenotobservedbetweenindividualcells.Itappearsthatthefloc- culantsarestilladsorbedtothesurface(Fig.5F),however,they locallycoverthecellsurfacewhichallowcellstointeractandform smallbridges.

InFig.6,theﬂocformationafteranadditionof60mg/LofChi- tosanis shown.Fig.6A, C,and Earepictures takenin brackish salinity(25g/L),andFig.6B,D,andFaretakeninverysalinecon- ditions(45g/L).

ThecontrolpictureinFig.6Aisthesamecontrolpicturethat wastakenin brackish salinitiesfor Fig.5. Fig.6C exhibitsthat, although60mg/LofChitosanwasadded,nolargeaggregatesare formedinbrackishconditions.Thereareseveralsmallaggregates

formed,butthosecontainnomorethanapproximatelythreeto fourcells.IncomparisonwithFig.6Carelativelylargeamountof non-adsorbedﬂocculantwasobservedintheformofwhitesmall aggregatesbetweenthealgalcells.

Thereweresimilarobservations inverysalineconditions.In Fig.6B,thesamecontrolthatwasdepictedinFig.5isshown.Also, smallalgalflocsaredepictedinFig.6DandF.Justaswasobserved inbrackishconditions,arelativelylargeamountofnon-absorbed flocculantremainsnexttothesmallflocs.

Inbothsalinities,thecationicpolymersofChitosanappeartobe moreentangledwitheachotherthanthoseofSynthoﬂoc5080H.

Despitethisentanglement,thepolymerswereadsorbedtothecell wall.Thisisinaccordancewiththeobservedbiomassrecoveries obtainedwithChitosan(Fig.1B).

Theobservations(Figs.5and6)correspondwellwiththeresults ofthe␨-potentialmeasurements.Itwashypothesizedthatpoly- mericflocculantsmustbeabsorbedtothecellwallbeforeinducing flocculation.After15minofmixing,alloftheSynthofloc5080H polymersappear tobe adsorbedsince whiteaggregates are no longerdetected.However,withChitosan,arelativelylargeamount ofnon-absorbedpolymersarestillobservedoutsidetheflocs.

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Fig.7.Biomassrecoveriesasafunctionofthechargedensity(Control,5025H;

5040Hand5080H).Allexperimentsareperformedinbiologicalduplicates.Syn- thoﬂoc5080HisadaptedfromFig.1.

Ourpreviouswork(‘tLam etal.,2015)mathematicallycon- firmedaproposedflocformingmechanismthat,justasinother, earlierstudies,assumespolymericadsorption(Vandammeetal., 2013).TheSEManalysisinthisstudysupportstheproposedmech- anismofadsorptionofaflocculantonacellwall.

Polymericadsorptiontoasurfacecanbeenhancedbycharge differences(BoltoandGregory,2007).Thelargerthechargediffer- encebetweenpolymersandthecellwall,thequickerthepolymer willbeadsorbed(Al-HashmiandLuckham2010;Tekinetal.,2010).

Theseresultsobtainedinotherstudiessuggestthenecessityofa highchargedifferencebetweenpolymerandsurface(inthiscase, themicroalgalcellwall).Ensuingfromthisconclusion,theresults reportedinFig.4suggestthatthedecreaseincationicchargecaused adecreasedefﬁciencyofcationicpolymersinelevatedsalinities.

Inadditiontoalowerdegreeofadsorptionofpolymersonthe cellwall,Tenneyetal.(1969)suggestedthatchargeneutralization playsaroleininducingflocformation.Whenchargeneutraliza- tionisactuallytakingplaceduringflocformation,apolymerwitha highercationicchargewillbemoreefficientinlocallyneutralizing thechargeofindividualcells.

Thedecreaseincationicchargethatcausedalowerdegreeof adsorptionincombinationwithadecreasedabilitytoneutralize cellwallchargesplausiblycausedthedecreasedﬂocculationofChi- tosaninelevatedsalinities(Fig.1).Itmayalsoexplaintheremaining amountof polymersthatwereobservedafter15minof mixing (Fig.6).

3.5. Flocculationatvariouscationicchargedensities

Toconfirmthatadecreaseincationicchargeduetoanincreas- ingsalinityiscausingadecreaseinflocculation,additionaltests wereperformedwithflocculantsfromtheSynthofloc50-series.By keepingthepolymericstructure(andsize)constantandvarying thechargedensityfromalowcharge(5025H)throughamoderate cationiccharge(5040H)uptoahighlychargedcationicpolymer (5080H),theeffectofcationicchargecouldbeconfirmed(Fig.7).

Theappliedsalinityinthisexperimentwas35g/L.

AccordingtoFig.7,witha flocculantdosageof 30mg/L,the flocculantwiththehighestchargedensity(5080H)wasthemost efficientinharvestingthebiomassinmarineconditions.Onaver- age, a 9% higher biomass recovery was obtained with 5080H comparedto5025H.Theseresultsdemonstratethatahighercharge densityresultsingreaterbiomassrecoveries.Thecombinationof theresultspresentedinFig.7withtheobserveddecreasein␨- potentialasafunctionofmediumsalinity(Fig.4)andapparent independenceofthebiomassrecoveryonthedegreeofcoilingof aflocculantsuggestthat,duetoadecreaseincationicchargein elevatedsalinities,flocculantsbecomelessfunctional.

Achangeinbiomassrecoveryasafunctionofthechargedensity, similartotheresultsinFig.7,waspreviouslyobservedbyRoselet et al. (2015).In theirstudy, thefreshwater microalga Chlorella vulgarisandtheseawatermicroalgaNannochloropsisoculatawere ﬂocculatedwithcationicpoly(acryl)amidesofthe‘Flopam’series.

Bymaintainingaconstantpolymericsizeandvaryingthecharge densityfrom0%to100%,theeffectofthecationicchargeonthe biomassrecoverywasdetermined.Thebiomassrecoveryincreased fromrecoverieslowerthan10%torecoverieshigherthan90%with bothmicroalgaeasafunctionofthechargedensity.

4. Conclusion

Thedecreaseinnettcationicchargeinelevatedsalinitiesincites decreasedfunctionalityofcationicpolymersandinducesfloccu- lationof N.oleoabundans.In highsalinities, theresultinglower chargecauseddiminishedefficiencyinformingpolymericbridges betweenindividualcells.Thisinsightresultedintheconclusionthat thecationicchargeisanimportantcriterioninselectingcationic polymersasaflocculantformarineapplicationswheretheappar- entpolymerlengthisofminorsignificance.Thisstudyalsorevealed that,inbothbrackishandmarineconditions,polymericbridgingis adominantmechanisminflocformationforcationicpolymers.

Acknowledgements

ThisworkisperformedwithintheTKIAlgaePARCBiorefinery programwithfinancialsupportfromtheNetherlands’Ministryof EconomicAffairsintheframeworkoftheTKIBioBasedEconomy under contract nr. TKIBE01009. The authors thank the depart- mentofFoodProcessEngineering(WageningenUniversity)and, inparticular,Jos Sewaltfor hisassistanceinanalysingthefloc- culants. Theauthors thankMarcel Giesbers of theWageningen ElectronMicroscopyCentreofWageningenUniversityforhissup- port with SEM imaging. The authors are grateful for receiving thepoly(acryl)amidicflocculantsfromSachtlebenWasserchemie GmbH(Germany).

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