VOL 83(1) (2018) COMMUNICATIONS IN AGRICULTURAL AND … · 2018. 12. 11. · VOL 83(1) (2018)...

VOL83(1)(2018)

COMMUNICATIONSINAGRICULTURALANDAPPLIEDBIOLOGICALSCIENCES

Publishers

PROF.GUYSMAGGHEPROF.STEVENSLEUTELPROF.JANBAETENS

DR.CHRISCALLEWAERT

Editorialaddress

Coupurelinks6539000Gent(Belgium)

ISSN1379-1176

ii

Theresultspublishedinthisbookofabstractsareunderthefullresponsibilityoftheauthors.Theorganizingcommitteecannotbeheldresponsibleforanyerrorsinthispublicationandpotentialconsequencesthereof.

PROCEEDINGS

23RDNATIONALSYMPOSIUMONAPPLIEDBIOLOGICALSCIENCES

FACULTYOFSCIENCE

ANDBIOSCIENCEENGINEERINGVUB

FEBRUARY8TH,2018

Websitehttp://www.vub.ac.be/events/nsabs2018-conference#nsabs-2018

ORGANIZINGCOMMITTEEWimDeMalsche(VUB),JulienCousin-Saint-Remi(VUB),KrisPauwels(VUB),ChristianHermans(ULB),BenoitScheid(ULB)VrijeUniversiteitBrussel–hostinstitute2018:WimDeMalsche,JulienCousin-Saint-Remi,KrisPauwelsUniversitéLibredeBruxelles–hostinstitute2018:ChristianHermans,BenoitScheid,DavidCannella,JulienLouvieuxUniversiteitGent:JanBaetens,ChrisCallewaert,StevenSleutelKULeuvenBarbaraDeConinck,BramVandenBergh,ElineVanuytrecht,LiesbethFrançois,MariyaPetrovaUniversiteitAntwerpen:JelleHofman,DieterVandenheuvelUniversitédeLiège:JérômeBindelle,HélèneSoyeurtUniversitéCatholiquedeLouvainValentinCouvreur,XavierDraye,GauthierLequeueUniversitédeMonsSylvainGabrieleREVIEWERCOMMITTEEKULeuven:BarbaraDeConinck,BramVandenBergh,ElineVanuytrecht,LiesbethFrançois,MariyaPetrova,TreesDeBaerdemaeker,NielsDeBrierUniversiteitAntwerpen:JelleHofman,TomTytgat,DieterVandenheuvel,ErikVanEynde,SammyVerbruggen,KarenWuytsUniversiteitGent:JanArends,JanBaetens,ChrisCallewaert,AislingDaly,TimDepraetere,NicoDeStorme,JoachimSchouteten,MajaSimpraga,StevenSleutel,MichielStock.UniversitéCatholiquedeLouvain:XavierDraye,ValentinCouvreur,SixtinePassot,GauthierLequeueUniversitédeLiège:HélèneSoyeurt,JérômeBindelle,AngéliqueLéonard,Jean-MicelRomuee,BenoitMercatoris,MagaliDeleu,GeorgesC.LognayUniversitéLibredeBruxelles:DavidCannella,FredericDebaste,BenoitHaut,ChristianHermans,BenoitScheid,GuillaumeSmitVrijeUniversiteitBrusselGeertAngenon,JulienCousin-Saint-Remi,WimDeMalsche,PierreGelin,FrederickLeroy,FrederickMatheuse,KrisPauwels,YannSterckx,IngeVanMolle,WimVranken

v

ACADEMICSUPPORTTheNationalSymposiumonAppliedBiologicalSciences(NSABS2018)isjointlyorganizedbythefollowinginstitutes:

- FacultyofScienceandBioscienceEgineering,VrijeUniversiteitBrussel- UniversitéLibredeBruxelles- FacultyofBioscienceEngineering,KULeuven- DepartmentofBioscienceEngineering,UniversityofAntwerp- FacultyofBioscienceEngineering,GentUniversity- FacultyofBioscienceEngineering,UniversitéCatholiquedeLouvain- EarthandLifeInstitute,UniversitéCatholiquedeLouvain- UniversitédeLiège

WEBSITEhttp://www.vub.ac.be/events/nsabs2018-conference#nsabs-2018ACADEMICANDGOVERNMENTALFINANCIALSUPPORTTheorganizingcommitteeofNSABS2018isgratefulforthefinancialandlogisticsupportbythefollowinginstitutesandtheirdoctoralschools:

- KULeuven- UAntwerp- VUB- UGent- ULB- UCL- ULg- UMons- UGentDoctoralSchools(Bioscience)Engineering- YouReCa- FNRSGraduateSchoolonEnvironmentalSciences,TechnologiesandManagement

(ENVITAM)- FNRSGraduateSchoolonAgriculturalSciencesandBiologicalEngineering(EDT-SCAIB)- FNRSGraduateSchoolinPlantSciences- Vlaamseoverheid- VlaandereninActie- RichtingMorgen(Vlaamseoverheid)

ENVITAM

vi

INDUSTRIALSPONSORSNSABS2018receivesthegeneroussupportoftheErnestSolvayFund(KingBaudouinFoundation)andoursponsors:

Gold sponsor

Silver+ sponsor

Silver sponsors

Bronze+ sponsor

Bronze sponsors

vii

PREFACE

With great pleasure the organizing committee presents you the proceedings of the 23rdNationalSymposiumonAppliedBiologicalSciences(NSABS2018).

ThissymposiumhasitsrootsinGhent,wherethefirstconferencewasorganizedin1995byGhentUniversity.Inthefollowingyears,otherBelgianuniversitieshavejoinedincontributingtotheconference.CurrentlyKULeuven,UAntwerp,UGent,ULg,UCL,UMONS,ULBandVUBare involved in reviewing, programming, promoting and financially supporting theconference.Themainpurposeoftheconferencehasbeenpreservedthroughouttheyears:to explore and to bring together the Belgian expertise in the field of Applied BiologicalSciences,focusingmainlyoncontributionsofPhDstudents.

Thisyears’NSABSisorganizedforthefirsttimeinBrussels,hostedbyboththeFlemishandFrenchspeakingFreeUniversityofBrussels,VUBandULB.Inthecurrenteditiontheemphasisis put on ‘crossing borders’ and a number of incentives have been taken to endorse thistheme.Next to best poster and presentation prizes, also thematic prizes are awarded tocontributions that specifically focus on and excel in multidisciplinarity, sustainability andvalorization.Thesetopicswillalsobeaddressedbyseveralrenownedspeakerswithinspiringstories,whilecareerperspectivesforyoungscientistswillbeputinthespotlightaswell.

Thecoreoftheconferenceremainsinthehandsofyoungresearchers,thefloorisgiventoPhDstudentsfor30oraland106posterpresentations.

We hope that you will enjoy NSABS 2018 and we look forward to a fruitful, interactive,informal,enjoyable,open-mindedandmemorableexperience.

Prof.Dr.ir.WimDeMalsche

Onbehalfoftheorganizingcommittee

viii

PROGRAMNSABS2018

23rdNationalSymposiumforAppliedBiologicalSciences(organizedbyVUBandULB)

08:30 Registrationandwelcomingcoffee(N.MandelaRoom)09:10 Openingconference(AulaA)

09:25PlenaryI(AulaA)

AlbertVandenBerg(UTwente,NL)“FromLabonaChiptoOrgansonChip”

Parallelsessions1AulaB AulaC AulaD

ChemistryandChemicalEngineering Biomathematics EnvironmentI

10:10 O1 O11 O2110:30 O2 O12 O2210:50 O3 O13 O2311:10 Coffeebreak(N.MandelaRoom)


AgricultureandFoodTechnologyI HumanHealthI Key-enabling

Technologies

11:35KeynoteI

TimStakenborg(IMEC)

KeynoteIIStéphanieDescroix(InstitutCurie,FR)

KeynoteIIIDavidCannella(ULB)

12:05 O4 O14 O2412:25 O5 O15 O2512:45 O6 O16 O2613:05 Lunchandpostersession1(N.MandelaRoom)

14:05PlenaryII(AulaA)

LuciaSmit(Braingain)"HowtomanageyourcareerasaPhD:challengesandopportunities"


AgricultureandFoodTechnologyII HumanHealthII EnvironmentII

14:40 O7 O17 O2715:00 O8 O18 O2815:20 O9 O19 O2915:40 O10 O20 O3016:00 Coffeebreakandpostersession2(N.MandelaRoom)17:00 PlenaryIII(AulaA)

FrédéricvanGansberghe(Galacticsa/nv)“Andnow,what’snext?”

17:30 Closingremarksandawards(AulaA)17:45 Networkingreception(N.MandelaRoom)

ix

ORALPRESENTATIONS

ChemistryandChemicalEngineering

O1.PierreGelin(VUB).UseofacousticsinmicrofluidicReactors.O2. Sander Stroobants (VUB).Anoveldevice for studyingnucleationof crystals andaggregates incontrolledshearflow.O3.HennieValkenier (ULB).Crossing theborder:organicmolecules transport chlorideacross lipidbilayers

AgricultureandFoodTechnologyI

Keynote I. Tim Stakenborg (IMEC). New Developments in Silicon Nanotechnology for HealthcareApplications.O4. Julie Vanderstraeten (UGent). Designer cellulosomics for a customized conversion oflignocellulosicbiomasstovaluablebulkandfinechemicals.O5. Kamal Belhaj (UniversityMohammed 1st,Morocco). The effect of tow traditionalMoroccan'scookingmethodsonOmega-6/omega-3andfattyacidshypo/hypercholesterolemicratiosofBeni-GuillambmeatbreedinginnortheastofMorocco.O6.IlseVanhoutte(UGent).Microbialdetoxificationofthemycotoxindeoxynivalenol.

AgricultureandFoodTechnologyII

O7.ElienLemmens(KUL).Theimpactofcontrolledsteepingandgerminationofwheatonhydrolysisofphytateandarabinoxylanandonmineralbio-accessibility.O8. Inga Tarasenko (UCL). The immediate response of tomato and rice to tephra deposition: anexperimentalstudy.O9.MarieVandermies(ULg).CatabolicselectablemarkereasesgenomeeditinginYarrowialipolytica.

Biomathematics

O11.MauricioMacossayCastillo(VUB).Proteinintrinsicdisorder:adouble-edgedsword?O12.ThomasSchalck (KUL). Identificationofcausalmutationsconferringhigh-ethanol tolerance inEscherichiacoli.O13.AuroreWoller (ULB).Metabolicsyndromeduetoconflictsbetween lightandfood:astoryoftwistedclocks.

HumanHealthI

KeynoteII.StéphanieDescroix(InstitutCurie,FR).Microfluidicsforbiomimetism:from3Dco-culturetogutonchip.O14.XeniaGeeraerts(VUB).Theimpactoflactateonthemetabolicphenotypeoftumor-associatedmacrophages.O15. Gaétan Kalala (ULg). Effect of a diet rich in prebiotic fibers of inulin type and behavior onintestinalhealthinobesepatientsinanhumaninvitrofermentationpatternsO16. Pieter De Bruyn (VUB). Biophysical characterization of a three-component toxin-antitoxinmodulefromthepathogenicbacteriumEscherichiacoliO157.

x

HumanHealthII

O17. Hans Gerstmans (UGent). High throughput generation of designer enzybiotics againstAcinetobacterbaumannii.O18.RobertAdamuShey(ULB).PredictionandvalidationofthestructuralfeaturesofOv-58GPCRasanimmunodominantantigenofOnchocercavolvulus:implicationsinonchocerciasiscontrolefforts.O19.FrédéricGoormaghtigh(ULB).Single-Cellimagingofbacterialpersisters.O20.AndreyKossarev(KUL).Shuttlevectorsystemforimprovedpestiviralreversegenetics.

EnvironmentI

O21.SteveHuysman(UGent).UHPLC-HRMSbasedtargetedanduntargetedscreeningofplasticizersinthemarineenvironment.O22.RosanneReitsema(UA).Effectsofclimatechangeonmacrophytegrowth,biomassallocation,physiologyandnutrientstoichiometry.O23.HeleenDeroo(UGent).ProcessesofCH4emission inpaddysoilunderwater-saving irrigationmanagement.

EnvironmentII

O27.ValérieVanEesbeeck (SCK-CEN/UGent).Assessing themicrobiologicaldiversity in thecoolingwatersofanuclearresearchreactor.O28.VereLeybaert(KUL).Quantifyingprecipitationerrorsviasoilmoisturedataassimilation.O29.FrancisVanryckeghem(UGent).ScreeningoftheBelgianPartoftheNorthSeatowardsemergingorganicmicropollutants–comparisonoftwoSPE-techniquespriortoUHPLC-Orbitrap-HRMSanalysis.O30. Myrsini Sakarika (UA). Bridging resource recovery to reuse: microbes as next-generationfertilizer.

Key-enablingTechnologies

Keynote III. David Cannella (ULB). Light powered enzymatic degradation of lignocellulose: a newphoto-bio-catalysisforfuelsandchemicals.O24.PaulineHerpels(KUL).PhotoreactivecrosslinkingrevealsnoveldirectinteractionpartnersofthepersisterregulatorOBG.O25.ShuntaFutagami(VUB).TheEffectoftheLayerThicknessonthePerformanceofPorousLayeredRadiallyElongatedPillarAllayColumns.O26. Marie Versaevel (UMons). MatrXcell: improving cell culture assays with tunable hydrogelrigidities.

xi

PLENARYTALKS

ALBERTVANDENBERG(UTWENTE,NL)"FROMLABONACHIPTOORGANSONCHIP"

AlbertvandenBergobtainedhisPhD inappliedphysics in1988at theUniversityofTwente

(UTwente,Netherlands).Afterwards,heworkedonminiaturizedchemicalsensorsattheCSEMcenter

andIMTinstituteinNeuchatel(Switzerland).From1993until1999hewasresearchdirectoratMESA

(UTwente). After a part-time professor position “Biochemical Analysis Systems”, in 2000 he was

appointed as full professor on Miniaturized Systems for (Bio)Chemical Analysis in the faculty of

ElectricalEngineeringandpartoftheMESA+InstituteforNanotechnology(UTwente).Heisco-author

ofmore than 400 peer reviewed publications (H=51) and obtained several prestigous awards and

honorssuchastwoERCAdvanced(2008,2015)andERCProofofConcept(2011,2013)grants,Spinoza

prize(2009)andboardmemberoftheRoyalDutchAcademyofSciences(KNAW,2011).

LUCIASMIT"HOWTOMANAGEYOURCAREERASAPHD:CHALLENGESANDOPPORTUNITIES"

Dr. Lucia Smit is founder of Braingain, organization sociologist and certified professional co-

active coach (CPCC). She started her career as management consultant for companies and

governm4ents. Afterwards, as a member of the OESO work group she studied the careers of

researchers. From2007, Lucia guides professionals at developping their ambitions into theirwork

environmentandjobmarket,withastartingpointbeingtheenhancementoftheirself-knowledge.

(Braingain,https://www.braingain.be/)

FRÉDÉRICVANGANSBERGHE“ANDNOW,WHAT’SNEXT?”

Mr.FrédéricvanGansbergheisCEOatGalacticSA/NV,ChairmanatCompagnieduBoisSauvage

SAandPresidentatGalactic,Inc.Hegraduatedaselectro-mechanicalengineerfromECAMandgota

post-graduatedegreeinbiotechnologyatUCL.

Lecturesession

CHEMISTRYANDCHEMICALENGINEERING

2

USEOFACOUSTICSINMICROFLUIDICREACTORS

PierreGelin1,DominiqueMaes1,WimDeMalssche1

1VrijeUniversiteitBrussel,Belgium

Microfluidicsisanupcomingtechnologyinawidevarietyoffieldsincludingbiology,chemistry

andmedicine.InthesesmallchannelstheReynoldsnumberisverylow(Re<1).AttheselowRevalues,

viscositydominatesover inertiaensuringpredictable laminar flows,whichprovidesanexceptional

controloverthehandledfluidstreams.Thismakesitpossibletocontinuouslyproducehighlyuniform

droplets and particles. Continuous and high throughput production of micron size particles is

becomingmoreandmorerelevantevenintheindustry.Particlematurationisoftenoneofthelimiting

factor.Fortheproductionofpoly(lactic-co-glycolicacid)(PLGA)microparticlesforexample,therate

limitingstepisthesolventextractionfromtheproduceddroplettoyieldthefinalparticle.

Theuseofstandingacousticwavesinmicrofluidicdevicesgivesthepossibilitytoenhancethe

masstransferratefarbeyondtherateofdiffusion.Acousticstreamingisaflowphenomenonoccurring

whenanacousticwavepropagatesthroughanenclosedfluid.Thesestreamingflowstaketheformof

vorticestransversetothemicrofluidicchannel.However,whenparticlesaresuspendedinaliquida

secondphenomenonoccurs,namelyacousticradiation.Acousticradiationarisesfromthescattering

of the soundwave on the particle, this pushes it towards the pressure node. Both phenomena,

radiationandstreaming,havebeeninvestigatedinthiscontribution.Forlargeparticles(5µm)radiation

dominates,whileforsmallparticles(0.5µm)streamingdominates.

CFDsimulationshavebeenperformedtogetinsightinthestreamingvelocityfieldsinsideahalf

wavelengthchannelforachanneldepthof100µm.Dependingontheappliedenergytothechannel,

velocitiesofapproximately50µm/scouldbereached,showingaveryfastmixingoftwostreams.This

has also been shown experimentally with fluorescence experiments. Tracking of small particles

revealed that thevortex flowsbehaveaspredictedandconform that theboundaryconditionsare

realistic. Large particle displacement distances and velocities have also been investigated

experimentallyasafunctionoftheappliedenergyandfrequency.Veryfastfocussingofparticleson

thepressurenodehasbeenshown.Theuseofboththesephenomena isofgreatrelevance inthe

productiononmicroparticles.Streamingcanbeusedtoenhancetheformationoftheparticleswhile

radiationenablestofocusandfurtherconcentratethedesiredparticles.

Comm. Appl. Biol. Sci, Ghent University, 83/1, 2018

3

ANOVELDEVICEFORSTUDYINGNUCLEATIONOFCRYSTALSANDAGGREGATESINCONTROLLEDSHEARFLOW

SanderStroobants1,ManlyCallewaert1,SudhaChinnu1,

WimDeMalsche1,DominiqueMaes1


Nucleation, the first step in any first-order phase transition, is one of the most secretive

processes in chemistry, physics, materials science, biophysics and biotechnology; predictions of

nucleation rates and yields often deviate from reality by several orders ofmagnitude. It iswidelyacceptedthatthenucleationofproteincrystalsandorderedaggregatesfollowatwo-stepmechanism:

inafirststep,clustersofdenseproteinliquidareformed,withinwhich,inthesecondstep,ordered

nucleiform[1,2].Becauseitisthefirstphaseofthecrystallization/aggregationprocess,nucleation

controlsnumerouspropertiesoftheemergingphase.NucleationalsounderliestheformationoffibrilsandaggregatesobservedindiseasessuchasAlzheimerandcataract.

Boththeory[3]andexperiments [4]have indicatedthatshear flowduringcrystallizationwill

affect the nucleation rate, the size of the emerging phase and even polymorphism. To further

investigatethisinfluence,wehavedevelopedanewsetupthatwillenableustocreateacontrolledconstantshearflowprofileinasolution.Theequipmentconsistsofastationaryglassplate;afused

silica wafer with microfluidics channels connected to a rotation stage and Peltier elements for

temperature control. Tomonitor the crystallization an invertedmicroscope setup is usedwith theoptionofaddingfiltersintheopticalpathway,enablingfluorescentmicroscopy.

Thewaferwillrotatewithrespecttothestationaryglassplate,thiswillresultinaconstantshear

flow in the channels. Simulation performed with COMSOL Multiphysics 5.0 [5] confirm these

assumptions and characterize the deviations from the ideal profile at the walls and inlets. Thenucleationandgrowthcanbecontrolledbychangingtemperature,assuchthenucleationphasecan

beseparatedfromthegrowthphase,enablingthestudyoftheeffectofshearflowduringbothphases.

References

[1]D.Maesetal.,“Doproteincrystalsnucleatewithindenseliquidclusters?,”ActaCrystallogr.Sect.FStruct.Biol.Commun.,vol.71,no.Pt7,pp.815–822,Jul.2015.

[2]M.A.Vorontsova,D.Maes,andP.G.Vekilov,“Recentadvancesintheunderstandingoftwo-stepnucleationofproteincrystals,”FaradayDiscuss.,vol.179,no.0,pp.27–40,Jun.2015.

[3]F.MuraandA.Zaccone,“Effectsofshearflowonphasenucleationandcrystallization,”Phys.Rev.E,vol.93,no.4,p.042803,Apr.2016.

[4]A.Penkova,W.Pan,F.Hodjaoglu,andP.G.Vekilov,“NucleationofProteinCrystalsundertheInfluenceofSolutionShearFlow,”Ann.N.Y.Acad.Sci.,vol.1077,no.1,pp.214–231,Sep.2006.

[5]COMSOLMultiphysicsReferenceManual,version5.0.www.comsol.com:COMSOL,Inc.

4

CROSSINGTHEBORDER:ORGANICMOLECULESTRANSPORTCHLORIDEACROSSLIPIDBILAYERS

HennieValkenier1,GlennGrauwels1,AnthonyDavis2,KristinBartik1

1UniversitélibredeBruxelles,Belgium2UniversityofBristol,UnitedKingdom

Absenceormalfunctionofmembraneproteinswhich formanion channels are the causeof

several diseases. Cystic fibrosis is for instance caused by a deficiency in transport of chloride and

restoringthistransport isan importantchallenge.Syntheticanioncarriers,whichareembedded inlipidbilayers,havethepotential todothis.Thesemoleculescanbindananion,extract it fromthe

aqueousphase,transportitthroughthelipophilicinteriorofalipidbilayer,tothenreleaseitonthe

othersideofthemembrane[1].Chemicalpotentialenergy,intheformofaconcentrationgradient,is

thedrivingforceforthistransportprocess.

Wewillshowhowthegeometryofthebindingsiteoftransportersplaysanimportantrole.The

typesofinteractionsanddegreeofencapsulationoftheaniondeterminetheselectivity[2],whilethe

binding site canbeeitherpreorganised,orundergo largeconformational changesuponbindingofchloride[3].

The potential of synthetic receptors as anion carriers to restore chloride transport will be

demonstrated:ourmostactivecarrierreachesratesoftransportupto800Cl-/sbyasinglecarrier,as

measuredinliposomes[4]andgiantunilamellarvesicles(GUVs)[5].Anothercompoundwasfoundtorestoresignificantchloridetransportinepithelialcellswithoutshowingtoxicity,makingitapromising

candidatefortherapeuticalapplicationsandbiomedicalresearch[6].

References

[1]P.A.Gale,J.T.Davis,R.Quesada,Chem.Soc.Rev.2017,46,2497.

[2]M.Lisbjerg,H.Valkenier,B.M.Jessen,H.Al-Kerdi,A.P.Davis,M.Pittelkow,J.Am.Chem.Soc.2015,137,4948.

[3]H.Valkenier,C.M.Dias,C.P.Butts,A.P.Davis,Tetrahedron2017,73,4955.

[4]H.Valkenier,L.W.Judd,H.Li,S.Hussain,D.N.SheppardandA.P.Davis,J.Am.Chem.Soc.,2014,136,12507.

[5]H.Valkenier,N.LópezMora,A.KrosandA.P.Davis,Angew.Chem.Int.Ed.,2015,54,2137.

[6]H.Li,H.Valkenier,L.W.Judd,P.R.Brotherhood,S.Hussain,J.A.Cooper,O.Jurček,H.A.Sparkes,D.N.SheppardandA.P.Davis,NatChem,2016,8,24.


5

Lecturesession

AGRICULTUREANDFOODTECHNOLOGY

6

KEYNOTEI

NEWDEVELOPMENTSINSILICONNANOTECHNOLOGYFORHEALTHCAREAPPLICATIONS

TimStakenborg

IMEC,Leuven,Belgium

TimStakenborggraduated in1998at theUniversityof Leuvenasamaster inengineering in

chemistryandbiochemistry.AfterbeingaprojectengineeratAlcon-NovartisandhisPhDinmolecular

biology,hejoinedIMEC.

AtIMEC,Timiscurrentlyaprincipalmemberofthetechnicalstaffandgroupleaderinthelife

sciencetechnologydepartment.Hehasbeenaprojectleadinseveralfundedandindustrialresearch

projectsespeciallyfocusingonmergingbiologywithtechnology.Inhiscurrentroleofgroupleader,he

is heavily involved in the transfer and integration of key technical components in fully functionaldevices.


7

DESIGNERCELLULOSOMICSFORACUSTOMIZEDCONVERSIONOFLIGNOCELLULOSICBIOMASSTOVALUABLEBULKANDFINECHEMICALS

JulieVanderstraeten1,M.Fonseca1,A.Thibau1,F.Hooghe1,Y.Briers1

1LaboratoryforAppliedbiotechnology,DepartmentofBiotechnology,GhentUniversity,

ValentinVaerwyckweg1,9000,Gent,Belgium

INTRODUCTION

Lignocellulose isamajorcomponentofplantcellwallsand ispresent ina largeamountofwasteandsidestreams.Efficientuseofthiscomponentasaresourceisoneofthemostimportantchallengesinthefieldofindustrialbiotechnology.

Inlignocellulosicbiomass,celluloseissurroundedandtightlyconnectedtohemicelluloseandthe hemicellulose itself forms a compact matrix with lignin. The crystallinity of cellulose, thehydrophobicity of lignin, the encapsulation of cellulose and the general non-uniform three-dimensionallignocellulosicstructureexplainsthehighresistancetowardsenzymaticdegradationandassuchtofurtherconversion.

Cellulosomesaremulti-enzymecomplexesproducedbyspecialistmicro-organismsthatfeedon plant cell wall carbohydrates. These ‘nanomachines’ consist of two complementary structuralmodules,alargescaffoldin,whichcomprisesdifferentcohesinmodulesanddockingenzymes,whichcomprise a dockerin module and a catalytic enzyme. A specific interaction between the cohesindomainsofthescaffoldinandthedockerindomainsofthecatalyticenzymesensuresthearrangementofdifferentcatalyticactivitiesonasinglebackbone.Assuch,allcatalyticmoduleswithcomplementaryfunctionsareclosetoeachother,enhancingtheirsynergism(Artzietal.,2016).

Becausecellulosomesarehighlyefficientindegradinglignocellulosicbiomass,theseenzymecomplexeshaveattractedtheinteresttoadaptandusetheminthelignocellulosicconversionprocess.Thegoalistousetheknowledgeaboutnaturallyoccurringenzymecomplexestocreateengineereddesigner cellulosomeswith thepotential todegradeall lignocellulosic componentsand to convertthese degradation products into valuable end products (Vazana et al., 2012). Unlike nativecellulosomes, the use of designer cellulosomes enables control over the composition andarrangementoftheselectedenzymes(Figure1).

Figure 1: Designer cellulosomes in the conversion process of lignocellulose. Designer cellulosomescompriseascaffoldinbearingdifferentdockingenzymes.Celluloseandhemicellulosecomponentsoflignocellulose are degraded to C5 and C6 monomers, which can be converted into valuable end-products.

8

Severalresearchgroupsaroundtheworldhavesuccessfullycreateddesignercellulosomes.However,theuseofstandardcloningtechniquesmakestheconstructionprocesstediousandallowsthecreationandanalysisofonlyoneorafewdesignercellulosome(s)atatime.OurlabhasdevelopedaDNAassemblymethod,whichenablesustocreatemodularproteinsatahighrate.

Below,wedescribethefirsthigh-throughputconstructionofdockingenzymesandscaffoldinsandtheinitialactivityanalysisoftheassembleddockingenzymes.Specifically,wehavefocusedonthe construction of the first galactomannan degrading designer cellulosome, termed themannanosome.Currentlydesignedcellulosomeshavemainlyfocussedonthedegradationofcellulose(cellulosome)andthepredominanthemicelluloseofhardwoods,xylan(xylanosome).However,thelargesthemicellulosefractioninsoftwood,mannan,hasnotbeencoveredyet.Mannansarestoredinendospermwallsandvacuolesofseedsandvegetativetissues(e.g.ivorynut,coconut,coffeebean).Astheseplantsarewidelyusedinthefoodindustry,severalmannan-richmaterialsarediscardedaswasteinthefoodsupplychain(Yamabhaietal.,2016).

MATERIALANDMETHODS

Cloning

The DNA encoding enzymatically active domains, dockerins, cohesins and carbohydratebindingmodule(CBM)wasamplifiedfromgenomicDNAusingsequencespecificprimersandclonedinentryvectorstocreatethebuildingblocks.Inasecondassemblyreaction,thebuildingblockswerecombinedinasinglereactiontubeandassembledinapre-definedorderinadestinationvector.

Expressionandpurification

Toexpressandpurify the recombinantproteins,E. coliBL21(DE3)RIL cells, containing thecorrectvector,weregrownat37°CinLB,supplementedwiththeappropriateantibioticsand2mMCaCl2.WhenA600reached0.6,isopropylthio-β-galactosidewasaddedtoafinalconcentrationof0.5mM. After overnight incubation at 16°C and 180 rpm, cells were harvested by centrifugation (5minutes,20000g)and resuspended inTris-buffered saline (TBS)pH7,4, supplementedwith2mMCaCl2.Cellswerethendisruptedbyaddinglysozymetoafinalconcentrationof1mg/ml,performingthreefreeze-thawcyclesandsonication.Proteinpurificationwasperformedbyimmobilizedmetal-ionaffinitychromatographyonaHisGraviTrap™column(GEHealthcare).

Enzymaticactivityassays

Mannanase and galactosidase activity was determined quantitatively by measuring thereducingsugarsreleasedfrommannanorgalactomannanbythedinitrosalicylicacid(DNS)method.An assay mixture consisted of 1 ml 0.5 % (w/v) mannan or galactomannan in 0.2 M Sørensen’sphosphate buffer (pH 7.3) and 1 µl docking enzyme (equimolar concentrations). At specific timepoints, 100 µl sampleswere taken,mixedwith 100 µl DNS reagent and incubated at 100°C for 5minutes. After cooling down to room temperature, the degree of enzymatic hydrolysis wasdeterminedspectrophotometricallybymeasuringtheabsorbanceat540nm.Thereleasedreducingsugarsweredeterminedagainstastandardcurveobtainedwithmannose.

RESULTS

Asour in-housedevelopedmodular assemblymethodallowsus to createDNA sequencesencodingmodularproteinsinhigh-throughput,wewereabletosystematicallyanalyseseveraldockingenzymeandscaffoldinparameters.TheworkflowshowninFigure2wasimplemented.

Thefirststepwastoconvertmodulesthatareusedtobuildupcellulosomesintocompatiblebuilding blocks (Figure 2, Step I). Four cohesin-dockerin pairs and one CBM were selected fromnaturallyoccurringcellulosome-producingorganismstoserveasthenon-catalyticmodules.Thethree


9

selectedcatalyticactivemodulesareall(galacto)mannanactiveenzymes.ß-1-4-mannanasecatalysesthe randomhydrolysis of ß-1,4 glycosidic bonds of themannanbackbone andß-1,4mannosidasereleasesmannoseresiduesfromß-1,4-linkedmannosides.BothenzymesoriginatefromThermobifidafusca, a natural plant cellwall degrader. α-(1,6)-linkedD-galactose side chains are released by α-galactosidase,originatingfromClostridiumcellullolyticum,anaturalcellulosomeproducer.

Thesecondstepwastoassemblethesemodulesintospecificscaffoldinanddockingenzymesequences(Figure2,StepII)).Weaimedfortheconstructionof24rationallydesignedscaffoldinsand19rationallydesigneddockingenzymesinonerun.ThescaffoldinsallcontainedonetofouroftheselectedcohesinsandoneornoCBM.Forthedockingenzymeswecombinedoneortwoofthethreedifferent catalyticmodules acting on galactomannanwith one out of the four different dockerinsavailableinourcollection.Cloneanalysisprovedthattheusedmethodhasa96%efficiency.

Afterassembly,allconstructswereexpressedinE.coliBL21(DE3)RILcellsandpurifiedbyHis-tagpurification.

Figure2:Adoptedworkflow.Westartfromaselectionofcellulosomebuildingblocks.Thesebuildingblocks are assembled into the coding sequences for docking enzymes and scaffoldins in high-throughput. Systematic parameter analysis then leads us to the determination of optimal dockingenzymeandscaffoldincombination.

Basedonourhigh flexibility in theconstructionofdifferentmodulardockingenzymes,wewereabletoinvestigatetheeffectofdifferentparametersontheperformanceofdockingenzymes(Figure2,StepIII).TheenzymaticactivitywasmeasuredbyDNSanalysis.Specifically,weanalysedtheeffectof thedockerinoriginand the specificorderof themodules. In addition,we compared the

10

activity of a bicatalytic docking enzyme to a mixture of two monocatalytic enzymes with thecorrespondingcatalyticmodules.Figure3showsaselectionofobtainedactivitypatterns.

Thetypeofdockerinthatischosentoconstructadockingenzymehasnosignificanteffectontheactivityofthedockingenzyme(p=0.1).Allvariantsreleasethesameamountofreducingsugarsovertime(Figure3A).

Figure3Bshowstheimportanceofthepositionofdifferentmodulesinsideafusionprotein.For thebicatalyticdockingenzymecombiningtheusedgalactosidaseandmannanaseenzyme, theoptimalconfigurationconsistsofgalactosidaseattheN-terminusandmannanaseattheC-terminus,linkedwithacentraldockerindomain(p<0.05).

Figure3Cindicatesthattwomonocatalyticdockingenzymeshaveahigheractivitythenonebicatalyticdockingenzymecomposedofthesameenzymaticallyactivedomains.

Figure3:Dockingenzymeactivityanalysis.Thecurvesshowdegradationbytheconstructeddockingenzymesrepresentedschematicallynexttoeachgraph.KeytotheusedsymbolscanbefoundinFigure2.A:Degradationpatternsforthreedockingenzymescomposedofthesamecatalyticmodules,butdifferentdockerins.B:Degradationpatternsfortwodockingenzymescomposedofthesamecatalyticmodulesanddockerin,butassembledindifferentorders.C:Activitypatternofabicatalyticdockingenzymecomparedtoamixtureoftwomonocatalyticdockingenzymeswiththecorrespondingcatalyticmodules.


11

DISCUSSION

Our first results indicate that preliminary research is crucial prior to the constructionof adesigner cellulosome. There is a high number of variables that can influence the performance ofdesignercellulosomes.Foreachspecificcollectionofenzymes,optimization isrequired inordertoassemblethemostoptimalsetofdockingenzymesontheidealscaffoldin.

Forexample,weobservedthatchangingthepositionofdifferentmoduleshasalargeeffectontheactivityofthefulldockingenzyme.Arrangementofdockingenzymesmustguaranteeoptimalfoldingofeachmoduletoensurethatdockerinsretainspecificbindingtotheirmatchingcohesinsandcatalytic modules preserve their hydrolytic properties. On top of that, earlier research hasdemonstrated the importance of the relative position of different docking enzymes within thescaffoldin(Sternetal,2015).

Theseessentialpreliminaryassays, inwhichanextensivenumberofdockingenzymes(andscaffoldins)arecompared,call foraquickandeasyassemblymethod.Ourmodularapproachwasproventobe96%efficient,thuslendingitselfperfectlyfortheconstructionofdesignercellulosomes,themostadvancedmodularproteinspresentinnature.

CONCLUSION

We have efficiently implemented a straight-forward workflow in which high-throughputassemblywasextendedtosystematicparameteranalysis.Assuch,wewereabletoanalyseseveraldockingenzymevariables.Thisisanimportantstepincellulosomeresearch,ascurrentlyusedcloningtechniquesallowtheconstructionandanalysisofonlyafewdockingenzymesorscaffoldinsatthesametime.

ACKNOWLEDGEMENTS

J. V. has received a doctoral scholarship of the Bijzonder Onderzoeksfonds of Ghent University(BOF17/DOC/086).

REFERENCES

ArtziL,BayerEA,MoraïsS.Cellulosomes:bacterialnanomachinesfordismantlingplantpolysaccharides.NatRevMicrobiol.2017;15:83-95

Stern J,KahnA,VazanaY,ShamshoumM,MoraïsS, LamedR,BayerEA.Significanceof relativepositionofcellulases indesignercellulosomesforoptimizedcellulolysis.PLoSONE.2015;10(5):e0127326

VazanaY,MoraïsS,BarakY, LamedR,BayerEA.Designercellulosomes forenhancedhydrolysisof cellulosic substrates.MethodsEnzymol.2012;510:429-52.

Yamabhay M, Sak-Ubol S, Srila W, Haltrich D. Mannan biotechnology: from biofuels to health. Crit Rev Biotechnol.2016;36:32-42

12

THEEFFECTOFTOWTRADITIONALMOROCCAN'SCOOKINGMETHODSONOMEGA-6/OMEGA-3ANDFATTYACIDSHYPO/HYPERCHOLESTEROLEMICRATIOSOFBENI-GUIL

LAMBMEATBREEDINGINNORTHEASTOFMOROCCO

BelhajK1,2,MansouriF1,SindicM2,FauconnierM-L3,BoukhartaM4,SerghiniCaidH.1,ElamraniA.1

1LaboratoiredeBiologiedesplantesetdesmicro-organismes,FacultédesSciences,

UniversitéMohamedIer,Oujda;Maroc.2LaboratoireQualitéetSécuritédesProduitsAlimentaires,GemblouxAgro-BioTech,

UniversitédeLiège;Belgique.3LaboratoiredeChimiegénéraleetOrganique,GemblouxAgroBio-Tech,

UniversitédeLiége;Belgique.4InstitutSupérieurIndustrielagronomique,Huy,Belgique.

INTRODUCTION

Meatcookingisessentialtoimprovemeatconsumer’spalatability.Infact,thermaltreatmentsimprovehygienicqualityofmeat(deactivationandeliminationpathogenicmicroorganisms),increaseitsshelflife,enhanceitsorganolepticqualityflavor,tendernessandcolor(Modzelewska-Kapitułaetal., 2012). However,meat cookingmodifies chemical composition (fatty acids, amino acids, …) bylosseswithaconsequentchangeofthemeatnutritionalvalue.Fatandfattyacidscompositionofmeatareaveryimportantcretariaforagoodhealth(Woodetal.,2004).Also,it'sveryimporanttoknowthatanimbalancedpolyunsaturatedfattyacids(PUFA)/saturatedfattyacids(SFA)andahighn-6/n-3ratiosmaycauseserioushealthproblemssuchascardiovasculardisease,obesityandcertaincancers(Simopoulos,2002,Simopoulos,2016).Moreover,thoseparametersareveryinfluencedbycookingmethods(Flakemoreetal.,2017).Theobjectiveofthisstudyistocompareanddeterminetheeffectof twoMoroccancookingmethods (BarbecueandTajine)onnutritionalqualityofBéni-guil's lambmeatespeciallytheomega-6/omega-3andhypo/hypercholesterolemicratios.

MATERIALANDMETHODS

ThetrialswereconductedontheLongissimusLumborum(LL)ofBéni-guilslambmeatobtained24hpostmortem(onemuscleforeachcarcass).ThepHultimewasanalyzedtoavoidanomalycarcass,byusingaportablepHmeter(pH/Cond340iWTW,Weilheim,Germany),equippedwithapenetrationelectrode.Then,wedividedthesamplesintothreegroupsaccordingtocookingmethods(30samplesforeachgroup).A totalof90 sampleswereobtained includingonegroupusedas control.Meats,denudedof external fatswere cooked, to a core temperatureof 70 °C, using twoMoroccanmeatcookingmethods:grilledusinganelectricalgriddle(drycooking)andTajineusing20mlofwater(moistcooking). After cooking the samples were freeized, lyophilised, crushed and stored at -20 C° forsubsuquentfattyacidsanalysis.Fattyacidsprofile

The intramusculare fat was extracted according to Bligh and Dyer (1959) using achloroform/methanol/watermixture(2/1/1;v/v/v).Thelipidextractwasmethylatedandconvertedtofattyacidmethylesters(FAME)accordingtothemethoddescribedby(BenMoumenetal.,2015).SeparationoftheFAMEwasperformedonanAgilentgaschromatograph(GC)(HP6890series,AgilentTechnologies,USA),equippedwithanOmegawaxcapillarycolumn(30m×0.25mm×0.25μmfilmthickness)fromSupelco(Bellefonte,PA,USA)andaflameionisationdetector(FID).Helium(99.999%,


13

AirLiquide,Liège,Belgium)wasusedasthecarriergas,ataflowrateof1.7mlmin-1.Thetemperatureoftheinjectoranddetectorweresetat150and250°C,respectively,andtheoventemperaturewasset at 210 °C. The injection volume was 1 µl, in splitless mode. A FAME standard, containing 37components(Supelco,Bellefonte,PA,USA),wasusedtoidentifytheindividualpeaks.Statisticalanalysis

Theanalysisofeffect'scookingmethods(BarbecueandTajine)onnutritionalvaluewascarriedouton triplicate foreachsampleandeachparameter.All statisticalanalysiswereconductedusingStatisticalPackagefortheSocialSciences(SPSSforWindows,version20,SPSSInc.,Chicago,IL,USA).ThenormaldistributionwasverifiedaccordingtoShapiroWilktest.One-wayANOVAstatisticalanalysisandDuncan'spost-hoctestwasusedformeanscomparison,thedifferencewasconsideredsignificant(atP<0.05).RESULTS

ThemeanvalueofpH24ofLLusedinthisstudywas5.66.Theresultsshowedthat100gofrawmeatcontains6.14%(table1)ofintramuscularFat(IMF).TheeffectofcookingmethodsshowedthatBarbecuecookingmethodpreserveditsIMFthanTajinecookingmethodwithacontentof5.31%and4.83%(table1)offreshmeat,respectively.TheeffectofcookingmethodsonfattyacidsratiosandsumsofBéni-guil'slambmeatproducedineasternMoroccoaresummarizedinTable1.Table1:Cookingmethodseffectoncooking loss, IMFandfattyacidsratiosandsumsofBéni-guil'slambmeat

Fattyacids(%) Rawmeat CookingmethodBarbecue Tajine

SFA 46,67a 42,21b 44,23cPUFA 9,47a 21,93b 16,36cUFA 53,32a 57,78b 55,76cUFA/SFA 1,14a 1,39b 1,27cPUFA/SFA 0,20a 0,54b 0,38cn-6 7,79a 18,70b 14,15cn-3 1,46a 2,69b 1,81an-6/n-3 5,70a 7,92a 11,45bh/H 1,62a 2,17b 1,98bCookingloss --- 21.14a 27.06b

IMF 6.14a 5.31b 4.83c

pH 5.66 --- ---Significantdifferencesareshownbydifferentletters(a-c)SFA:Saturatedfattyacids;PUFA:Polyunsaturatedfattyacids;UFA:Unsaturatedfattyacids;n-6:n-6PUFA;n-3:n-3PUFAh/H=(18:1n9c+18:2ω6+20:4ω6+18:3ω3+20:5ω3+22:5ω3+22:6ω3)/(14:0+16:0).

DISCUSSION

Theresult forcooking lossshowedthatthermaltreatments inducedan internal juice loss inmeat.Thesmallest internal juicelossvaluewasrecordedinbarbecue(21.14%)thanTajinecookingmethod (27.06%). So, the first cookingmethod holds back its internal juice than the second one.Similarresultwasreportedbyotherauthors(Lorenzoetal.,2015).Also,thebarbecuecookingmethodallowshighpreservationofIMFthanTajinewith6.14%,5.31%and4.83%forfreshmeat,barbecue

14

and Tajine respectively. Consequently, Barbecuemethod guaranteed a better juiciness than Tajinemethod.GC-FIDanalysis revealedasignificanteffectofcookingmethodson fattyacidsprofileandratios(Table1).Thesaturatedfattyacids(SFA),polyunsaturatedfattyacids(PUFA),andunsaturatedfattyacids(UFA)percentageswereveryinfluencedbycookingmethods.BarbecuecookingmethodsimprovesmeatnutritionalvaluebyincreasingPUFAfraction.So,theincreaseofPUFA/SFAratiorangedbetween0.20,0.38and0.54forRawmeat,Tajineandbarbecuerespectively.Similaroutcomeswerefind by other authors (Ramamurti, 1986, Domínguez et al., 2015). This implies an increase, ofhypocholesterolemicfattyacidsandthereforeanincreaseoftheh/Hcholesterolemicratiowhichhelpsreducecardiovasculardisease.Theh/HratiofoundforBarbecuecookingmethod(2.17)washigherthan those reported inSpanish (Campoetal., 2013))andAustralian (Flakemoreetal., 2017) lambmeats.

Thisresultcanbeexplainedbycookinganddriploss,becausetheproportionalchangeinfattyacidscompositionandthisisconsistentwiththeresultsreportedbyRamamurti(1986).Additionally,then-6/n-3ratiowasaffectedbycookingmethods.Theresultsofn-6/n-3ratioinourstudyishigherthanthosereportedinbeef(Alfaiaetal.,2010)andinfoal(Domínguezetal.,2015)meats.

CONCLUSION

ForthefirsttimewestudiedtheeffectofdifferentMoroccan'scookingmethodsonnutritionalquality of the Béni-Guil Protected Geographical Indication meat from eastern Morocco and weconcludethat froma taste,nutritionalanddietaryqualitypointofviewbarbecuecookingmethodallowsbetterpreservationofmeatnutritionalqualitythanTajineanditisthecookingmethodtoberecommended.REFERENCESALFAIA,C.M.,ALVES,S.P.,LOPES,A.F.,FERNANDES,M.J.,COSTA,A.S.,FONTES,C.M.,CASTRO,M.L.,BESSA,R.J.&PRATES,

J.A.2010.Effectofcookingmethodsonfattyacids,conjugatedisomersoflinoleicacidandnutritionalqualityofbeefintramuscularfat.MeatScience,84,769-777.

BENMOUMEN,A.,MANSOURI,F.,RICHARD,G.,ABID,M.,FAUCONNIER,M.L.,SINDIC,M.,ELAMRANI,A.&SERGHINICAID,H.2015.Biochemicalcharacterisationoftheseedoilsoffoursafflower(Carthamustinctorius)varietiesgrowninnorth-easternofMorocco.InternationalJournalofFoodScience&Technology,50,804-810.

BLIGH,E.G.&DYER,W.J.1959.Arapidmethodoftotallipidextractionandpurification.Canadianjournalofbiochemistryandphysiology,37,911-917.

CAMPO,M.,MUELA,E.,OLLETA,J.,MORENO,L.,SANTALIESTRA-PASÍAS,A.,MESANA,M.&SAÑUDO,C.2013.InfluenceofcookingmethodonthenutrientcompositionofSpanishlightlamb.Journaloffoodcompositionandanalysis,31,185-190.

DOMÍNGUEZ,R.,BORRAJO,P.&LORENZO, J.M.2015.Theeffectofcookingmethodsonnutritionalvalueof foalmeat.JournaloffoodCompositionandAnalysis,43,61-67.

FLAKEMORE, A. R.,MALAU-ADULI, B. S., NICHOLS, P. D. &MALAU-ADULI, A. E. O. 2017. Omega-3 fatty acids, nutrientretentionvalues,andsensorymeateatingqualityincookedandrawAustralianlamb.Meatscience,123,79-87.

LORENZO,J.M.,CITTADINI,A.,MUNEKATA,P.E.&DOMÍNGUEZ,R.2015.Physicochemicalpropertiesoffoalmeatasaffectedbycookingmethods.Meatscience,108,50-54.

MODZELEWSKA-KAPITUŁA,M.,DĄBROWSKA,E., JANKOWSKA,B.,KWIATKOWSKA,A.&CIERACH,M.2012.Theeffectofmuscle,cookingmethodandfinalinternaltemperatureonqualityparametersofbeefroast.Meatscience,91,195-202.

RAMAMURTI,K.1986.Effectsofcooking,freezingandfrozenstorageonthefattyacidPatternofgroundbeef.LWTFoodSci.Technol.,19,481.

SIMOPOULOS, A. P. 2002. The importance of the ratio of omega-6/omega-3 essential fatty acids. Biomedicine &pharmacotherapy,56,365-379.

SIMOPOULOS,A.P.2016.Anincreaseintheomega-6/omega-3fattyacidratioincreasestheriskforobesity.Nutrients,8,128.

WOOD,J.,RICHARDSON,R.,NUTE,G.,FISHER,A.,CAMPO,M.,KASAPIDOU,E.,SHEARD,P.&ENSER,M.2004.Effectsoffattyacidsonmeatquality:areview.Meatscience,66,21-32.


15

MICROBIALDETOXIFICATIONOFTHEMYCOTOXINDEOXYNIVALENOL

IlseVanhoutte1,KrisAudernaert1,LeenDeGelder1

1GhentUniversity,Belgium.

Mycotoxins are secondarymetabolites producedby fungi.When these fungi arepresent on

agricultural commodities, they canposeahigh risk toanimalandhumanhealth.As theEuropean

Commissionimposesstrictregulationsonmaximumlevelsofseveralmycotoxins,itisaprerequisitefor farmers tomeet these regulations toavoidcomplete lossofacontaminatedbatch. Inanagro-

ecosystem, farmers try to avoid fungal infection and the occurrence of mycotoxins in their

commoditiesviapreventionandinterventionapproaches,butoftenfailincompletelyeliminatingthe

risk. Therefore, detoxification strategies are needed such as the application of binders or theapplicationofmicroorganismsabletodetoxifythesemycotoxins.

Our project focuses on the microbial detoxification of deoxynivalenol (DON), a worldwide

prevalent mycotoxin. Microorganisms were screened for their biotransformation capacity of DONthroughnon-targetedLC-MS/MS.Furthermore,abioassayusingtheaquaticplantLemnaminorwas

developedtoassesstheresidualtoxicityofDONmetabolitesafterbiotransformation.Pursuingthis

approach,twomixedcultureswereobtained,originatingfromsoilandactivatedsludge,capableof

biotransformingDONintotheepimerofDONandtheepimerofDOM-1,bothconveyingnoresidualtoxicityfortheLemnaminorplant.

16

THEIMPACTOFCONTROLLEDSTEEPINGANDGERMINATIONOFWHEATONHYDROLYSISOFPHYTATEANDARABINOXYLANANDONMINERALBIO-ACCESSIBILITY

ElienLemmens1,NielsDeBrier1,PeterGoos2,ErikSmolders3,JanDelcour1

1Laboratoryofchemistryandbiochemistry,KULeuven.

2DivisionofMechatronics,BiostatisticsandSensors,KULeuven.3DivisionofSoilandWaterManagement,KULeuven.

Wheatwholegrain foodproductsdeserve tobean importantpartofahealthydietas they

reducetheriskofdevelopingdiet-relateddisorderssuchasobesity,typeIIdiabetes,cardiovascular

diseaseandcancer.Theirhealthbenefitsaremainlyascribedto thehighconcentrationsofdietaryfiberandotherbio-activessuchasBvitaminsandminerals inbran.However,thesebio-activesare

entrappedincellswithrigidwallswhichwithstandconventionalmillinganddigestionbythehuman

enzymearsenal.Moreover,wheatphosphorusismainlystoredasphyticacidwhichchelatesdivalentcationssuchasironandzinc.Asaconsequence,thebio-accessibilityofwheatmineralsonlyamounts

to5to25%.Hydrolyticenzymessuchasxylanaseandphytaseareactivatedanddenovosynthesized

inwheataleuronecellstofuelseedlinggrowth.Xylanaseshydrolyzeandsolubilizearabinoxylan(AX),

themainaleuronecellwalldietaryfibre(DF)component.Atthesametime,theincreaseinphytaseactivityduringgerminationmakesphosphate,mineralelementsandmyo-inositolavailableforplant

growthanddevelopment.Inafoodsystemcontext,thisisimportantsinceopeningwheataleurone

cellwallsandbreakingdownphytatestructuresby(partial)germinationmaywellresultinagreater

wheatmineralaccessibility inthehumangastro-intestinaltract.WeherestudiedhowsteepingandgerminationaffectthewheatDFcomplexandincreasethebio-accessibilityofminerals.

Duringsteeping(29h)andgermination(120h)ofwheatgrainsat15°Cthexylanaseandphytase

activity levels increasedabout five times.As a result, the contentofwater extractableAX (WEAX)

doubledduringsteepingandgermination[from0.5%to1.0%ofinitialdrymatter(idm)].However,thephytatecontentdecreasedonlyfrom0.96%to0.81%ofidm.Theironbio-accessibilityincreasedfrom

7% in regularwheat to 15% in germinatedwheat. In a next step,we used an I-optimal design of

experimentstomaximizetheimpactofcontrolledsteeping(3-36h;15-50°C)andgermination(6-120h;15-30°C)onAXandphytatehydrolysisinwheatand,hence,improvethelevelsofsolubleDFand

elemental bio-accessibility. The WEAX content was mainly determined by the main effect of

germinationtimeandthequadraticeffectofsteepingtemperature.Whenwheatgrainsweresteeped

at24°Cfor36handgerminatedat22°Cfor120h,theWEAXcontentinwheatincreasedfrom0.5%to1.3%of idmandthephytatecontentdecreasedfrom0.96%to0.66%of idm.Weconcludethat

controlled steeping and germination of wheat offers novel raw materials which can be used for

manufacturing cereal products with different DF properties and an improved bio-accessibility of

minerals.


17

THEIMMEDIATERESPONSEOFTOMATOANDRICETOTEPHRADEPOSITION:ANEXPERIMENTALSTUDY

IngaTarasenko1,StanleyLutts2,PierreDelmelle1

1EnvironmentalSciences,EarthandLifeInstitute,UniversitéCatholiquedeLouvain,

Louvain-la-Neuve,Belgium.2Agronomy,EarthandLifeInstitute,UniversitéCatholiquedeLouvain,Louvain-la-Neuve,Belgium.

During a volcanic explosive eruption, tephra particlesmay interact with terrestrial plants in

variousways,possiblyresultingindetrimentaleffectssuchasalterationofleafphysiologyandreducedplantgrowth.Inagriculturalareas,tephradepositioncanleadtoseverecropdamagesandsignificant

economic losses. Themagnitude of tephra impacts on vegetation ismodulated by several tephra

propertiesactingincombination;theseincludethickness,porosity,grainsizedistribution,colourand

composition.Whilemanystudieshighlightthemechanicalimpactofheavytephrafallonvegetation,littleisknownaboutthephysiologicalresponseofplantleavestoathintephradeposit.

Here, we present the results of an experimental study aimed at better understanding the

physiologicalconsequencesoftephra-leaf interaction.Tephrafromthe1991Mt.Pinatuboeruption

wasappliedtoriceandtomatoplantsgrownincontrolledconditionsinaphytotron.Twoscenariosoftephradepositionwereconsidered:aone-eventscenario(S1)inwhichagivenamountoftephrawas

appliedtoplantsonce,andamultiple-eventscenario(S2) inwhichtephrawasappliedthreetimes

overaperiodoftwoweeks. Inbothcases,thetotalamountoftephrareceivedbytomatoandrice

plants was ~90 g and ~180 g, respectively. Each experiment lasted two weeks and involved sixreplicates.

Applicationoftephraontomatoandriceplantsresultedinleafwilting,twistingandfolding.A

colourchangewasalsonoticedandplantgrowthwasaffected.Inbothtephradepositionscenarios,theshootbiomassoftomatoandriceattheendoftheexperimentwasreducedbyupto~30and

~50%,respectively,whencomparedtocontrolplantsthatdidnotreceivetephra.InS2,thetephra-

coveredriceplantsshoweda~30%lowerrateofphotosyntheticactivitythanthecontrolplantsthat

didnotreceivetephraduringthetwoweeksofexperiment.InS1,therateofphotosyntheticactivityinricedecreasedby~40%withinonedayoftephraapplication;itremainedsignificantlyloweruntil

the firstweek of the experiment. The rate of photosynthetic activitymeasured for tomato leaves

coveredbytephrawasalwayslower(byafactorofupto2)thanthatmeasuredforcontrolplants.Leaf

temperature in both the rice and tomato experiments increasedby ~10%.Our preliminary resultsalreadysuggestvariousphysiologicaleffectsintomatoandriceplantsaffectedbyatephradeposit.

Additionalmeasurementsonleafchlorophyllandsugarcontents,plantwateruseefficiency,stomatal

conductance,internalCO2concentrationandtranspirationratewillbepresented.

18

CATABOLICSELECTABLEMARKEREASESGENOMEEDITINGINYARROWIALIPOLYTICA

MarieVandermies1,OliviaDenies1,Jean-MarcNicaud2,PatrickFickers1

1UniversitédeLiège2INRA-AgroParisTech

Selectable markers are a central component of genome edition technologies. In the yeast

Yarrowia lipolytica, thesemarkersaretraditionallybasedonantibioticresistance(hygromycinB)orauxotrophy (e.g., leucine, uracil). However, the use of the former is impaired by a high level of

spontaneous resistance, and the use of the latter by continuous complementation of the culture

mediumorrestorationofprototrophytothestrains.Asanalternative,genesrelatedtothecatabolism

ofcarbonsources,or“catabolicselectablemarkers”,presenttheadvantageofnotbeinginvolvedinessentialmetabolicpathways.TherecentlyidentifiedEYK1,encodingerythrulosekinase,canserveas

anefficientcatabolicselectablemarkerforgenomeeditinginY.lipolytica.Comparedtoauxotrophic

markerssuchasURA3andLEU2,EYK1increasesthegrowthrateoftransformantsonselectivemedium

andtheefficiencyofgenomeedition.TheutilityofthemarkerEYK1inareplicativevectorwasalsodemonstrated. Besides, the cloning-free strategy developed here simplifies the construction of

disruptioncassettesforgenomeeditinginY.lipolytica.


19

Lecturesession

BIOMATHEMATICS

20

PROTEININTRINSICDISORDER:ADOUBLE-EDGEDSWORD?

MauricioMacossayCastillo1,MainakGuharoy1,GiulioMarvelli1,PeterTompa1,2,ShoshanaJ.Wodak1

1VIB-VUBCenterforStructuralBiology,VrijeUniversiteitBrussel

2InstituteofEnzymology,HungarianAcademyofSciences

Intrinsicallydisorderedproteins and regions thereof (IDPs/IDRs) adopthighlyheterogeneous

anddynamicconformationalstates.Thesepropertiesenablethemtoperformimportantregulatory

andsignalingrolesinthecell.Moreover,IDPs/IDRswereshowntoengageinweakinteractionswithotherproteins.Onemaythereforewondertheextenttowhichevolutionoperatestolimitthenon-

functionalinteractionsthatIDP/IDRswouldforminthecell.Sinceaccordingtothelawofmassaction,

such interactions should increase with the protein abundance level, we investigated how various

intrinsic properties of S. cerevisiae proteins, in particular those of IDPs/IDRs, vary with proteinabundance.Inaddition,weanalyzetherelationsbetweentheintrinsicpropertiesoftheproteins,their

abundancelevelsandthepatternsofphysicalandfunctional interactionstheyform,asdetectedin

oneoftherecenthighconfidenceprotein-proteininteractionnetworkinvolvingyeastsolubleproteins.

ResultsshowthatIDRsofmoreabundantproteinstendtobemorepolar,lesshydrophobicand

lesspronetobindtootherproteins,thanthoseoflowabundanceproteins,justasforsurfaceresidues

inglobularproteins.Incontrast,theseproteinsfeaturealargernumberofdistinctstructured(Pfam)

domains.ThissuggeststhatevolutionreducesthelikelihoodofhighabundanceproteinstoengageinspuriousinteractionsbyincreasingthesolubilityofIDPs/IDRs,whilelikelyallowingthemtoengagein

morefunctionallydiverseinteractionsthroughtheirstructureddomains.Analysisoftheyeastprotein-

interaction network then confirmed earlier findings that the number of interacting partners per

protein,increaseswithitsabundancelevel.Furthermore,usingasemanticsimilaritymeasureofthegeneontology(GO)annotations,andgeneco-expressionlevels,wefindthathigh-abundanceproteins

tendtohavemorefunctionallydiverseinteractionpartnersthatarealsolesscoherentlyexpressedbut

that IDPs/IDRs were notmore prone to having such partners than globular proteins. Our current

interpretationofthesefindingsisthatevolutionstrikesadelicatebalancebetweenenablingdiversefunctionalroleswhileminimizingspuriousinteractions.Itdoessonotonlybyregulatingtheprotein

abundance levels and their cellular localization, but also by modulating the intrinsic molecular

propertiesoftheproteins,inbothglobularandintrinsicallydisorderedregions.


21

IDENTIFICATIONOFCAUSALMUTATIONSCONFERRINGHIGH-ETHANOLTOLERANCEINESCHERICHIACOLI

ThomasSchalck1,2,T.Swings1,2,C.Bonte1,B.VandenBergh1,2&J.Michiels1,2

1CentreofMicrobialandPlantGenetics,K.U.Leuven,KasteelparkArenberg20,Heverlee,Belgium2VIB-KULeuvenCenterforMicrobiology

INTRODUCTION&RESEARCHSCOPE

Fordecades,fossiloilwasdrivingthedevelopmentofmanyindustries.Asaresult,petroleumoveruse not only depleted our oil reserves to alarmingly low levels but also lumbers us withenvironmentalproblems.Fuelingvehiclesandsupplyingtheindustrywithessential,chemical‘buildingblocks’canhowever,alsobedonebyrelyingonrenewableenergysourceswhichwouldavoidtheadverseeffectsofcrudeoil.Oneofthesefossiloilalternativesthatisalreadywidelyintegratedinoursociety is bioethanol. Mainly, because this alcohol has extraordinary properties that meet therequirementsforusageasafuelandasabuildingblock inpetrochemical industry.Theproductionprocess of bioethanol involves the fermentation of sugar-rich plant-or algae-derived biomass byethanologenic microorganisms, including the yeast Saccharomyces cerevisiae or bacteria, such asEscherichiacoliandZymomonasmobilis.Inbrief,thisconversionofsugarstoethanolhasanetzerocarbonfootprintasthealgaeorcrops, includingmaize,wheatandswitchgrass,photosyntheticallycapture the carbon dioxide released during combustion of bioethanol in vehicles and duringproductionofplasticsinthepolymerindustry.

Traditionally,microbial fermentationprocessesarewellestablished inthefood industrytoproducealcoholicbeveragessuchasbeerorwine.Incontrast,industrialbioethanolcanbeconsideredasabulkproductanddiffersfromdrinkingalcoholintermsofprice(around70UScents/liter),volumeandgrade(sometimesupto99%foranhydrousbioethanol)(Fairley,2011).Therefore,highethanoltitersjustaftermicrobialfermentationarepreferredasenergyconsumptionduringdistillationcouldbe limited to keep the overall bioethanol production process profitable. However, themaximumethanol content in the fermentationbatch is inevitably determinedby themicrobe’s tolerance toethanol. Indeed,ethanolandotheralcoholsexert toxiceffectson themicrobial cell envelopeandothercellularprocesses.Therefore,strategiestoprotectmicroorganismsagainstlethalethanolstresscouldholdthecluetoimprovebioethanolyields(Dunlop,2011andLingetal.2014).Althoughthisrelationship between productivity and tolerance may seem trivial at first glance, the geneticbackground underlying the tolerance phenotype is extremely complex and remains incompletelyunderstood.

ANEVOLUTION-DRIVEN INSILICOMODELTOUNDERSTANDTHEMECHANISMSBEHINDETHANOLTOLERANCE

Inpreviouswork,ourlabusedexperimentalevolutiontoincreaseethanoltoleranceinE.coli.The long-term evolution experiment under gradually increasing ethanol concentrations yieldedseveral highly ethanol tolerant strain. Subsequently, these evolved strains were sequenced andrevealedaglimpseofthemajor,cellularmechanismsresponsiblefortheincreaseinethanoltolerance.Aidedby thesedata,wenow start tounderstandhow themodel bacterium,E. coli, adapts to anextremelystressfulenvironment(Swingsetal.2017a).Indepthstudyofthesehypertolerantstrainsrevealedastrongcorrelationbetweenmutation frequencyandthepropensity to increaseethanoltoleranceinawaythatonlystrainsthataccumulatedamutatormutationwereabletoadapttohighethanollevels.Asaresultofahypermutationstate,hundredsofmutationsaccumulatedthroughoutthe entire genome (some evolved strains harbor even up to thousandmutations). Someof those

22

mutations,classifiedas‘passenger’,settledinthepopulationbyhitchhikingwithanotherbeneficialmutation and tend tohave aneutral or evendeleterious effects.Others, called ‘drivers’, improvefitnessandcontributetothetolerancephenotype,butoftenremainedhiddenunderthenoiseofthepassenger mutations (Bozic et al. 2010 and Lenski, 2017). Thus, retaining the ethanol tolerance-associateddrivermutationsanddiscardingthepassengersisasuitablestrategytodefineaframeworkforstrainengineeringinthecontextofethanolproduction.Ideally,thisindustrialstrainshouldharbortheminimumsetofdrivermutationstoprovideprotectionagainstacertainethanolconcentrationwithout affecting critical industry-related, microbial parameters like growth rate and productionproperties.

Todistinguishbetweendriverandpassengermutations,weidentifiedcorepathwayswhicharedirectlylinkedtoimprovedethanoltoleranceinE.colithroughabioinformaticsnetworkanalysistool, called Identification of Adaptive Mutations in Evolutionary Experiments or shortly IAMBEE(Swingsetal.2017b).Thismethodappliesacombinationofwhole-genomesequencinginformationfrom individually, evolved E. coli populations together with preexisting network databases.Morespecifically, the algorithm focuses on genomic data at selective sweeps, corresponding to ethanolthresholds at which favorable adaptive mutations take the upper hand thereby purging geneticdiversitywithinabacterialpopulation(Cohan,2016).Asaconsequence,mutationswhichoccurredduringtheseparticularevolutionarytimepointsarelikelytobeadaptiveunderethanolconditionsandthus provide thebackbone for subsequent network analysis. Finally, the in silico study revealed avariety of subnetworks involved in different cellular processes, ranging from cell envelopehomeostasis to transcription. Some identified pathways, such as a pathway involved in plasmamembranefattyacidcomposition,werealreadyreportedinliterature,whereasothers,suchastheAcrAB-TolCeffluxpumpcomplex,werenotlinkedtoethanoltoleranceyet.

FROMANINSILICOTOWARDSANINVIVOCONTEXTHere, we sought to verify IAMBEEs outcome and estimate the relative contribution of

common point mutations within the key IAMBEE-identified subnetworks in vivo. To study thesepolymorphisms individually, it is clear that the mutation of interest has to be isolated from thecorresponding,evolvedcellpopulationandtransferredtotheoriginal,ancestralbackground.Forthispurpose,wedevelopedamodifiedtwo-plasmidCRISPR/Cas9approachwhichenablesto introduceanypointmutationinanygeneofinterestinastraightforwardandconvenientworkflow(Swingsetal. 2018).Moreover, this genome editing technique allows to combinemultiple single nucleotidepolymorphisms(SNPs)inonesinglecelltoinvestigatetheeffectofepistasisintheethanoltolerancecontext.Aftergenomemodification,thefitnessoftheCRISPR-mutantsarecomparedtowild-typeE.coliintermsofgrowthdynamicsandcellsurvivalinethanol-richmediumusingopticaldensitydevicesandclassicalplatingtechniques.Importantly,thesemicrobiologicaldatacanprovideaninvivorealitychecktoeitherprovetheevolutionaryrelevanceofinsilicomodelsor,incaseofinconsistencies,tofine-tune the prediction power of computational tools.Moreover, besides being a research tool,CRISPR/Cas9 can also serve as a useful method for (tolerance) engineering purposes in order todevelopsuperiorethanol-producingstrains.

Inasecondpart,weareplanningtoset-upaninvivophagetransduction-basedscreeningset-upfortheinvivoidentificationofethanoltolerance-improvingmutations(Nehringetal.2015).Inanutshell,thisconceptwillallowustoexchangegenomiclocifromthewild-typeancestortoanevolvedstrainortheotherwayaround.Inthefirstcase,ethanoltoleranceisexpectedtodropwhendrivermutationsareremovedbyphagetransductionwhereas,inthesecondsituation,theintroductionofadvantageous,adaptivemutationsinthewild-typebackgroundshouldimprovegrowthcharacteristicsandsurvivalunderethanolstress.Bothtransductionapproachesshouldprovideagenome-widescan,indicatingthetrueadaptivemutations,whichshouldincludethegenesandpathwaysidentifiedbyIAMBEEandlikelysupplementthelistofcausalmutationswithnewethanoltolerance-associatedloci.


23

The previous paragraphs described a pipeline for in silico and in vivo identification ofmutations conferring ethanol tolerance as well as a methodology to integrate individual or acombinationofSNPs intoanyE. colibackground.Obviously, creating superiorE. coli fermentationstrains using the obtained list of ethanol-associated driver mutations in combination with theCRISPR/Cas9editingtoolshouldbeinreachatthispoint.Subsequently,weareplanningtoevaluatethemicrobialperformanceoftheseengineeredstrainsintermsofethanolproductiontoestablishaclearlinkbetweentoleranceandproductivityinafollow-upstudy.Inaddition,theseeditedstrainswillbesuitableresearchsubjectstounderstandhowmutationscanalterthefunctionofaspecific,cellularpathwaytopushthecell’sethanoltolerancelimit.

CONCLUSIONTo summarize, this project covers the entire research flow from the identification of

tolerance-associatedmutationstowardsapplicationofelementaryknowledgeinstrainengineering.In addition, fundamental insights into the ethanol tolerance mechanism could offer a deeperunderstandingonhowlivingorganismsareabletocopewithextremestressesthroughoutevolution.Finally,duetotheapplicablenatureofthisresearchtopic,strainimprovementofindustriallymorerelevant production strains, such as Z. mobilis or S. cerevisiae, could be tackled by focusing onhomologouspathwayswhichappearedpredominantforethanoltoleranceinourmodelorganismE.coli.

REFERENCESBozic,I.etal.Accumulationofdriverandpassengermutationsduringtumorprogression.ProcNatlAcadSci107,18545–

18550(2010).Cohan,F.M.DispatchesBacterialSpeciation:GeneticSweepsinBacterialSpecies.Curr.Biol.26,R112–R115(2016).Dunlop,M.J.Engineeringmicrobesfortolerancetonext-generationbiofuels.Biotechnol.Biofuels4,32(2011).Fairley,P.Nextgenerationbiofuels.Nature474,S2–S5(2011).Lenski,R.E.Experimentalevolutionandthedynamicsofadaptationandgenomeevolutioninmicrobialpopulations.Int.Soc.

Microb.Ecol.11,2181–2194(2017).Ling,H.,Teo,W.,Chen,B.,Leong,S.S.J.&Chang,M.W.Microbialtoleranceengineeringtowardbiochemicalproduction:

Fromlignocellulosetoproducts.CurrentOpinioninBiotechnology(2014).Nehring,R.B.etal.Anultra-denselibraryresourceforrapiddeconvolutionofmutationsthatcausephenotypesinEscherichia

coli.NucleicAcidsRes.(2015).Swings,T.etal.AdaptivetuningofmutationratesallowsfastresponsetolethalstressinEscherichiacoli.Elife6,(2017).Swings,T.etal.Network-basedidentificationofadaptivepathwaysinevolvedethanol-tolerantbacterialpopulations.Mol.

Biol.Evol.34,2927–2943(2017).Swings,T.,MarcianoD.etal.CRISPR-FRT:Targetingsharedsitesinaknock-outcollectionforoff-the-shelfgenomeediting.

NatureCommunications(inreview).

24

METABOLICSYNDROMEDUETOCONFLICTSBETWEENLIGHTANDFOOD:ASTORYOFTWISTEDCLOCKS

AuroreWoller1,DidierGonze1

1UniversiteLibredeBruxelles,Belgium

Circadianrhythmsarebiologicalprocessesthatoscillatewithaperiodofabout24hours.These

rhythmsareubiquitous,endogenousandcanbeentrainedbyexternalperiodicevents(suchaslight-

darkcycleandfeeding-fastingcycle).Themammaliancircadiansystemiscomposedofmultipleclocksthroughoutthebody.Theselocalclocksareentrainedbynutrient,neuralandendocrinecuesanddrive

diversephysiologicalfunctionsincludingmetabolism.Inparticular,theclockofthepancreaticbetacell

rhythmically regulates the transcription of genes involved in glucose-stimulated insulin secretion.

Perturbationsof this fine-tunedoscillatorynetwork increase the susceptibility todiseases.Besideschronicjetlagandshiftwork,commonperturbationsareill-timedeatingpat-ternswhichcanleadto

metabolictroubles(suchhypoinsulinemia).Wehavebuiltamathematicalmodeldescribingtheclock-

dependentpancreaticregula-tionofglucosehomeostasisinrodents.Aftercalibratingthemodel,wehaveinvestigatedtheeffectofrestrictingfoodaccesstothenormalrestphase.Oursimulationshave

shownthattheresultingconflictbetweenthe light-darkcycleandthefeeding-fastingcyclecreates

differential phase shift in the expression of core clock genes (consistent with experimental

observations).Ourmodelfurthershowsthatthisinducesanon-concomittancebetweennutrientcuesand clock-controlled cues controlling metabolic outputs: this results in hypoinsulinemia,

hyperglycemiaaswellasinalossoffoodanticipation.


25

Lecturesession

HUMANHEALTH

26

KEYNOTEII

MICROFLUIDICSFORBIOMIMETISM:FROM3DCO-CULTURETOGUTONCHIP

StéphanieDescroix

MacromoleculesandMicrosystemsinBiologyandMedicine,InstitutCurie(MMBM),France

Stéphanie Descroix has an initial background in biochemistry and she obtained her PhD in

AnalyticalChemistryfromtheUniversityPierreandMarieCurie in2002.ShewashiredbyCNRSas

researcheratESPCI(Paris)in2004todevelopmicrofluidicdeviceforbioanalyticalapplication.In2011,

she joined the lab PhysicoChemistry Curie at Institut Curie, a unique interdisciplinary and clinicalenvironment.Her research focuseson thedevelopmentofmicrofluidicdevices forbiophysicaland

biomedicalapplications.Shehasauthoredmorethan70papersand5patents.Shehasbeenawarded

in 2013 by the French Chemical Society. Since 2013, she is head of the CNRS French Micro andNanofluidicNetwork.Sheisalsoco-founderofINOREVIAlaunchedin2016.


27

THEIMPACTOFLACTATEONTHEMETABOLICPHENOTYPEOFTUMOR-ASSOCIATEDMACROPHAGES

XeniaGeeraerts1,JoVanGinderachter1,SarahMariaFendt2

1LaboratoryofCellularandMolecularImmunology,VrijeUniversiteitBrussel,Brussels,Belgium

2LaboratoryofCellularMetabolismandMetabolicRegulation,VIBCenterforCancerBiology,VIB,Leuven,Belgium

One of the outstanding challenges in macrophage biology is to understand how particular

microenvironmental cues translate into macrophage functions and ultimately in the outcome of

macrophage-regulatedpathophysiologicalprocesses.Averyrelevantdiseaseinthisrespectiscancer.Tumor-associatedmacrophages(TAM)areamongstthemostabundantinflammatorycellsintumors

andasignificantcorrelationwas foundbetweenhighTAMdensityandaworseprognosis formost

cancers.Theco-existenceofdistinct,specializedTAMsubpopulationswithinthesametumorhasbeendefinedbeforeandindicatedthepresenceofprotumoralM2-likeMHC-II(lo)TAMandantitumoralM1-

likeMHC-II(hi)TAM.Therefore,strategiesthatreprogramprotumoralM2TAMintoanantitumoralM1

phenotype,arecurrentlyhighlyinvestigated.

Inthiscontext,ourworkhasfocusedonunravellingthelargelyunknownmetabolicphenotypeof TAM subsets in a murine lung carcinoma model. Interestingly, we found that the metabolic

phenotypeofMHC-II(hi)TAMandMHC-II(lo)TAMshowssimilaritieswithinvitrostimulatedM1and

M2macrophages,characterizedbyaerobicglycolysisfollowedbyatruncatedTCAcycleandoxidative

metabolism,respectively.13Ctraceranalysisrevealedlactate,whichisknowntohighlycontributetotumoracidosis,asacarbonsourceforMHC-II(lo)TAMwhichcanbefueledintotheTCAcyclecoupled

toOXPHOS.Ourdatasuggestthatlactatestronglyaffectsmacrophagemetabolism,asMHC-II(hi)TAM

metabolismisdisturbedandforcedtowardsanintactTCAcycleinthepresenceoflactate.

Overall,ourfindingssuggestthatlactatemightbeanimportantregulatorofTAMmetabolism.Sincemetabolism is shown to be inextricably connected to immune cell function, interferingwith

(lactate)metabolismcouldbeanelegantwaytometabolicallyrepolarizeTAMtowardsanantitumoral

phenotypeandsubsequentlyaffecttumorgrowthandmetastasis.

28

EFFECTOFADIETRICHINPREBIOTICFIBERSOFINULINTYPEANDBEHAVIORONINTESTINALHEALTHINOBESEPATIENTSINANHUMANINVITROFERMENTATION

PATTERNS

GaétanKalala1,2,4,B.Kambashi2,N.Everaert2,Y.Beckers2,A.Richel2,B.Pachikian3,A.M.Neyrinck3,N.M.Delzenne3,J.Bindelle2

1UniversitédeKinshasa,DepartmentofAnimalProduction,Kinshasa-X,DRCongo

2UniversitédeLiège,GemblouxAgro-BioTech,B-5030,Belgium3UniversitécatholiquedeLouvain,MetabolismandNutrition,B-1200,Brussels,Belgium

4WallonieBruxellesInternational,B-1080,Brussels-Belgium.

Obesityanditsassociatedpathologieshaveseriousconsequencesonpatients’lives,leadingto

high costs for society. Consumption of dietary fiber (DF) and prebiotics remains essential for the

modulationofthegutmicrobiotatopreventthesediseases(Delzenneetal.2013).Inthisregard,theFood4Gutmultidisciplinaryresearchprojectisinvestigatingtheuseoffiber-richvegetables,especially

fructans, in order to observe their positive effects on intestinal health. In this framework, obese

patients are subjected for 3months tooneout of twopossible dietary treatments: onebasedon

fructan-richvegetablesandonebasedonvegetablespoorinfructans(placebo).Fecesweresampledand used as inoculum in a dual in vitro model of the gastro-intestinal to study the changes in

fermentationpatternsofthefibrefractionofvegetablesaccordingtothetreatmentthepatienthad

received.Sixvegetablesweresampledintriplicates(N=3)andsteamedfor20to30min:Jerusalemartichoke, salsify, asparagus, pumpkin, fennel and swede (Kalala et al.2017). They were chosen

becausetheydisplayavarietyofcontentsinfructans,soluble(SDF)andinsolubledietaryfiber(IDF).

Steamedvegetable sampleswerehydrolyzed invitrowithporcinepepsinandpancreatin tomimic

digestionintheuppergut.Undigestedfiberresidueswererecoverusinga6kDadialysismembraneand fermented in vitro with the fecal inoculums collected before and after the treatment.

Fermentationkineticsover24haswellasshort-chainfattyacidproductionandprofileswerecompared

accordingtotheindividualdonorandthevegetablespecies.Theextentandtherateoffermentation

were strongly related to the content and fiber profile of vegetables,with high variability betweenindividuals. This variability included a significant difference in the SCFA profile due to the dietary

treatmentwithahigherproportionofbutyrateaftertheinterventionwithfructan-baseddiets.Itcan

be concluded that fermentation potential of fecesmicrobiota of obese patients is highly variable

between individual and that a dietary interventionwith fructans for 3months positively shift thefermentationpatternofvegetablesfibre.

References

DelzenneNetal.2013.Gutmicrobiotaandmetabolicdisoder.BJN

KalalaGetal.2017.Characterizationoffructansanddietaryfiberprofilesinrawandsteamedvegetables.InpressIJFSN


29

BIOPHYSICALCHARACTERIZATIONOFATHREE-COMPONENTTOXIN-ANTITOXINMODULEFROMTHEPATHOGENICBACTERIUMESCHERICHIACOLIO157

PieterDeBruyn1,RemyLoris1


Bacteriahaveextraordinarywaystodealwithstressingeneralandantibioticsinparticular.The

use and over-use of antibiotics in Europe and the USA has resulted in the rapid development of

resistancetomultipletypesofantibioticsinpathogenic.Asidefrominheritableantibioticresistance,multidrugtolerancecanalsobelinkedtoanon-inheritabletraitcalledpersistence,thesurvivalofa

certain percentage of bacteria in a stressful environment by entering a dormant state. Because

antibiotics preferably work on metabolically active cells, these bacteria are capable to withstand

differentantibioticsduringthetimeoftreatment.

Toxin-antitoxinmoduleshavebeenlinkedtoinducingpersistenceviaamechanismthatinvolves

theactivityofthetoxin.ThepaaR2-paaA2-parE2operonisauniquethree-componenttoxin-antitoxin

module present in pathogenic Escherichia coli O157. Aside from the toxin (ParE2) and antitoxin

(PaaA2),thereisanadditionalregulator(PaaR2)thatisinvolvedintherepressionofthetranscriptionoftheoperon.TheobjectiveistounderstandhowPaaR2autoregulatesthetranscriptionoftheoperon

andhowthisregulationislinkedtotheonsetofpersistence.Viaacombinationofstructuralbiology,

biochemistryandbiophysicalexperiments,amechanisticmodelfortheautoregulationofthepaaR2-paaA2-parE2operonwasconstructedtounravelitsroleinpersistence.

30

HIGHTHROUGHPUTGENERATIONOFDESIGNERENZYBIOTICSAGAINSTACINETOBACTERBAUMANNII

HansGerstmans1,2,RobLavigne2,YvesBries1

1UniveristeitGent,Belgium

2KatholiekeUniversiteitLeuven,Belgium

Overthepastfewyears,antimicrobialresistancehasevolvedfromarareeventtoaneverydayoccurringproblem.Managingthesebacteriainhealthcareiscomplicatedasthelackofnewantibioticsandtheongoingspreadofmulti-andevenpandrug-resistantstrainsleaveuswithareducednumberofpotentialantibiotics,especiallyagainstGram-negativepathogens1.Fallingshortofnewtreatmentswould lead to thedeathof10millionpeopleeachyearworldwide 2, anda cumulativeworldwideeconomiclossupto$124trillionby20503.Moreover,carbapenemresistantAcinetobacterbaumanniiisrecentlycategorizedasoneofthemostdangerouspathogensbytheWHO4.

Enzyme-basedantimicrobialsorenzybioticsexploitthenaturalantibacterialactivityofenzymes.Specifically,weusedendolysinsthatarefoundinbacterialviruses,alsoknownasbacteriophages5.Byprotein engineering we proved in the past that we can customize the antibacterial properties ofendolysinsandwewerethefirsttomakethemhighlyfunctionalagainstGram-negativebacteria(i.e.Artilysins)forwhichmostantibioticsdonotwork6–8.

In2016,pipelineportfolioreviewaboutalternativestoantibioticshighlightedenzybioticsasthealternative class of antibacterials with the greatest potential, based on their clinical impact andtechnicalfeasibility9.Enzybioticshaveseveraluniqueattributes:(1)theirfast(secondstominutes)6,mode-of-actionthatisindependentofanactivehostmetabolism(e.g.bacterialpersisters)6,7(2)theirspecificity against both Gram-positive and Gram-negative pathogens (including antibiotic resistantstrains)5,8(3)thelimitedresistancedevelopmentreportedsofar6.Recently,wehavedevelopedandgradually optimized designer enzybiotics against the four major epidemiological strains ofAcinetobacterbaumannii.

References

1.WHO,WHO’sfirstglobalreportonantibioticresistancerevealsserious,worldwidethreattopublichealth.2014.2.Neill,J.O.,AntimicrobialResistance:Tacklingacrisisforthehealthandwealthofnations.2014.3.Taylor,J.etal.Estimatingtheeconomiccostsofantimicrobialresistance.2014.4.Kahlmeter,G.etal.GlobalPriorityListOfAntibiotic-ResistantBacteriaToGuideResearch,DiscoveryAndDevelopmentOf

NewAntibiotics.2017.5.Schmelcher,M.etal.Bacteriophageendolysinsasnovelantimicrobials.FutureMicrobiol.2012.6.Defraine,V.etal.EfficacyofArtilysinArt-175againstResistantandPersistentAcinetobacterbaumannii.AntimicrobAgents

Ch.2016.7.Briers,Y.etal.Art-175IsaHighlyEfficientAntibacterialagainstMultidrug-ResistantStrainsandPersistersofPseudomonas

aeruginosa.AntimicrobAgentsCh.2014.8.Briers, Y. et al. Engineeredendolysin-based ‘Artilysins’ to combatmultidrug-resistant gram-negativepathogens.MBio

2014.9.Czaplewski,L.etal.Alternativestoantibiotics—apipelineportfolioreview.LancetInfect.Dis.2016.


31

PREDICTIONANDVALIDATIONOFTHESTRUCTURALFEATURESOFOV-58GPCRASANIMMUNODOMINANTANTIGENOFONCHOCERCAVOLVULUS:IMPLICATIONSIN

ONCHOCERCIASISCONTROLEFFORTS

RobertAdamuShey1,2*,FerdinandNgaleNjume1,2,LeaOliveTchouateGainkam1,PhilippePoelvoorde1,LeonMutesa4,AnnieRobert5,PerrineHumblette2,Jean-Pierre

Munyampundu4,JosephKamgno3,LucVanHamme1,StephenGhogomu2,JacobSouopgui1

1UniversiteLibredeBruxelles,Belgium

2UniversityofBuea,Cameroon3CRFilMT,Cameroon

4UniversityofRwanda,Rwanda5UniversitéCatholiquedeLouvain,Belgium

Onchocerciasisisadevastatingyetneglectedtropicaldiseasethatcreatesaremarkablestigma,generates and perpetuates poverty, and is a major impediment to socioeconomicdevelopment/progressofaffectedcommunities.Itisthesecondleadinginfectiouscauseofblindnessworldwide,affectingabout15.5millionpeopleincluding12.2millionwithskindisease,1.025millionwithvisionloss[1]andanadditional172millionpeopleinneedpreventivechemotherapy.Currently,Ivermectinistheonlydrugusedformasstreatmentagainstthedisease[2].Ivermectin,howeverhaslimited effects on adult parasites, so treatments must be expanded over at least 12-15 years,correspondingtothereproductivelifespanoftheadultwormwhenexposedtodrugpressure,hopingnofurtherinfectionoccurswithinthistime.

TheWHO’sambitiousgoalofeliminatingthediseaseby2025willnotbeachievedwithoutthedevelopmentofnewtools [3].Measures tocertifydiseaseeliminationandsurveillanceareofdireneedforhealthsystemsincountrieswhereonchocerciasisisapublichealthproblem.

Here,wereportthat(i)O.volvulusantigenOv58isaG-proteincoupledreceptorconservedinnematodes, (ii) synthetic peptides predicted in the extracellular domain (ECD) of Ov58GPCR elicitpositiveresponsestoserafromonchocerciasispatients,(iii)syntheticpeptidecocktailsdiscriminateserafrominfected,treatedpatientsandnormalAfricancontrols,(iv)polyclonalantibodiesagainsttherecombinantECDrevealedasinglebandonblotsofO.volvulustotalextracts,correspondingtotheexpectedsizeoftheendogenousnativeantigen,(v)Ov58GPCRistranscriptionallyactivatedinboththe larvaeandadult parasite, (vi) humoral immune responsesdepictdeclineswith IVM treatmentcompliance.Hence,weconcludethattheECDofOv58GPCRharborsantigenicfeaturesofinterestinthecontextofonchocerciasisdiagnosisandcontrolefforts.

References1.Lustigman,S.,etal.,Onchocercavolvulus:TheRoadfromBasicBiologytoaVaccine.TrendsParasitol,2017.2.Kuesel,A.C.,ResearchfornewdrugsforeliminationofonchocerciasisinAfrica.IntJParasitolDrugsDrugResist,2016.

6(3):p.272-286.3.Hotez,P.J.,etal.,EliminatingtheNeglectedTropicalDiseases:TranslationalScienceandNewTechnologies.PLoSNeglTrop

Dis,2016.10(3):p.e0003895.

32

SINGLE-CELLIMAGINGOFBACTERIALPERSISTERS

FrédéricGoormaghtigh1,LaurenceVanMelderen1,TatjanaSchlechtweg1

1UniversitéLibredeBruxelles,Belgium

Bacterialpersistersarerarephenotypicvariantsthataretoleranttolethaldosesofantibiotics.

Onthecontrarytoresistance,persistenceisatransientphysiologicalstatethatdoesnotrelyongenetic

mutationsandisthereforenotheritable.Persistercellsarethoughttoenteradormantstateinwhichthey are protected against the lethal effect of antibiotics. Two types of persister cells have been

identifiedsofar:typeIpersisters,originatingfromthepreviousstationaryphaseandtypeIIpersisters,

whichareformedstochasticallyduringexponentialgrowth.Uponremovaloftheantibiotic,persister

cellsareabletoresumegrowthandgiverisetoanewbacterialpopulation,whichwillbeassensitiveto the antibiotic as the initial population. Their low frequency (± 1/10,000) associated with their

transientnaturemakethephenomenonparticularlydifficulttocharacterizeusingclassicalpopulation

approaches.Asaresult,thephysiologyofthesephenotypicvariantsremainsmostlyuncovered.

Inthiswork,bycombiningmicrofluidicsandfluorescencemicroscopy,wewereabletoidentify

and track E. coli persisters to ofloxacin (a fluoroquinolone). For the first time, persister cellswere

observedthroughouttheirlifecycle:exponentialgrowth,antibiotictreatmentandgrowthresumption

afterofloxacinremoval.Morphology,growthrate,nucleoidslocalizationandSOSresponseinductionweremonitoredforbothpersisterandsensitivecells.Interestingly,wefoundthatwhiletypeIpersister

cellsarenon-dividingduringthegrowthperiod,typeIIpersistersdodividenormallywithinthesame

period. Interestingly,persistersdosustainDNAdamageasshownbythecompactionof theirDNA,

induction of SOS response and appearance of membrane perturbations. Upon removal of theantibiotic,persistercellsstarttoelongate,formingverylongfilaments(44±23µm)andinducethe

SOS response, allowing them to repair DNA damages caused by the ofloxacin treatment. DNA

replicationthenoccurs,resultingintheformationofmulti-nucleatedfilaments.Finally,divisionevents

takeplaceatmultiplelocationswithinthefilamentandanewbacterialpopulationisgenerated.

Asaconclusion,thisisthefirsttimetypeIIpersistercellswereobservedandcharacterizedat

the single-cell level before, during and after the antibiotic treatment. Such an analysis provides a

powerful tool for studying persister cells physiology and would be valuable to most populationanalysesperformedpreviouslyinthefield.


33

SHUTTLEVECTORSYSTEMFORIMPROVEDPESTIVIRALREVERSEGENETICS

AndreyKossarev1,JanPaeshuyse1

1LaboratoryofHost-PathogenInteractions,DepartmentofBiosystems,KULeuvenUniversity

PestivirusesareubiquitousRNAviruses,generaoftheFlaviviridaefamily,whichpredominately

affectlivestockandresultinseriouseconomiclosses.Someprominentmembersincludebovineviral

diarrhoea (BVD) and classical swine fever virus. On the level of pathogenicity, pestiviruses arecharacterizedbyhighgeneticvariabilityandagradientinvirulence.DuetotheinherentnatureofRNA

viruses,theirinstabilitypresentsachallengeasgeneticengineeringiscumbersometasktoachieve.

Contemporary methods in reverse genetics circumvent this problem through generation of

complementaryDNA (cDNA),whichallows fordirectmanipulationsandpropagationasabacterialcDNAplasmidclone.Furthermore,suitableRNApolymerase(RNAP)promotorspermitgenerationof

infectious genomic viral RNA by in vitro transcription.Modern strategies combine stable bacterial

artificialchromosomes(BAC)witheukaryoticorviralpromotersforinvivo/invitrotranscriptions.

Our prime objective is to establish an improved reverse genetics system with simplified

workflow and provide rigorous tools for molecular studies of pestiviruses. For this purpose we

engineered a BAC shuttle vector system, partially inspired by the KU Leuven PLLAV system

(WO2014174078A1),withasetofregulatoryelementsasafirststeptowardsthisgoal.Currentshuttlevectorversioniscomposedofcopycontrolregulatoryelementsandyeastoriginofreplication(ori).

Thefirstelementfurtherincreasestheoverallvectorstabilitybyreducingtheplasmidcopynumberto

aminimum.A switch from single copyori to high copyori is achieved through arabinose induced

activationoftransactingtrfAelement,increasingthecopynumberupto50times.Yeastorielementallows for propagation and maintenance of the shuttle vector in various yeast strains while

simultaneouslyexploitingyeast’shighlyefficienthomologousrecombinationgeneticmanipulationsof

characterized and uncharacterized pestiviral strains. This step is performed in combination with

CRISPR-Cas9 technology,providingmoreefficientgeneticengineeringmethods.Furthermore,eachviral cDNAclone ispositionedunder the controlofT7promoterand terminator.However, in vitro

transcriptionstepisomittedthroughutilizationofstableT7RNAPexpressingMDBKcell lines.Such

strategyeliminatestheneedforexpensiveT7RNAPkits,significantly increasestheoverallreactionyieldsandmaximizesthechancesforsuccessfulinitiationofviralinfectiouscycle.Lastly,generationof

genuine3’endsandeliminationofpotentialrun-offeffectisrealisedbyflankingviral3’UTRdirectly

withaHepatitisDeltavirusribozyme.Ongoingeffortsareaimedatenhancingcurrentshuttlevector

withstableeukaryoticepisomalretentioncapabilitiesandaninduciblebiotypeswitchforBVDstrains.Finalizedsystemwillbepresentedandpublishedforscientificuseinreversegeneticsofpestiviruses

andRNAvirusesingeneral.


35

Lecturesession

ENVIRONMENT

36

UHPLC-HRMSBASEDTARGETEDANDUNTARGETEDSCREENINGOFPLASTICIZERSINTHEMARINEENVIRONMENT

SteveHuysman1,LievenVanMeulebroek1,FrancisVanryckeghem2,HermanVan

Langenhove2,KristofDemeestere2,LynnVanhaecke1

1GhentUniversity,DepartmentofVeterinaryPublicHealthandFoodSafety,LaboratoryofChemicalAnalysis(LCA)

2GhentUniversity,DepartmentofSustainableOrganicChemistryandTechnology,ENVOC

Thecumulativeamountofplasticsenteringouraquaticenvironmentispredictedtoincreaseby

an order of magnitude by 2025, resulting in the extensive entree of plasticizers. Plasticizers, i.e.

phthalateacidestersandalkylphenols,areeasilyreleasedintotheaquaticenvironmentasaresultoftheirweakphysicalbondingtoplasticpolymers.As ithasbeenshownthatcontinuousexposureto

plasticizers results inaplethoraofadverseecologicaleffects,monitoringphthalateacidestersand

alkylphenolsinthemarineenvironmentisofgreatimportancetoprovideinsightinthewidespreadoccurenceofthesecompoundsinouraquaticecosystems.

Therefore, this study engaged on the analysis of the abovementioned compound groups in

seawater samples obtained from different locations in the Belgian Part of the North Sea during

differentseasons(2016-2017).Theseawatersampleswereanalyzedusinganin-housedevelopedandvalidatedultra-high-performanceliquidchromatographichigh-resolutionmassspectrometric(UHPLC-

HRMS) method. This novel analytical method was specifically designed to minimize false-positive

detectionsresultingfromlaboratorycontaminationofplasticizers.Performingthetargetedanalysisof

27plasticizersrevealedtheprevalenceof16uniqueplasticizersintheseawatersamples.Alkylphenolsandphthalateswererespectivelydetectedatconcentrationsrangingfrom24to8000ngL-1and20to

2580ngL-1.Moreover,multivariatestatisticsdisplayedthehighestconcentrationsofethylphenoland

dibutyl phthalate in the harbor of Ostend. Next to the native plasticizers, also primary plasticizer

metabolites(mono-phthalates)wereubiquitouslydetectedwithconcentrationsrangingbetween25and2550ng L-1. Subsequent to the targeted screening,untargetedanalysisof the sameseawater

samples revealed 3688 different unknown compounds. Further data analysis for identification

purposesoccurredthroughonlineenvironmentaldatabasesearch,includingMarineLit,MarineDrugs,EPA, drugsbank, ACTor and EAWAG biodegradation. In conclusion, the ubiquitous prevalence of

plasticizers inourmarinewaterssuggeststhatplasticizerswillhavereacheddifferenttrophic levels

throughoutouraquaticecosystemandmaythuscontributetoadverseecologicaleffectsthreatening

thequalityofourmarineenvironment.

Acknowledgments: The authors acknowledge the Belgian Science Policy (BELSPO) for funding the

NewSTHEPSproject(BR/143/A2/NEWSTHEPS;www.newstheps.be)inwhichthisworkfits.


37

EFFECTSOFCLIMATECHANGEONMACROPHYTEGROWTH,BIOMASSALLOCATION,PHYSIOLOGYANDNUTRIENTSTOICHIOMETRY

RosanneReitsema1,PatrickMeire1,JonasSchoelynck1

1EcosystemManagementResearchGroup(Ecobe),DepartmentofBiology,

UniversityofAntwerp,Belgium

Freshwater macrophytes are faced with the effects of climate change. Increased CO2 andnutrientsinthewaterandperiodsofhigherflowvelocityduetoincreasedprecipitationintensityarethreeimportanteffectsinfreshwaterecosystems(Shortetal.,2016).Itisnotfullyunderstoodhowmacrophytesrespondtothisandwhetherthereisaninteractionbetweentheeffects.InaracetrackflumeexperimentshootsofBerulaerectawereexposedtolow(400ppm)andhigh(1000ppm)CO2concentrations under low (0.04m s-1) and high (0.4m s-1) flow conditions. The experimentwasexecutedtwice:undermesotrophicandundereutrophicconditions.After10weeks,biomassofeachplantwasdetermined(roots, leavesandstemsseparately);morphologywasmeasured(numberofstems,leavesandstolons,lengthanddiameterofthelongeststem,andtotalandaverageleafarea);chlorophyllcontentofasubsetofplantswasmeasured;andfinallynutrientstoichiometry(C:N:P:Siratio)willbeanalysed(theresultswillbeinbeforethepresentation).

TheresultsshowthatplantsgrownunderhighCO2concentrations(HC)comparedtolowCO2concentrations (LC) hadmore and thicker stems,more leaves,more stem- leaf- and belowgroundbiomass,ahigherleaf:stemratioandroot:shootratio,morestolons,ahigherRGRahighertotalleafarea, a lower SLA and a higher chlorophyll content. Flow velocity (FV) also had an effect onplantgrowth:plantsgrewtaller,buthadfewerstems.TherewerealsointeractioneffectsbetweenFVandCO2:HighFV reduced stemand leafbiomassunderhighHC,butenhanced stemand leafbiomassunderLC.PlantsgrownunderlowFVandHCalsohadthehighestaverageleafsurfacearea.Undereutrophicconditions,similarpatternswereseen:morebiomasswasproducedunderHC.However,theplantsweresmallerthaninthefirstexperiment.Clearly,eutrophicationhadanegativeeffectonplantbiomass.Thiswasprobablyduetothegrowthofalgaethatcoveredtheplantsandreducedlightavailability.

It is important to understand howmacrophyte quantity and qualitywill respond to climatechange because living macrophytes play an important role in stream structure and functioning(Schoelyncketal.,2012)andmacrophytedetritusaffectsthedecomposersfoodweb(Polunin,1984)andtheaquaticcarboncycle (Regnieretal.,2013).Thisexperimenthasshownthat increasedCO2concentrations can lead tomoremacrophyte biomass, that this effect can be influenced by flowvelocity,butthattheeffectismuchsmallerundereutrophicconditions.Theseresultshighlyrelevanttohelppredictingandunderstandinghowmacrophyteswilladapttonewconditionsunderclimatechange.

References

Polunin,N.V.C.(1984).InAdvancesinEcologicalResearch.Ac.Press,NewYork,115-166Regnier,P.etal.(2013).NatureGeoscience,6(8),597–607.Schoelynck,J.etal.(2012).Ecography,35,760–768.Short,F.T.etal.(2016).AquaticBotany,135,3–17.

38

PROCESSESOFCH4EMISSIONINPADDYSOILUNDERWATER-SAVINGIRRIGATIONMANAGEMENT

HeleenDeroo1,MasudaAkter1,ElizabethVerhoeven2,DanielSaid-Pullicino3,Marco

Romani4,PascalBoeckx1,StevenSleutel1

1GhentUniversity,Belgium2ETHZurich,Switzerland3UniversityofTurin,Italy4EnteNazionaleRisi,Italy

Owingtothedominantanaerobicconditionsinirrigatedricefields,considerablemethane(CH4)

productioninsoil(witheitheracetateorCO2assubstrate)canresultinsubstantialCH4emissiontothe

atmosphere.Management to limit such CH4 emissions during the rice growing season is possible,providedthatthecontrollingprocessesareunderstood.Inparticular,itisnotcleartowhatextenta

reduction of CH4 emissions under water-saving irrigation management may be the resultant of

enhancedCH4oxidation,duetothedifficultytoreliablyquantifyCH4oxidation.Weassessedrelativecontributions of CH4 production and oxidation to its emission in rice fields under water-saving

Alternate Wetting and Drying irrigation (AWD) in comparison with the conventional Continuous

Flooding(CF)inanItalianricefieldin2015and2016.Therefore,wemeasuredsoilCH4emissionusing

closedfluxchambersinthefieldalongsideinsituassessmentofCH4oxidationbytwodistinctmethods.Firstly,CH4oxidationwasmeasuredthroughselectiveinhibitionofCH4oxidiserswithdifluoromethane

(CH2F2).Secondly,CH4oxidationandcontributionsofbothCH4productionpathwayswereestimated

bymeansofstableCisotopemeasurementsinaerobicandanaerobicincubations,plantaerenchym

andemittedCH4.

As it turnsout,acetatewas themain substrate forCH4production in theendof the season

(theoretical2:1ratio),withthecontributionofthispathwaybeingsignificantlylowerunderAWDin

the beginning of the growing season. Furthermore, lower CH4 emissions in fields under AWD (in

average2.9mgCH4h-1m-2versus14.0underCFin2015;and5.4versus9.3underCFin2016)resultmainlyfromlowerCH4production,andtoalesserextentfromprobablyhigherrelativeCH4oxidation

in thebeginningof thecropping season.Caution is required in the interpretationofCH4oxidation

resultsthough,sincebothmethodswerebiased.Forthefirstmethod,weassumethatCH2F2interactedunintentionallywithCH4production,asoftenCH4emissionswerehigherwithoutthanwithaddition

oftheCH4oxidation inhibitortotheclosedchambers’headspace.Concerningthe isotopemethod,

severalincubationsampleswereprobablytoomuchdisturbed–asindicatedbytheiroftenphysically

impossibleresults.Inconclusion,water-savingirrigationmanagementleadstolowerCH4production,andmaybe–buttoalesserdegree–tohigherCH4oxidationinthebeginningoftheseason.


39

ASSESSINGTHEMICROBIOLOGICALDIVERSITYINTHECOOLINGWATERSOFANUCLEARRESEARCHREACTOR

ValérieVanEesbeeck1,MohamedAhmed1,RubenProps2,NatalieLeys1,CorinneRivasseau3,

JacquesMahillon4,PieterMonsieurs1

1SCK-CEN,Belgium2GhentUniversity,Belgium

3CEA,France4UniversitéCatholiquedeLouvain,Belgium

TheBR2nuclearresearchreactorcomprisesdifferentwateryenvironments:theprimarycoolingcircuitconsistsofaclosedloopcontainingwaterthatcoolsdownthefuelrodsinthereactorcore.Anopenbasinsurroundsthereactorvessel,andwheneverfuelisloadedontoorunloadedfromthecorematrix, anexchangeoccursbetween thebasinand theprimary circuitwater. Spentnuclear fuel isstored in the contiguous spentnuclear fuelpool (SNFP) inorder to cooldownbeforebeing safelydisposed.Remarkably, despite the low-nutrient environment combinedwith thehighly radioactivecharacterofthewaterandthepresenceofdissolvedradioactivemetals,microbialgrowthisnotfullyprevented. Indeed, several microbes appear to be able to survive and thrive in such conditions.Microorganismsidentifiedinthoseenvironmentsthusprovideauniqueopportunitytoacquirenewinsightsintosurvivalstrategiesandradiation-resistancemechanisms.

The objective of thiswork is to explore the bacterial communities present in the describedenvironmentsoftheBR2nuclearresearchreactor.Inparallel,thisresearchalsofocusesonfollowingupthesecommunitiesovertimeduringandoutsidereactoroperationtomonitorthelong-termeffectofionizingradiation.Finally,thisprojectalsoaimsataphenotypicalcharacterizationoftheprevailingspecies.

Forthecharacterizationandthefollow-upofthebacterialcommunities,a16SrRNAampliconsequencingapproachwasadopted.Resultsfromalong-termfollow-upexperimenthighlightedaclearshiftinthebacterialcommunityprofileduringandoutsidereactoroperation,bothforthebasinandthe primary water. More specifically for the basin water, the profiles observed across multipleshutdownperiodsappearedtobequitesimilar.Incontrasttothis,thecommunityinterestinglyshoweddrasticvariationwhencomparedacrossdifferentreactoroperationcycles.Thiscouldbeduetothechangeinphysico-chemicalparametersthatthesewatersundergowhentransitioningfromonestatetotheother.

Withregardtothephenotypicalcharacterization,strainswereisolatedfromthebasinandtheSNFP and subsequently subjected to ionizing radiation in a gamma irradiation facility to test theirradiationresistance.Thisexperimentshowedthatall12testedstrainstoleratedadoseof300Gy,butonly8%ofthestrainswasabletocopewithadoseof2100Gy,indicatinglargevariabilityinradiationresistancebetweendifferentstrains,andassuchnotnecessarilyahighradiationtolerancetosurviveinthebasinandthespentnuclearfuelpool.

40

QUANTIFYINGPRECIPITATIONERRORSVIASOILMOISTUREDATAASSIMILATION

VereLeybaert1

1KatholiekeUniversiteitLeuven,Belgium

The partitioning of the water budget is of key importance to global climate and weather

forecasting,andtothemonitoringofagriculturalandothersocioeconomicphenomena.Soilmoisture

iscrucialinthewaterbudgetattheinterfacebetweenlandandatmosphere.Thebestestimatesofsoilmoisturecanbeobtainedbycombiningsatelliteobservationswithlandsurfacemodelsviadata

assimilation. However, by improving soil moisture estimates, some inexplicable imbalances in the

waterbudgetareintroduced.ThisPhDresearchwill,forthefirsttime,usethemostrecentsatellite

data for soil moisture and precipitation estimation in combination with a quasi-operational landsurfacemodelinordertolinksoilmoisturedataassimilationincrementstoerrorsinprecipitationor

othermodelingerrors,andcontributetomoreaccurateestimatesofthewaterbalancecomponents.

Apreliminaryanalysisof theoperationalSoilMoistureActivePassive (SMAP)Level4SoilMoistureproduct (L4_SM) and in situ precipitationmeasurements over theMurrumbidgee area in Eastern

Australia will be presented. The link between precipitation errors and SMAP L4_SM soilmoisture

incrementswillbeanalyzedusingtemporalandspatialplots.


41

SCREENINGOFTHEBELGIANPARTOFTHENORTHSEATOWARDSEMERGINGORGANICMICROPOLLUTANTS–COMPARISONOFTWOSPE-TECHNIQUESPRIORTOUHPLC-

ORBITRAP-HRMSANALYSIS

FrancisVanryckeghem1,S.Huysman2,H.VanLangenhove1,L.Vanhaecke2&K.Demeestere1

1GhentUniversity,Dept.ofGreenChemistryandTechnology,ResearchGroupEnvironmentalOrganicChemistryandTechnology(EnVOC),CoupureLinks653,9000Gent,Belgium

2GhentUniversity,Dept.ofVeterinaryPublicHealthandFoodSafety,LaboratoryofChemicalAnalysis,Salisburylaan133,9820Merelbeke,Belgium

INTRODUCTION

Alargevarietyoforganicchemicals–includingpharmaceuticals,personalcareproductsandpesticides–areusedineverydaylifeacrosstheglobe.Manyofthesechemicalsaredischargedintothe sewer system, either in their unchanged form or as metabolites, and will eventually enterwastewater treatment plants (WWTP). Yet, WWTP are not designed and most often not able tocompletely remove these so-called micropollutants (Vergeynst et al., 2015). As result of WWTPeffluentdischarge, incorrectusage, improperdisposaland industrialprocesses, these traceorganiccompoundsareemerginginouraquaticenvironment.Asmanyofthechemicalsareknowntohavebiological effects, they areof great environmental concern and their occurrence shouldbe closelymonitored. Presently, knowledge about theprevalence andbehaviour of these compounds iswelldocumented for wastewater, riverine water, groundwater and drinking water (Stuart et al., 2012;Caldasetal.,2013;Battetal.,2017).Marinewaters,onthecontrary,havereceivedmuchlessattentionso-farregardingtheoccurrence,fateandeffectsofemergingorganicmicropollutants.Mainreasonscanbefoundin,a.o.,theanalyticalchallengeswithrespecttosamplingandtheirprevalenceatultra-trace (≤ppb-levels) concentrations. Suitable sample preparation techniques are thus of paramountimportancetobeabletodealwiththeselowconcentrationsprevailinginthemarineenvironment.Next to that, highly sensitive and selective instrumental analytic techniques are needed. In thiscontext, modern high-resolution mass spectrometry (HRMS) enables the quantification of knownmicropollutantsatenvironmentalconcentrationsandthescreeningforavirtuallyunlimitednumberofunknownmicropollutantsinasinglerun.Therefore,thisstudyfocussedonthecomparisonoftwosolid-phase extraction (SPE) techniques followed by a newly developed and validated UHPLC-Q-Orbitrap-HRMSmethodfortargetedscreeningofthemarineenvironmenttowardsalargesetofmulti-classpharmaceuticals,personalcareproductsandpesticides.Next,untargetedscreeningfollowedbymulti-variateanalysiswasperformedasaproofofconceptandtoenablethediscriminationbetweenthetwoSPE-methods.MATERIALANDMETHODSSamplinglocations

Triplicategrabsampleswerecollectedduringtwosamplingcampaignsi.e.inwinter2016andspring2017atthreedifferent locations intheBelgianPartoftheNorthSea(BPNS); i.e.harbourofZeebrugge(51.340467°,3.203364°),atopenSeainfrontofZeebrugge(51.360494°,3.113614°),andtheharbourofOostende(51.226300°,2.935556°)(Figure1).Grabsamplesweretakenat2mdepthusing5LNiskinbottlesandwerestoredinpre-rinsedamberbottles.

42

Figure1.DetailedmapofthegrabsamplesthatweretakenintheBelgianPartoftheNorthSea,i.e.harbourofZeebruggeⓐ,openseainfrontofZeebruggeⓑandharbourofOostendeⓒ.Samplepreparationandsolid-phaseextraction

Samplepreparationconsistedof filtering thesamplesuponarrivalat the labusing2.7µmWhatmann filters to remove sediments and debris. To prevent microbial growth during samplestorage(maximum4daysat4°C),thesampleswereacidifiedtopH3withformicacid.Priortosolid-phase extraction (SPE), the samples were neutralized (pH 7) and Na2EDTA.2H2O was added.Thereafter, the samples were extracted in parallel on Oasis® HLB cartridges and on Bakerbondhydrophilicdivinylbenzene(DVB)Speedisks(toallowforacomparison).TheSPE-procedureonOasisHLBandDVBSpeedisksconsistedrespectivelyofloadingavolumeof200mLand1000mLonpre-conditioned cartridges. After washing and drying the cartridges, elution was performed usingmethanol/acetonitrile (50/50, v/v). The eluent was collected in pre-rinsed test tubes, evapo-concentratedandreconstitutedin1mLofmethanol/water(10/90,v/v)with0.1%(v/v)formicacidand0.01%Na2EDTA.2H2O.Finally,theglasstubesweresonicated,vortexedandcentrifuged,andthesupernatantwastransferredintoanHPLCvial.Instrumentation

SamplesoriginatingfrombothOasisHLBandSpeediskextractionwereanalysedusinganin-housedevelopedUHPLC-HRMSmethod.Briefly, thechromatographicanalysiswascarriedoutonareversedphaseHypersilGoldcolumn(50mmx2.1mm,1.9µm).Themobilephaseconsistedofamixtureofwaterandmethanol,bothacidifiedwith0.1%formicacidforanalysisinpositiveionisationmode and 0.01% for negative ionisationmode. The detection was done by a High Resolution Q-ExactiveTMOrbitrapmassspectrometer,equippedwithaheatedelectrosprayionization(HESI)sourceand operating in full scan to allow measurement of known (target) and unknown (untargeted)compounds.Validation

Both analytical methods, i.e. the Oasis HLB and DVB Speedisk-based method, were fullyvalidated using artificial seawater and according to the standard addition approach described byMatuszewskietal.(2003)andVergeynstetal.(2017).Briefly,artificialseawatersamplesspikedbefore(pre-spiked)andafter(post-spiked)extractionwereanalysedintriplicatetogetherwithnon-spikedsamples.Dailyexternalstandardcalibrationwasperformedtoconvertpeakareastoconcentrations.Method precision was determined as relative standard deviation (RSD) of the calibratedconcentrations,measuredintriplicatesamplesonthreedifferentdays.Linearity,methoddetectionlimits,analyterecovery(RE),matrixeffects(ME)andoverallprocessefficiency(PE)weredeterminedasdescribedinVergeynstetal.(2017).

ⓐ ⓑ

ⓒ Oostende Zeebrugge

2 km 1 mi

500 m 1000 ft


43

Dataprocessing

For targeted analysis, the identification of the compounds was confirmed based on theiraccuratemassandchromatographicretentiontime.ThechromatogramsweremanuallyprocessedusingExactFinder2.0(ThermoFisherScientific,SanFrancisco,USA).Basedonlegislation(EUDirective,2015)andscientificliterature(Willeetal.,2011;Tanwaretal.,2015;Vergeynstetal.,2015),atargetlistwascompiledconsistingof89emergingorganicmicropollutants,including52pharmaceuticals,9personalcareproductsand28pesticides.Untargetedscreeningwasperformedasaproofofconceptmaking use of Compound Discovery 2.1 (Thermo Fisher Scientific, San Francisco, USA) for dataprocessing,followedbymulti-variateanalysisusingSIMCA2.2(Umetrics,Malmo,Sweden).RESULTSANDDISCUSSIONValidationparameters

Methodprecision(RSD,n=3)wasbetterthan20%foratleast80%and97%ofthesamplesextractedwithOasisHLBandSpeediskcartridges,respectively.Methoddetectionlimitswerelowerthan10ng.L-1for67%oftheextractedtargetcompoundswithOasisHLB,whereasthiswasthecasefor80%oftheextractedtargetcompoundswithDVBSpeedisk.LowerMDLsobtainedwithSpeediskextractionclearlyresultfromthelargerextractionvolume(1000mLvs200mL)andpre-concentrationduringsamplepreparation.

The PE values ranged from 0.06% to 103% and from 1.4% to 76% forOasis HLB andDVBSpeediskvalidationsamples,respectively.AdetailedinspectionofthePEanditsallocationintoREandMErevealedthatthedifferencesinPEbetweenthetwoextractionproceduresmainlyoriginatedfromthedifferencesintheextractionrecovery(RE)ratherthanthematrixeffects(ME).Overall,RE-valueswere lower for Speedisk extraction, which could be caused by partial breakthrough for somecompounds. Polar compounds showed higher affinity for Oasis HLB, whereas apolar compoundsshowedhigherrecoverywiththeSpeedisks(Error!Referencesourcenotfound.).

Figure2.SPErecoveries(RE),obtainedwith(●)OasisHLBand(○)DVBSpeediskextraction,asfunctionofpolarity(LogP)forthetargetcompounds.ForallplottedREvalues,thestandarddeviationsare<20%.

44

Targetedscreening

The targeted screening was performed on grab samples collected during two samplingcampaignsonthreedifferentlocations.Intotal,60compounds–outofthe89targetcompounds–were quantified in at least one sample. Concentrations ranged between 0.13 - 370 ng.L-1 for thepharmaceuticals,0.65-43ng.L-1forthepersonalcareproducts,and0.10-68ng.L-1forthepesticides.SamplesoriginatingfromtheharbourofOostendeweresignificantlydifferent(p<0.05)fromtheotherlocations.Duetothelowerdetectionlimits,upto62%moretargetcompoundswerefoundingrabsamplesextractedwithSpeediskcomparedtoOasisHLB.Untargetedscreening

Untargetedscreeningrevealedthepresenceofupto1300so-farnon-identifiedcomponentsinasinglesample,i.e.chromatographicpeakswithapeakarealargerthan500000a.u.towhichanaccurate mass could be attributed. The amount of components found in Speedisk extracts wassignificantlylarger(uptoafactorof2.6)thanthosefoundinOasisHLBextracts.Furthermore,multi-variate (discriminative) analysis on all acquired untargeted screening data clearly showed adiscriminationbetweenthetwousedextractiontechniques.CONCLUSION

ThisstudydemonstratestheapplicabilityofanewlydevelopedUHPLC-HR-Q-OrbitrapTM-MSmethodincombinationwithsolid-phaseextractionforbothtargetedanduntargetedscreeningofthemarine environment towards emerging organic micropollutants. The analysis of 18 grab samplescollectedatthreedifferentlocationsintheBelgianPartoftheNorthSea,revealedthedetectionof60pharmaceuticals,personalcareproductsandpesticidesatconcentrationsrangingfrom0.10to370ng.L-1.AlthoughtheextractionrecoveryislowerwithDVBSpeedisksthanwithOasisHLBcartridges,largersamplevolumesandlowerdetectionlimitsoffertheopportunityofdetectingmorecompoundsintargetedanalysisandtracingmorecomponentsinuntargetedanalysis,showingthemeritofthisSPEmethodformonitoringthemarineenvironment.ACKNOWLEDGEMENTS

The authors like to acknowledge the Belgian Science Policy (BELSPO) for funding theNewSTHEPSproject (BR/143/A2/NEWSTHEPS).Thefinancialsupport fromtheHerculesFoundation(Flemish Government; AUGE/11/016) and from the Ghent University Special Research Fund(01B07512) is acknowledged for the UHPLC-Q-ExactiveTM and the automated SPE equipment,respectively.REFERENCESBatt,A.L.,Furlong,E.T.,Mash,H.E.,Glassmeyer,S.T.andKolpin,D.W.(2017)‘Theimportanceofqualitycontrolinvalidating

concentrationsofcontaminantsofemergingconcerninsourceandtreateddrinkingwatersamples’,ScienceoftheTotalEnvironment.ElsevierB.V.,579,pp.1618–1628.doi:10.1016/j.scitotenv.2016.02.127.

Caldas,S.S.,Bolzan,C.M.,Guilherme,J.R.,Silveira,M.A.K.,Escarrone,A.L.V.andPrimel,E.G.(2013)‘Determinationofpharmaceuticals, personal care products, and pesticides in surface and treated waters: Method development andsurvey’,EnvironmentalScienceandPollutionResearch,20(8),pp.5855–5863.doi:10.1007/s11356-013-1650-9.

EUDirective(2015)‘CommissionimplementingDecision(EU)2015/495establishingawatchlistofsubstancesforUnion-widemonitoringinthefieldofwaterpolicypursuanttoDirective2008/105/ECoftheEuropeanParliamentandoftheCouncil’,OfficialJournaloftheEuropeanUnion,L78.

Matuszewski, B. K., Constanzer, M. L. and Chavez-Eng, C. M. (2003) ‘Strategies for the assessment of matrix effect inquantitative bioanalytical methods based on HPLC – MS/MS’, Analytical Chemistry, 75(13), pp. 3019–3030. doi:10.1021/ac020361s.

Stuart, M., Lapworth, D., Crane, E. and Hart, A. (2012) ‘Review of risk from potential emerging contaminants in UK


45

groundwater’,ScienceoftheTotalEnvironment.ElsevierB.V.,416,pp.1–21.doi:10.1016/j.scitotenv.2011.11.072.Tanwar,S.,DiCarro,M.,Ianni,C.andMagi,E.(2015)‘OccurrenceofPCPsinNaturalWatersfromEurope’,inM.S.Díaz-Cruz,

D.Barceló(Eds.),TheHandbookofEnvironmentalChemistry,pp.41–53.doi:10.1007/698.Vergeynst, L., Haeck, A., De Wispelaere, P., Van Langenhove, H. and Demeestere, K. (2015) ‘Multi-residue analysis of

pharmaceuticals in wastewater by liquid chromatography-magnetic sector mass spectrometry: Method qualityassessment and application in a Belgian case study’, Chemosphere. Elsevier Ltd, 119, pp. S2–S8. doi:10.1016/j.chemosphere.2014.03.069.

Vergeynst,L.,K’oreje,K.,DeWispelaere,P.,Harinck,L.,VanLangenhove,H.andDemeestere,K.(2017)‘Statisticalproceduresforthedeterminationoflinearity,detectionlimitsandmeasurementuncertainty:AdeeperlookintoSPE-LC-Orbitrapmassspectrometryofpharmaceuticalsinwastewater’,JournalofHazardousMaterials.ElsevierB.V.,323,pp.2–10.doi:10.1016/j.jhazmat.2016.05.077.

Wille, K., Claessens,M., Rappé, K., Monteyne, E., Janssen, C. R., De Brabander, H. F. and Vanhaecke, L. (2011) ‘Rapidquantificationofpharmaceuticalsandpesticidesinpassivesamplersusingultrahighperformanceliquidchromatographycoupledtohighresolutionmassspectrometry’,JournalofChromatographyA.ElsevierB.V.,1218(51),pp.9162–9173.doi:10.1016/j.chroma.2011.10.039.

46

BRIDGINGRESOURCERECOVERYTOREUSE:MICROBESASNEXT-GENERATIONFERTILIZER

MyrsiniSakarika1,JanneSpanoghe1,EvaWambacq1,MaartenMuys1,AbbasAlloul1,MarcSpiller1,GustavoGomesdeSousa1,VeerleDerycke2,LutgardStragier3,HarmeenVerstraete3,

KoenFauconnier4,OliverGrunert5,GeertHaesaert2,SiegfriedVlaeminck1

1UniversityofAntwerp,Belgium2GhentUniversity,Belgium

3AvecomNV,Belgium4AgrAqua,Belgium

5GreenyardHorticultureBelgiumNV,Belgium

Organic fertilizers (OF) are conventionallyproduced fromanimal andplantderivedmaterial.

Theirstrikingfeatureisthatnitrogen(N)andpartlyphosphorus(P)arepresentinorganicformandthus are releasedgradually throughdecompositionbymicrobial activity in the soil or the growing

medium.A special typeofOF,microbial biomass (MB), canupgrade inorganic nutrients to ahigh-

qualityOF.Experimentswereconductedusing3differenttypesofMBgrownon(realandsynthetic)

non-feacaly contaminated side-streams: (i) consortium of aerobic bacteria (CAB), (ii) Rhodobactersphaeroides(purplenon-sulfurbacteria)and(iii)Arthrospiraplatensis(Spirulina).Testsusingstruvite

asarecoveredP-sourcerevealedthat theP-releaserates (0.32–4.69gP/L/d)arehigher thanthe

microbial P-uptake rates, indicating that struvite can be directly dosed into the bioreactors as a

sustainable, low-costalternative forcommercialPandNsources.Mineralization testsshowedthatcomparedtothereferencefertilizer (RF) (basedonacombinationofanimalandplantmaterial),N

mineralizationinMBoccursgraduallyduringthefirstweeksofincubation,while25-35%oftheNis

mineralizedafter42daysinallMB(individualandblends)tested.Interestingly,MBblendsprovedto

be useful for steering the NH4-N mineralization. Subsequently, pot trials were performed usingperennial ryegrass (Lolium perenne), parsley (Petroselinum crispum) and tomato (Lycopersicon

esculentum).TheseshowedpromisingresultsastheMB,usedinavarietyofblends(75%CAB:25%

phototrophsto90%CAB:10%phototrophs),performjustaswellastheRFintermsofplantlengthanddryweightincrease,whileduringthediseaseresistancetestsperformedintomatoplantsthepresence

of Spirulina led to a markedly reduced sensitivity to a biotrophic fungus (mildew). In terms of

fertilizationperformance,MBcancompetewithatypicalOF,andCABisfornowthemostcost-efficient

novelsolution.Lifecycleassessment(LCA)resultsindicatethatCABfertilizershaveacomparableoreven lower environmental impact than alternative OF blends (i.e. horn, blood and bone meal).

Efficiency gains in downstream processing and use of renewable energy can even reduce the

environmentalimpact(i.e.greenhousegassemissions)belowthatofmineralfertilizers.Theresearch

wasfundedbyMIPiCleantechfortheMicroNODproject(http://www.micronod.be/).


47

Lecturesession

KEY-ENABLINGTECHNOLOGIES

48

KEYNOTEIII

LIGHTPOWEREDENZYMATICDEGRADATIONOFLIGNOCELLULOSE:ANEWPHOTO-BIO-CATALYSISFORFUELSANDCHEMICALS

DavidCannella

PhotobiocatalysisUnit,UniversitéLibredeBruxelles,Belgium

E-mail:[email protected]

EverytimeasinglephotonhitsplanetEarth,acomplexsetofreactionsdesignedforcapturing

itsenergystartsrunning,ignitingachainofeventswhoseoutcomesare:theoxygenicphotosynthesis,

thegrowthofbiomassthroughreductionofcarbon,andultimatelytheoriginoflife.Tocloseitscycle,carbonneedstobeoxidizedandreturnedtotheatmosphere.Recentlyhasbeenreportedanefficient

light-drivenenzymaticsystemtooxidizecarbohydratesfromcellulose,whichisthemajorfractionof

the photosynthetically fixed carbon. The system discovered is based on photosynthetic pigmentchlorophyll,andtheubiquitousenzymelyticpolysaccharidemonooxygenase(LPMO)thatdegrades

carbohydrateslikeplantcellwall,starch,andchitinfoundinallecosystems.Sinceitconsumesoxygen

andutilizesthechlorophyllandenergyoflighttodegradetheproductofphotosynthesis(cellulose),it

hasbeencalledinpopularterm“ReversePhotosynthesis”.

This breakthrough provides us with monooxygenases, a major and powerful class of redox

enzyme,asabasictoolto initiateandexpandresearchonthebiggestchallengeswithinchemistry,

physicsandbiology:acompletelyrenewable/greentechnologycapableofconverting light’senergy

into chemical energy (i.e. unique oxidized oligosaccharides, phenols, organic acids, methanol andethanol). Such a technology could indeedmake a real contribution to the 21st century biobased

society.


49

PHOTOREACTIVECROSSLINKINGREVEALSNOVELDIRECTINTERACTIONPARTNERSOFTHEPERSISTERREGULATOROBG.

PaulineHerpels1,ElenLouwagie2,JosepRayoCompany3,AnastassiosEconomou3,

NatalieVerstraeten1,JanMichiels1

1VIB,KatholiekeUniversiteitLeuven,Belgium2KatholiekeUniversiteitLeuven,Belgium

3Rega,KatholiekeUniversiteitLeuven,Belgium

Chronicinfectionsthatarerecalcitranttotreatmentwithantibioticsareposingamajorthreat

tohumanhealth.Thisrecalcitranceispartlycausedbyso-calledpersistercells.Persisterscomprisea

smallfraction(typically<0.1%)oftransientlyantibiotic-tolerantcellsthatareabletosurviveprolongedexposuretoantibiotictreatment.Althoughitsclinicalimportancehasbeenelaboratelydocumented,

theprecisemolecularmechanismsunderlyingpersistencearenotfullyunderstood.Previousresearch

inourgrouphasdemonstratedacentralrolefortheconservedGTPaseObginmediatingpersistence.

Unraveling the precise functioning of Obg-mediated persistence will shed light on this transientphenomenon and identify possible targets for anti-persister therapies. To identify novel direct

interactionpartnersofObg,aninnovativephoto-crosslinkingtechniquewasused.Thismethodallows

covalent binding of direct binding partners in vivo, which is an important feature concerning thetransientnatureofpersistence.Theunnaturalphoto-reactiveaminoacidp-benzoyl-L-phenylalanine

(pBpa)isincorporatedatspecificlocationsoftheObgprotein.UnderinfluenceofUVradiation,the

carbonyloxygenofpBpacrosslinkstoanycarbon-hydrogenbondofmoleculeswithinaradiusof3

angstrom. We used this method to incorporate pBpa in Obg from Escherichia coli (ObgE). Byexperimentaloptimization,wewereabletoefficientlyincorporatepBpaatdifferentresidueslocated

onthesurfaceofObgEandObgED246G(mutantformdeprivedofthepersisterfunction).Crosslinking

ofObgEwithpossibleinteractionpartnerswasdetectedandtheinteractingmoleculeswereidentified

usinghighresolutionliquidchromatography-massspectrometry.Resultsarecurrentlybeingconfirmedand complemented using a bacterial-two hybrid approach. Further testing of the identified direct

interaction partners will result in a selection of genes specifically involved in the Obg-mediated

persistencepathway.The fullunderstandingof thispathwaywill contribute to thedevelopmentof

anti-persister therapies.We hypothesize that targeting Obg-mediated persistencewill significantlyreducethenumberofpersistercellsinbacterialpopulations,therebyfacilitatingclearingofinfections

byconventionalantibiotics.

50

THEEFFECTOFTHELAYERTHICKNESSONTHEPERFORMANCEOFPOROUSLAYEREDRADIALLYELONGATEDPILLARALLAYCOLUMNS.

ShuntaFutagami1,TakeshiHara2,HeidiOttevaere1,HermanTerryn1,GinoV.Baron1,Gert

Desmet1,WimDeMalsche1

1VrijeUniversiteitBrussel,Belgium2KyushuUniversity,Japan

Vast progress in the column efficiency of pillar array columns has been achieved with theadoptionof radiallyelongatedpillars (REPs)asanalternativetoclassicalcylindricalpillars,andthispillar shape can provide a sub-micron minimum plate height. We recently reported that themesoporous silica layerprepared in aREP columnby sol-gel processingdramatically increases theretentioncapacitywhilekeepingitshighcolumnefficiency[1].Inthiscontribution,aREPcolumnwithathickermesoporoussilicalayerisreportedanditsperformanceiscomparedwithamesoporoussilicalayeredREPcolumnpreparedwiththepreviouslyreportedrecipe.

MesoporoussilicalayerwaspreparedinaREPcolumn(1mmwide,18μmdeep,and16.5cmlong)witha sol-gel feed solution,which consistedof amixtureof tetramethoxysilane (TMOS)andmethyltrimethoxysilane(MTMS),polyethyleneglycol(PEG),urea,andaceticacidaqueoussolution.Inordertoprepareathickerlayerthantheonepreviouslyreported[1],thefeedsolutioncontainedmoreTMOS/MTMSmixture, and less PEG to allow for slower gelation. After the C18modification, theretentioncapacityandthecolumnefficiencywereexaminedbychromatographicmeasurementswithalkylphenones.Scanningelectronmicroscopy(SEM)wasappliedtovisuallyinspectthemesoporoussilicalayers.

SEMimagesofamesoporoussilicalayeredREPcolumnshowedstraightanduniformdepositionofthe layer.FromtheSEMimages,thethicknessofthe layerwasdeterminedtobe220nm, . It isnoteworthythatthepreparationconditionforthethickerlayercoveredtheglasslidwithasthickalayerasthatonsilicon. Incontrast, itwasnotthecasewiththepreviouslyreportedcondition. It isassumedthatinthethickerlayercondition,coveringtheglasslidismorefavoredthangrowinganeventhicker layer on silicon. The retention capacity of the PLREP columns was compared by LCmeasurements with alkylphenones with mobile phases of 60, 70, 80 and 90% acetonitrile. ThemeasurementsrevealedthatthePLREPcolumnhavingathickerlayerhas1.3timeslargerretentioncapacity than thePLREP columnhaving a thinner layer. Comparisonof theH-uplotsof thePLREPcolumnsforoctanophenonedemonstratedthattheincreaseintheminimalplateheightof0.94µmto1.43µmwasaccompaniedbytheoptimalvelocityshift from0.10mm/sto0.24mm/swhengoingfromathinnertothickerlayer.Theseverylowplateheightsoutperformclassicalpackedbedcolumnsandhavegreatacademicandindustrialpotential.

References

[1]Futagami,S.etal.J.Chromatogr.A2017,1523,234–241.


51

MATRXCELL:IMPROVINGCELLCULTUREASSAYSWITHTUNABLEHYDROGELRIGIDITIES

MarieVersaevel1,SylvainGabriele1

1UniversityofMons,Belgium

The development and testing of drug candidates require large amount of upstream in vitro

screening assays. These tests are usually performedwith primary or immortalized cells grown on

culturedishesmadeofrigidpolystyreneorglass,whicharerarelyfunctionalizedwithaspecificproteincoating.Inaddition,plasticandglassculturedishesarecharacterizedbyaveryhighstiffness(~10^9

Pa), far from the softness of physiological tissues (10^2-10^5 Pa). However, many works recently

demonstrated that cells are able to sense themechanical properties of their environment and to

respondbyamodulationoftheirshape,internalstructureandbiologicalfunctions.Asaconsequence,rigid culture substrates can alter the cell sensitivity to drug candidates, that can lead to

misinterpretations in in vitro testings.Cell sensitivity to chemotherapeuticagents forexamplewas

clearlydemonstratedtobedependentonsubstratestiffness.

Indeed, accumulating evidence suggests that the physico-chemical properties of the cellular

microenvironmentmodulateimportantcellularfunctions(e.g.proliferation,migration,differentiation,

…).Weaddressedthischallengebydevelopinginnovativesofthydroxy-PAAmculturesubstratesthat

enableaprecisecontrolofboththestiffnessandthebiologicalcoating(patentUS20150104812andEP2854885).Thisstrategypermitstoobtaintailor-madeculturesubstratesthatmatchthephysico-

chemicalenvironmentofthetissueconcerned.Hereweshowthatbraincells(neuronsandastrocytes)

grownontheseoptimizedculturesubstratespresentamorerelevantphenotypeandoptimalcellular

functionscomparedtoclassiccellcultureconditions.WeaiminlaunchingtheMatrXcellspin-offthatproducesandcommercializesinnovativecelltype-optimizedculturesubstrates.

WeacknowledgetheFIRSTSpin-offprogramoftheWalloonRegionforthefundingofthisproject.


53

POSTERSESSIONS

54

Unevennumbers–PosterSession1Evennumbers–PosterSession2

ChemistryandChemicalEngineering

P1.NathanielBerneman(VUB).Nano-PrecisionConstructionEngineeringatthe1-MicronScale:NewHierarchicalMaterialsandNewApplications.P2. Agata Kryj (VUB). Development of novel column support structures in microchips forelectrochromatographicseparations.P3.MartynaBaca(VUB).Achievingapeakcapacityof1500usingan8mlongchromatographicchip.P4.Benoit Thienpont (VUB).Colorfulmicrospheresmadeof crystallizednanospheres inapolymermatrix.P5. IlseDeLeersnyder(UGent).The influenceofmediacomponentsonthestabilityandtoxicityofsilvernanomaterials.P6.ThomasVirdis(VUB).Non-IdealityinTheAdsorptionOfEthanol/EthylAcetate/WaterMixturesOnZIF-8MetalOrganicFramework.P7. José Luís Dores-Sousa (VUB). Mixed-mode retention mechanisms and selectivity of reversed-phase/weak-anion-exchangecolumnsinliquidchromatography.P8.ChristinaTiflidis(VUB).ElectrochemicalStudyofITOasaWorkingElectrodeforaTransparentChip.P9.JoséLuísDores-Sousa(VUB).Prototypingofmicrofluidicthermoplasticchipsforhigh-performanceliquid-chromatography.P10.AtirujTheppawong(UGent).Synthesis,BiologyandStructure-ActivityRelationshipsofNovelβ-enaminone-,aza-aromatic-andthiazepane-basedCurcuminoids.

AgricultureandFoodTechnology

P11.JoseC.Jimenez-Lopez(EEZ–CSIC,Spain).Flavonoidssyntheticpathwayenzymesasbiomarkersfor the study of biochemical and physiological changes occurring during narrow-leafed lupin seedgerminationandseedlingdevelopment.P12. Oyakhilomen Oyinbo (KUL). Maize Farmers’ Preferences for Site-specific Extension ServiceDeliveryinNigeria:InsightsfromaChoiceExperiment.P13.BrackeJolien(UGent).Nondestructiveopticalcropsensingtomonitornitrogencontentinwoodyornamentals.P14.AliSedaghatDoost(UGent).Formulationofnovelbiopolymernanoparticlesfortargeteddeliveryofbioactivecompounds.P15. Claire Chigwedere (KUL). Influence of storage on the cooking quality of Canadian wondercommonbeans (Phaseolus vulgaris): insight intomechanismof softeningandevolutionof volatilecompounds.P16. Wim Geeraerts (VUB). Lactic acid bacteria species diversity on a selection of cooked meatproductsintheBelgianretail.P17.VincentGreffe (KUL). Improvingtheshelf lifeof rhizobia inoculants:apangenomicsapproachinvestigatingstress-resistanceinSinorhizobiummeliloti.P18.VictorMoreto(KUL).Calibrationofasugarcanecultivarinaquacropmodel.P19.FlorentAudrit(ULB).Effectoftheoxidativeactivityoflaccaseonfoodproducts.


55

P20.MaremeNiang(CERAAS,Sénégal).Germinationresponsestodroughtstressamongstbaobabs(AdansoniadigitataL)inSenegal.P21.ElsDebonne(UGent).Assessmentoftheantifungalpreservationpotentialofnaturalwateroroilsolublecompoundsinbothin-vitroandbreadbakingtrials.P22.CatherineMermans (UGent).AG326Esubstitution in theglutamate-gatedchloridechannel3(GluCl3) of the two-spotted spider mite Tetranychus urticae abolishes the agonistic activity ofmacrocycliclactones.P23. Niels Langenaeken (KUL). Contribution of small and large barley starch granules to maltoseproductionduringmashing.P24.WillyIrakoze(UCL).Comparativeeffectsofchlorideandsulfatesalinitiesontwocontrastingricecultivars(OryzasativaL.)attheseedlingstage.P25.LauraDeMets(UGent).Crossingborderstomitigatemycotoxins:wheremicrobiology,poly-omicsandcropprotectionmeet.P26.EmielVanReckem(VUB).Theinfluenceofprocessingconditionsonthecommunitydynamicsofcoagulase-negativestaphylococciduringmeatfermentation.P27. Marko Verce (VUB). The composition and functional potential of water kefir fermentationmicrobiotaasrevealedthroughshotgunmetagenomics.P28.WannesVanBeeck(UA).Fermentedcarrots:Anovelfoodmatrixforprobioticinterventions.P29. Louise Vermote (VUB). Cheese brines harbour both halophilic/halotolerant and cheeseingredient-associatedmicroorganisms.P30.BiancaDrepper(KUL).NewweatherrelatedinsurancemodelsfortheFlemishhardfruitsector.P31. Savio Rodrigues (KUL). Hairy Root Disease: Unravelling the molecular interactions betweentomatorootsandrhizogenicagrobacteria.P32.RayangnéwendéAdèleOuédraogo (UCL).Evaluationdedifférentes formulationsdecompostsassociéesounonauTrichodermaetauxchampignonsmycorhiziensarbusculairessurlerendementdelatomate(Solanumlycopersicum)etsurlesolenconditionssemi-contrôlées.P33.PhuongNguyenNhu(VUB).AnovelstressresponsetriggeredbyERD14.P34. Manon Demulder (VUB). Chemical inhibitors of the SOG1 transcription factor to promotealuminiumtoleranceinmaizeandbarley.P35. Anne-SophieMathieu (UCL). Impact of high temperatures on sugarmetabolism and yield inCichoriumintybusL.varsativum.P36.MarieVandermies(ULg).VersatileregulatedpromoterforYarrowialipolytica.P37.AlexisFernandez(ULg).Doespearlingprocessconcentrateβ-Glucansinbarleygrain?P38.AmauryGérard(ULg).WhatisthelandscapeofartisanalcheeseproductioninBelgium?P39.HassinaHafidaBoukhalfa(MohamedKhiderUniversity–Biskra,Algeria).Improvementofsprayretentiononbarleyleaves.P40.QuentinRivière(ULB).Roleofthecircadianclockintheresponseoftheplant-modelArabidopsisthalianatomagnesiumdeficiency.P41.SoundousEl-Hajjaji(ULg).InventoryoftherawmilkbutterproductioninWallonia(Belgium).P42.AmauryGérard(ULg).StudyofthegrowthpotentialofListeriamonocytogenesincheesesmadebyBelgianfarmers.P43. Phuc LeHo (KUL). Changeof electrolyte leakageof dragon fruit (Hylocereus undatus) duringstorage.P44.RedaMelhaoui(UniversitéMohammedPremierOujda,Maroc).TocopherolanalysisinalmondoilsproducedineasternMorocco.

56

P45. Ali Sedaghat Doost (ULg). Fabrication of thymol nano-emulsions as a potential naturalantioxidant.P46.KahramenDeghnouche(MohamedKhiderUniversity–Biskra,Algeria). InventoryandstudyofthenutritionalvalueofspontaneousforageplantsinanaridregionofAlgeria.P47.FinguMabolaJuniorCorneille(ULg). Impactofendophyticentomopathogenicfungusonplantattractivityinaphid-virus-plantinteractions.P48.HassinaHafida Boukhalfa (MohamedKhiderUniversity – Biskra, Algeria). Evualution of sprayretentionvariabilityofhydrophobicplantleaves.P49. Kahramen Deghnouche (Mohamed Khider University – Biskra, Algeria). Evaluation of growthperformanceOuledDjellallambsaccordingtosex,sizeofthelitterandtheparityinasemi-aridregionofAlgeria.P50.JulienLouvieaux(ULB).Below-groundapproachestoimprovenitrogenuseefficiencyinoilseedrape.

Biomathematics

P51.FlorisDalemans(KUL).ApredictionmodelfortheseedandoilyieldofPongamiapinnata.P52.GlebGoussarov(UGent).WholeGenomeStatisticsforComparingBacterialStrains.P53.NicolasDierckxsens(ULB).Targetedassemblyandvariancecallingfromwholegenomedata.P54.JanisBaeten(UGent).Asimplemodelforaerobicgranularsludge.P55.JoëlleDeCaluwé(ULB).Modelingthephotoperiodicentrainmentoftheplantcircadianclock.P56.FrançoisAncien(ULB).Predictionandinterpretationofdeleteriouscodingvariantsintermsofproteinstructuralstability.P57.TamasLazar(VUB).Biophysicalvalidationoffullydisorderedproteinensembles.P58.StijnWittouck(UA).ComparativegenomicsofLactobacillusbacteria.

HumanHealth

P59. Jone Paesmans (VUB). Revealing the function of TBC1D24mutations in epilepsy and relatedneurologicaldiseases.P60.JoseC.Jimenez-Lopez(EEZ–CSIC,Spain).Bluelupinseedβ-conglutinproteinsreversebacktheinducedinsulinresistanceinpancreaticcells.P61.HungNguyen(VUB).Validationofdenovodesignedcalpaininhibitors.P62.MohamedMusrati(VUB).Ly6ClowMonocytesPromoteLiverRegenerationduringDrug-InducedLiverFailure.P63.MateKiss(VUB).Non-canonicalIL-1βreleasebymyeloidcellspromotestumorprogressionandresistancetoimmunotherapy.P64.EvangeliaBolli(VUB).Nanobody-baseddepletionofprotumoraltumor-associatedmacrophagesasnovelcancertherapy.P65.AnnegreteVisnapuu(KUL).Studyingancientmechanismtosynthesizenovelanti-biofilmdrugs.P66.TingWu(UGent).Anti-inflammatoryeffectofAroniapolyphenolsincombinedmicrobialinvitrodigestion/cellculturemodels.P67. KennyAppermans (KUL).Gradients of antimicrobials influence the development of antibioticresistance.


57

P68.ShahbazBashir(KUL).Quantificationofimmuneresponseinskinswellingtestusinganimage-basedplatform.P69.AnaRitaPomboAntunes(UGent).Dissectingtheheterogeneityandfunctionsoftheglioblastomaimmunecompartment.P70.LaureVerstraete(KUL).InvitroandinvivoidentificationofpersisterdeterminantsinnaturalandclinicalPseudomonasaeruginosastrains.P71. Laetitia VanWonterghem (KUL). Identification and characterization of bacterial host factorsinvolvedinconjugativehorizontalgenetransfer.P72. Bram Lories (KUL). Responses to ecological competition link biofilm formation, virulence andantibioticresistanceinSalmonella.P73.MiekeDeBuck(KUL).COOH-terminalSAA1peptidesfailtoinducechemokinesbutsynergizewithCXCL8andCCL3torecruitleukocytesviaFPR2.P74. Helena Van Damme (VUB). The CCL8/CCR8 axis maintains an immunosuppressive tumormicroenvironment.P75. Hanne Vissenaekens (UGent). Cellular quercetin uptake, localization and metabolism understressedconditions.P76. Khaoula Toumi (ULg). Assessment of exposure of workers to pesticide residues in tomatogreenhouses.P77.HassinaHafidaBoukhalfa(MohamedKhiderUniversity–Biskra,Algeria).Operatorexposurerisksinphytosanitarypractices,caseofBiskra–Algeria.P78. Sara Abouelasrar Salama (KUL). Proteolytic cleavage of serum amyloid A (SAA) 1 by matrixmetalloproteinasesfine-tunestheinflammatorycapacityofthisacutephaseprotein.

Environment

P79.HassanMaklad (VUB). Phosphorylationof theTetR transcription factor FadR in thearchaeonSulfolobusacidocaldariusaffectsitsligandbindingability.P80.ZeLiu(UGent).CombinationofO3/H2O2andUVtoassessdegradationofatrazineinaqueoussolution.P81.VereLeybaert(KUL).QuantifyingPrecipitationErrorsviaSoilMoistureDataAssimilation.P82.HuiXu(UGent).Greatercontributionofbelowgroundthanabovegroundmaizebiomasstothestablesoilorganiccarbonpool.P83.MichielMaertens(KUL).TheimpactofvegetationchangesonsoilmoistureovertheDryChacoP84.TessGoessens(UGent).Environmentalscreeningofmulti-classantimicrobialdrugresiduesinsaltandfreshwaterecosystemsusingtargetedanduntargetedhighresolutionmassspectrometry.P85. Nina Ricci Nicomel (UGent). Development of a high-throughput automated platform forstandardizedmetalbiosorptionstudies.P86. AbuakriWumbei (UGent). Pesticides Residue Analysis in Yam from SelectedMarkets AcrossGhanaandBelgium:AnEvaluationoftheQUECHERSMethod.P87.AnuYadav(SCK-CEN).Radiationresistanceinthecyanobacteriumarthrospira.P88. Gilles Olive (Ecole Industrielle et Commerciale de la Ville de Namur). Can Medauroideaextradentata (Brunner von Wattenwyl, 1907) and/or Haaniella echinata (Redtenbacher, 1906)(Phasmatodea:Phasmatidae)helpustofightFallopiajaponica((Houtt.)RonseDecr.,1988)?P89.TriTruong(UGent).DevelopmentawatermonitoringsystembasedonbiologicalindicatorswithBenTreriversystem-thelatestbranchofMekongRiverBasinbeforegoingtothesea.

58

P90. Kamilia Farhi (Mohamed Khider University – Biskra, Algeria). Estimation of the Black belliedsandgrousegroup’ssizeinwateringsiteinSouth-EastofAlgeria.P91.ElenaBorregánOchando(UA).IncorporatingtheUSEPA’s“CompTox”dashboardandthe“For-Ident”platforminworkflowsfortheidentificationandpreliminaryassessmentoforganicemergingmicropollutantsinwater.P92.Marlies Volckaert (UGent). Supply chain of forest products supported by spatial informationsystems.P93. Liesbeth Lemmens (VUB). Transcription factor-mediated gene regulation in response to heatshockstressinthermoacidophilicarchaea.P94.EdelbisLópezDávila(UGent).PesticidesknowledgeandusebyCubanfarmersfromSanctiSpíritusregions.

Key-enablingTechnologies

P95.FréderickMatheuse(VUB).LiquidChromatographyandadvanced3Dprinting:amatchmadeinheaven?P96.LisaTripodi(KUL).Plasticbasedhydrophilic-in-hydrophobicmicrowellarraysfordigitalbioassays.P97.JolienBreukers(KUL).DevelopmentofamicrofluidicplatformforscreeningandsortingofsingleBcells.P98.DavidSybers(VUB).DevelopmentoffluorescentreportersforthermoacidophilicarchaeaP99.KarenVen(KUL).DNA-onlydigitalbioassayP100. Bernd Peeters (KUL). AuNP-labelled FO-SPR sensors to develop innovative DNAzyme-basedbioassayconcepts.P101.MarineLuciano(UMons).Decipheringtheroleofthecurvatureinfoldedepithelialtissues.P102. Laura Alaimo (UMons). Collective cell migration in geometric confinements imposed bymicrochannels.P103.DriesVloemans (KUL). Integrationof passive SAT-basedheating systemonPOCmicrofluidic(i)SIMPLEplatform.P104. Iene Rutten (KUL). DNA origami nano-architectures for well-defined and robust biosensinginterfaces.P105.Michiel Van Tendeloo (UA). Promising nitrogen removal performancewith theMainstreamAnammoxSystem(MAS)forsewagetreatment.P106.SteveSchoonooghe(VUB).Nanobodies:fromresearchtoolstodiagnostics&therapeutics.

POSTERCONTRIBUTIONS

Extendedabstracts

60

FLOWERING AND FRUITING OF YELLOW PASSION FRUIT (PASSIFLORA

EDULIS, VAR F. FLAVICARPA.DEG) AS INFLUENCED BY DIFFERENT FERTILIZER APPLICATIONS IN KIAMBU AND EMBU COUNTIES, KENYA

MWIRIGIPETER1,JOSEPHGWEYI-ONYANGO1ANDMAINAMWANGI1

1DepartmentofAgricultureScienceTechnology,KenyattaUniversity,P.O.Box43844-00100,Nairobi-Kenya

INTRODUCTIONInKenyatheyellowpassionfruit(Passifloraedulis,varf.flavicarpa.deg)varietyhassourandsweetcultivarswhicharemostlysuitedtolowaltitudes.Thevarietyalsothriveswellinmidaltitudesofupto1500mabovesealevelandlatelytherehasbeenincreasedproductionofthisfruitinsemi-aridregionsofEmbu,Meru,TharakaNithiandKirinyagacounties(HCDA,2013).ThestrengthofyellowpassionisthatitisthoughttobemoretoleranttoFusariumwilt,adiseasethathasseverelyreducedpurplepassionfruitproductivity.Worldwide,Brazil isconsideredthecenterofdiversityforthegenusPassiflora,andtheyellowpassionfruit(PassifloraedulisSimsf.flavicarpaDeg.)isthespeciespredominantlygrown(Oliveiraetal.,2012).Brazilisalsothelargestproducerandconsumerofpassionfruitintheworld(JanickandPaul,2008).

ThefruithasemergedasanimportanthighvaluehorticulturalcropinKenyaandhasgainedwideadoptioninMbeere,EmbuCountyduetoitsapparentadaptationtothehotaridconditionsandareadymarketforthefruit.ThefruitismainlyconsumedlocallyandalsoexportedbysomeKenyacompanies(HCDA,2013).Nevertheless,thecurrentproduction levelsofyellowpassion fruit are lowdue to poor agronomic and fertilizermanagement, declining soil fertility levels and erratic rainfallpatterns.

Analysisofsoilandassessmentofplantbehaviorundervaryingamountofnutrientsisrequisiteinoptimizationofnutrientrequirementsforpassionfruitplants.Inaddition,basedonthefactthateachnutrienthasaspecificroleinthephysiologicalfunctionsofplants,thenimbalancesoftenresultsto lowyieldandfruitsofpoorquality. Inadditiontowaterdeficit,theMbeereregionofEmbufacehighsalinity,usuallyduetothehighevapotranspirationdemand,reducedprecipitationandpoormanagementoftillage(DoneenandWestcot,1988).Pooruseoffertilizersbyfarmersfurtherexacerbatesthisproblem.AccordingtoAyersandWestcot(1999),yellowpassionfruit ishighlysensitivetotheactionofsaltshenceirrigationwithsalinewaterandincorrectfertilizerapplicationmayproduceundesirableeffectsinthephysicochemicalqualityofthefruit(Costaetal.,2001;Freireetal.,2010;Diasetal.,2011).Allthesefactorsaffectthefruitformindexleadingtoshriveling,malformationsanddeformedfruits;importantattributesinclassificationandstandardizationhenceaffectingtheacceptanceandjudgmentoftheproductinconsumermarkets.

Themainobjectiveofthisstudywastoassessflowerinductionandfruitformationofyellowpassionundervaryingfertilizertreatmentsintwodifferentecologicalstudysites.

MATERIALSANDMETHODSSixty-fourplantsbelongingtoKPF4varietyweregrownintwositesatKenyattaUniversity(KU)FarmandatUgwerifarmer’sorchardinEmbu.ExperimentaldesignTheexperimentwaslaidoutinaFactorialCompleteRandomizedBlockDesignwithplantingandtopdressingfertilizersasthemainfactors.TheplantingfertilizerswereFarmyardmanureandDiammoniumphosphate(DAP)whilethetopdressingwasCalciumammoniumnitrate(CAN)andNitrabor.Thetreatmentsincluded100gDAP+20kgManure+50gNitrabor;100gDAP+10kgManure+50gNitrabor;100gDAP+50gCAN;100gDAP+50gNitrabor;10kgManure+50gCAN;10kgManure+50gNitrabor; 20kgManure+50gCAN; 20kgManure+50g Nitrabor; 50g DAP+10kgManure+50g CAN, 50g DAP+50g CAN, 50gDAP+50gNitraborandControl(nofertilizer).DatarecordingandanalysisDatarecordingbegunattheonsetofthefirstflowerbudandthenumberofunopenedflowerbuds,openflowersandyoungunripefruits(fruitsformed)wasrecordedonaweeklybasisfortwoconsecutiveseasonsofDecember2016andApril2017.Thedatacollectionwascontinueduntilapproximately80%oftheflowershadformedfruits.AnalysisofvarianceusingSASsoftwareversion9.1wasperformedonthedata.Thedatawascodedasfollows:

Treatment Code Treatment Code100gDAP+20kgManure+50gNitrabor 1 20kgManure+50gCAN 7100gDAP+10kgManure+50gNitrabor 2 20kgManure+50gNitrabor 8


61

100gDAP+50gCAN 3 50gDAP+10kgManure+50gCAN 9100gDAP+50gNitrabor 4 Control 10

10kgManure+50gCAN 5 50gDAP+50gCAN 1110kgManure+50gNitrabor 6 50gDAP+50gNitrabor 12RESULTSANDDISCUSSIONSResultsindicatethatthereweresignificantdifferences(≤0.05)amongthetreatments(Table1).Table1:AnalysisofthevariousdependentvariablesforfertilizertreatmentinKenyattaUniversityFarmandUgweriorchard

DependentVariable:Flowerbudinitiation

Source DF SumofSquaresMeanSquare FValue Pr>F

Model 16 68076.8636 4254.804 15.61 <.0001

Error 278 75782.133 272.5976

CorrectedTotal 294 143858.9966

DependentVariable:Openflower


Model 16 19701.77955 1231.36122 17.06 <.0001

Error 278 20062.54588 72.16743

CorrectedTotal 294 39764.32542

DependentVariable:Fruitsformed


Model 16 116328.2391 7270.5149 12.57 <.0001

Error 278 160753.0965 578.2485

CorrectedTotal 294 277081.3356 Additionally,fortheflowerbudinitiationandopenflowers,thereweredifferencesforthetwositeswithEmbuhavingthehighestaveragenumberofflowerbudsandopenflowers(Figure1).

Fig1:ComparisonofthemeannumberofflowerbudsandopenflowersinthetwostudysitesThecombinedtreatmentsof100gDAP+10kgManure+50gNitraborand20kgManure+50gNitraborshowedthebestresultsintermsofflowerandfruitformationforthetwosites(Figure2).ProbablythecommercialNitraborfertilizer(15.4%N+

0

5

10

15

20

25

30

35

40

1 3 5 7 9 11

Meannu

mbe

rofflowerbud

s

Treatment

Embu

KU

0

5

10

15

20

25

30

35

1 3 5 7 9 11

Meannu

mbe

rofo

penflo

wers

Treatment

Embu

KU

62

25.9%CaO+0.3%B)hadaneffectonthefloweringpossiblyduetoitsenhancedboroncontentandtheimprovedsolubilityofcalcium.Ontheotherhand,treatments9,10and11whichwerelackingtheNitraborshowedlessnumberofopenedflowersandfruitsformed.ThisstudyfurtheragreeswithfindingsbyFreitasetal.(2006)whichdemonstratedthatnitrogendeficit influences fruitquality, reducing fruitnumberperplant, thepercentageof seed in fruit, °Brixand thevitaminCcontentinthejuice,inadditiontoincreasingskinpercentageandfruitthickness.

Fig2:MeannumberofopenflowersandfruitsformedatthetwositesofstudyThe number of fruits formed increased over the season and had differences in the seasons but there were no majorobservationaldifferencesbetweenthetwosites.However,ahighnumberoffruitdropandespeciallyinEmbuwasobservedwhicharoseasaresultoftheprolongeddryspell.CONCLUSIONThis study has demonstrated that varying fertilizer application can have significant impact on the flowering and fruitformationofyellowpassionwhicharekeydeterminantsofthepotentialyieldofanorchard.REFERENCESAyers,R.S.;Westcot,D.W.(1999).Aqualidadedaáguanaagricultura.CampinaGrande:UFPB,pp.1-158,(FAO:Drainagepaper,29).Costa,J.R.M.;Lima,C.A.A.;Lima,E.D.A.;Cavalcante,L.F.;Oliveira,F.K.D.(2001).Caracterizaçãodosfrutosdemaracujáamareloirrigadoscom

águasalina.RevistaBrasileiradeEngenhariaAgrícolaeAmbiental,5(1):143-146.Doneen,L.D.andD.W.Westcot(1988).Irrigationpracticeandwatermanagement.IrrigationandDrainagePaperNo.1,Rome,FAO.Freitas,M.S.M.,P.H.Monnerat,L.G.R.Pinho,andA.J.C.Carvalho.(2006).Mineraldeficiencyofmacronutrientsandboroninsweet

passionfruit:fruitquality.RevistaBrasileiradeFruticultura28:492–496.[Crossref]).HorticulturalCropDevelopmentAuthority(HCDA).NationalHorticultureValidatedReport.2013.Janick,J.,Paull,R.E.,(2008).TheEncyclopediaofFruitandNuts.CABI,London.Oliveira, E.J., Dias, N.L.P., Dantas, J.L.L., (2012). Selection of morpho-agronomic descriptors for characterization of papaya cultivars.

Euphytica185,253–265.

0

5

10

15

20

25

30

1 2 3 4 5 6 7 8 9 10 11 12

Mea

n nu

mbe

r of

flow

ers

Treatment

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12M

ean

num

ber

of fr

uits

form

ed

Treatment


63

QUANTIFICATION OF IMMUNE RESPONSE WITH A SKIN SWELLING TEST USING AN IMAGE-BASED PLATFORM

SHAHBAZBASHIRA,BERNICELIMA,B,ALIYOUSSEFALIAMERB,TOMASNORTONB,JANPAESHUYSEA

AHostPathogenInteractionLaboratory,DepartmentofBiosystems,FacultyofBioscienceEngineering,KasteelparkArenberg30,3001

Heverlee,KULeuvenUniversity,Belgium.BPrecisionLivestockfarmingLaboratory,DepartmentofBiosystems,FacultyofBioscienceEngineering,KasteelparkArenberg30,3001

Heverlee,KULeuvenUniversity,Belgium.Correspondence:ShahbazBashir([email protected])

INTRODUCTIONEcoimmunologyaddressesthecoreconceptofbiologicalorganizationatthelevelofanindividual´simmunesystemanditsevolution in a given ecological and life history context. Since its emergence, it has widened our knowledge about thedevelopmentofimmunesystemfrommechanismatmolecularleveltoitsphysicaloutcome.Butstilltoday,howtomeasuretheindividualimmunityremainsitscentralconundrum(Brock,Murdock,&Martin,2014).Despiteofanastoundinglargenumber of immunological tests available, complex andmultifaceted nature of the immune systemmakes it difficult tounderstandandencompassitsdiverseanddynamicalaspects(Demas,Zysling,Beechler,Muehlenbein,&French,2011).Inthe field of ecoimmunology and ecotoxicology, Systematized examination of individual's immunocompetence followingexperimentalelicitationofimmuneresponsethroughmitogeninjectionisconsideredasastandardapproach.Skinswellingtest is a cheap and simple approach allowing researcher the opportunity to estimate the cell-mediated immuneresponsivenessofthe individualbyusingdifferentmitogens.Thistest isbasedonthemeasurementoftheskinswellingresponse at specific times following subcutaneous or intradermal administration of experimental pro-inflammatorystimulant(Vinkler,Bainová,&Albrecht,2010).Inthisregards,phytohaematagglutinin(PHA),aplantlectinextractedfromPhaseolusvulgaris,isconsideredasauniversalmitogenofchoice.Forinvivoapplications,PHA-P,acombinationofPHA-L(Leukoagglutinin)andPHA-E(Erythroagglutinin),ispreferred(Kennedy&Nager,2006).SkinswellingresponseofPHAhasmostlybeeninvestigatedinbirds(Adelman&Ardia,2014;Martinetal.,2006;Tollingtonetal.,2015),amphibians(Brown,Shilton,&Shine,2011)andreptiles(Fingeretal.,2013).Incontrast,PHAproinflammatoryresponsehasrarelybeenexploredinmammals,particularlycow.Giventhecomplexityoftheimmuneresponse,theimmunologicalbackgroundmechanismsunderpinningtheskinswellingaftermitogenapplicationisstillnotwellunderstood.Theobjectiveofthestudyathandistousean image-basedplatformto investigateandquantify the inflammatoryresponseandunderlayingthermaldynamicalchanges basedon thewarmth generatedby increasedblood flow at the site of PHA injection in a cow. To best of ourknowledge,thisisthefirststudytounderstandtheinvivothermaldynamicsofanimalskininflammatoryresponseprovokedbyanymitogen.MATERIALANDMETHODSInfraredthermographicimagingandFLIROne™Electromagneticwavelengthspectrumofinfraredradiationrangesfrom700nanometer(nm)to1millimeter(mm).Allbodiesabovezerokelvin(K)emitinfraredradiationsinvisibletonakedhumaneyebetweenelectromagneticwavelengthspectrumrangeof3-15micrometer(μm)andcouldbecapturedbythesensorofthermalcamera.Eachpixelofthegeneratedimagecorresponds to the infrared radiation energy. Total radiation energy emitted from a body follows Stefan-Bolztman Law(Stefan-Bolzmannequation)andisdirectlyproportionaltothebody’ssurfaceareaanditsemissivity(Jiji,2006).

Stefan-Boltzmannequation𝑷=𝑨𝛆𝛔𝑻𝟒P=powerofradiation,A=surfaceareaofanobject,ε=emissivityoftheobject’ssurface,σ=Stefan-BoltzmannconstantandT=absolutetemperatureoftheobject.

To capture the thermal images of the skin swelling response, amobile and inexpensive commercial smartphone basedinfraredthermalcameraFLIROne™(Figure1)wasusedinthisstudyhavingthetemperaturedetectionrangefrom-20to120°Cwithasensitivityof0.1°C.FLIR®systemssoftwarereleasedsoftwaredevelopmentkittoaccesstherawdataofthecameraandcreateacustomsmartphone-basedapplication.

64

ExperimentalanimalandtreatmentinoculaThestudywasapprovedbyUniversityEthicalCommitteeforAnimalExperimentationKULeuven(projectnumberP069-2017)andconductedonHolsteincowatZoötechnischCentrum(ZTC)KULeuveninApril,2017.Bothsidesoftheneckwereshavedproperlyandprickedintradermallywith2.5mg/mlPHA-P(Sigma-Aldrich,L8754)inDPBS(Sigma-Aldrich,D5652),5mg/mlHistamine((Sigma-Aldrich,H7125)inDPBSaspositiveandDPBSasnegativecontrol.ExperimentalDesignAspecialcameraframe(Figure2)of15×15×20cmdimensionswasmadefrompolyvinylchloridetosupportthecameraandmobileandtoensuretheconstantdistanceofcameraperpixelfromtheskinatalltimesduringimagecapturing.Intensivethermographic imagingwith FLIROne™ and physicalmeasurementswith digital vernier caliper (POWERFIX®, Lidl)wereperformedat0hourpre-injectionandevery15minutesduringthefirsthourofpostinjections,everyhouruntil15hourspostinjectionsandthenat19,21,24,30and48hourspostinjectiontonoticethethermaldynamicsofinflammation.Thediameteroftheskinswellingareaanddoubleskinfoldthicknessweremeasuredasapartofphysicalmeasurements.ThermalimagescapturedbyFLIROne™werefurtherprocessedinMATLABR2016bbyusingtherawdata.RESULTSSkinswellingresponsewasrapidfollowingallinjections.Maximumswellingdiameterwascausedbyhistamine(80.58mm)followedbyDPBS(35.36mm)andPHA(34.98mm),respectively(Figure3).Themaximumswellingdiameterofhistaminewasmorethan2timesthanthatofcausedbyPHAandDPBS.HistamineelicitedmorerapidandaggressiveswellingascomparedtothegentleswellingofPHA.Theinterestingthingwasthetimingwhenmaximumswellingdiameterwasachieved,whichwasat3hourspostDPBSinjectionandat5hoursposthistamineandPHAinjection.Doubleskinfoldthicknessmeasurementis themost commonmethod to estimate the swelling response.Histamine caused themaximumdouble fold thickness(21.04mm)withalmost350%increment,whilePHAandDPBS(15.65and15.04mm)showedsimilartrendindoubleskinfoldthicknesswithjust150%increment(Figure4).Thermalimagingrevealedthatintradermalinjectionofhistamine(Figure6B)andDPBS(Figure5B)causedaquickreactionandachievedamaximumtotalsurfaceareaoftemperatureincrementwithinfirst1.5hourspost-injectionascomparedtothedelayedreactionshownbyPHA(Figure7B)6hourspostinjection.Thearea of temperature increment caused by PHAwas larger than that of achieved by histamine, despite of the fact thathistaminecauselargerskinswellingandthickness.

Figure 1. FLIR One™ infrared thermal camera for Android

Figure 2. Camera frame

Figure 3. Skin swelling diameter

Figure 4. Double skin fold thickness increment with each hour


65

AnotherinterestingfeatureobservedwasthefactthatPHAswellingshowedlocalizedtemperatureincrement(Figure7),whilehistamine(Figure6)andPBS(Figure5)swellingshowedaspreadoutwardtrendoftemperatureincrementfromthesiteofinjectionwithalmostbeingattheborderofthethermalimagesat30hourspostinjection.DISCUSSIONThe physical measurements and imaging was done by the same person to minimize the error, thus enhancing therepeatabilityandprecision.Generally,Skinswellingtestsarebasedontheswellingasaresultofinflammationatthesiteofallergen/mitogeninjection.However,swellingisnottheonlysignofinflammation,heating/warmthcausedbytheincreasedbloodflowtothesiteofinjectionisalsoamongstthecardinalsigns.OurstudyutilizestheproinflammatorypotentialofPHAtounderstandthethermaldynamicsofinflammatoryresponse.Skinswellinganddoublefoldthicknessareininlinewiththestudiesconductedpreviously(Demasetal.,2011;Fingeretal.,2013).LowswellingresponseinourstudytoPHAmightbeduetothefactthatcowwasnotsensitizedtoPHApreviously.ObservationlocalizedtemperatureincrementtrendincaseofPHAanddisseminatedtrendincaseofhistamineandPBSisaninterestingpointtolookfor.

Thisstudyshowsthatobservationoftemperatureincrement,asaresultofallergicreactionatthesiteofmitogeninjection,couldbeapracticaltoolinquantifyingandunderstandingtheimmuneresponseofskinswellingtest.ACKNOWLEDGEMENTSTheauthorwouldliketoacknowledgeDVMWouterMerckxandallthestaffofZoötechnischCentrum(ZTC)KULeuvenfortheirkindcooperationandhelpduringthisstudy.TheauthorwouldalsoliketothankLudoHappaertsforbuildingthecameraframe.REFERENCESAdelman,J.S.,&Ardia,D.R.(2014).Chapter22–Ecoimmunology.InAvianImmunology(pp.391–411).https://doi.org/10.1016/B978-0-

12-396965-1.00022-4

Figure 5. Area of temperature increment processed in MATLAB R2016b provoked by I/D DPBS injection: A) immediate post-injection, B) maximum at 1-hour post injection and C) 30-hours post injection

Figure 6. Area of temperature increment processed in MATLAB R2016b provoked by I/D Histamine injection: A) immediate post-injection, B) maximum at 1.5-hour post injection and C) 30-hours post injection

Figure 7. Area of temperature increment processed in MATLAB R2016b provoked by I/D PHA-P injection: A) immediate post-injection, B) maximum at 6-hour post injection and C) 48-hours post injection

66

Brock,P.M.,Murdock,C.C.,&Martin,L.B.(2014).Thehistoryofecoimmunologyanditsintegrationwithdiseaseecology.InIntegrativeandComparativeBiology(Vol.54,pp.353–362).https://doi.org/10.1093/icb/icu046

Brown,G.P., Shilton,C.M.,&Shine,R. (2011).Measuringamphibian immunocompetence:Validationof thephytohemagglutinin skin-swellingassay in thecane toad,Rhinellamarina.Methods inEcologyandEvolution,2(4),341–348.https://doi.org/10.1111/j.2041-210X.2011.00090.x

Demas,G.E.,Zysling,D.A.,Beechler,B.R.,Muehlenbein,M.P.,&French,S.S.(2011).Beyondphytohaemagglutinin:Assessingvertebrateimmunefunctionacrossecologicalcontexts.JournalofAnimalEcology.https://doi.org/10.1111/j.1365-2656.2011.01813.x

EFSAPanelonAnimalHealthandWelfare. (2012).ScientificOpinionontheuseofagamma interferontest for thediagnosisofbovinetuberculosis1.EFSAJournal,10(12),2975.https://doi.org/10.2903/j.efsa.2012.2975.

Finger,J.W.,Adams,A.L.,Thomson,P.C.,Shilton,C.M.,Brown,G.P.,Moran,C.,…Isberg,S.R.(2013).Usingphytohaemagglutinintodetermine immune responsiveness in saltwater crocodiles (Crocodylus porosus). Australian Journal of Zoology, 61(4), 301–311.https://doi.org/10.1071/ZO13041

Jiji,L.M.(2006).Heatconvection.HeatConvection.https://doi.org/10.1007/978-3-540-30694-8Kennedy,M.W.,&Nager,R.G.(2006).Theperilsandprospectsofusingphytohaemagglutinininevolutionaryecology.TrendsinEcology

andEvolution.https://doi.org/10.1016/j.tree.2006.09.017Martin,L.B.,Han,P.,Lewittes,J.,Kuhlman,J.R.,Klasing,K.C.,&Wikelski,M.(2006).Phytohemagglutinin-inducedskinswellinginbirds:

Histological support for a classic immunoecological technique. Functional Ecology, 20(2), 290–299. https://doi.org/10.1111/j.1365-2435.2006.01094.x

Tollington,S.,Greenwood,A.,Jones,C.G.,Hoeck,P.,Chowrimootoo,A.,Smith,D.,…Groombridge,J.J.(2015).Detailedmonitoringofasmallbutrecoveringpopulationrevealssublethaleffectsofdiseaseandunexpectedinteractionswithsupplementalfeeding.JournalofAnimalEcology,84(4),969–977.https://doi.org/10.1111/1365-2656.12348

Vinkler,M.,Bainová,H.,&Albrecht,T.(2010).Functionalanalysisoftheskin-swellingresponsetophytohaemagglutinin.FunctionalEcology,24(5),1081–1086.https://doi.org/10.1111/j.1365-2435.2010.01711.x


67

BLUE LUPIN SEED Β-CONGLUTIN PROTEINS REVERSE BACK THE INDUCED INSULIN RESISTANCE IN PANCREATIC CELLS

ELENALIMA-CABELLO1,SONIAMORALES-SANTANA2,VICTORALCHE3,ALFONSOCLEMENTE4,

JUAND.ALCHE1,JOSEC.JIMENEZ-LOPEZ1,5*

1Dept.Biochemistry,Cell&MolecularBiologyofPlants,and4Dept.PhysiologyandBiochemistryofAnimalNutrition;EstacionExperimentaldelZaidin,SpanishNationalResearchCouncil(CSIC),Granada,Spain

2CIBERofFragilityandHealthyAging(CIBERFES),EndocrinologyUnit,EndocrinologyDivision,andProteomicResearchService;BiomedicalResearchInstituteofGranada“IBS.Granada”,UniversityHospitalSanCecilio,Granada,Spain

3AndalusianHealthSystem,HealthCenter"VillanuevadelasTorres",Granada,Spain5TheUWAInstituteofAgricultureandSchoolofAgricultureandEnvironment;TheUniversityofWesternAustralia,Perth,WA,Australia

*Correspondingauthor:[email protected]

INTRODUCTION

Dietscontaininghighlevelsofsaturatedfatsandfructoseplayanimportantroleinobesitydevelopment.Obesityinducesinsulinresistanceintheliver,skeletalmuscles,andadiposetissues,stronglydevelopingintotype2diabetesmellitus(T2DM).Diabetesmellitusisoneofthemostprevalentchronicdisordersworldwide,andtype2diabetesaccountsforover90%ofdiabeticcases.Type2diabetesisfeaturedbyinsulinresistance,whichoccurswhentheinsulin-responsivetissues,mainlyskeletalmuscle,adiposetissue,andtheliver,cannotrespondtoinsulinproperly. Insulinpromotestheglucoseuptakeofinsulin-sensitive tissues and inhibits glucose export from the liver; thus, insulin resistance causes hyperglycaemia andstimulatespancreaticβcellstosecretemoreinsulinformaintaininganormallevelofbloodglucose,whichwillresultinβ-celldamageandthedevelopmentoftype2diabeteswithoutprompttreatment.

Furthermore,insulinresistanceisalsoariskfactorforadiversityofdiseases,includingcardiovascularandkidneydiseases,several cancers and cognitivedisease, increasing themortality risk. Theprevalenceof T2DMhas increaseddramaticallyworldwidenotonlyinadults,butalsoinadolescentsandchildren.

Narrow-leafed lupin or NLL (Lupinus angustifolius L.), a legume crop belonging to the Fabaceae family, is a worldwideimportantpulse,whichdisplaysawiderangeofbenefitsforagriculture.Atmolecularlevel,lupinseedproteinsmayalsobeinvolvedinpossibleplantpathogenssuppression(Jimenez-Lopezetal.,2016).Therefore,NLL,withlowcontentinalkaloids,is attracting great attention lately because of their nutritional attributes and human health benefits (nutraceuticalproperties).Thesepropertiesareassociatedtothehighprotein,dietaryfibercontent,absenceofgluten,helpingtoreducebloodpressuretheriskofcardiovasculardisease,andcontributingtoreductionofglucoseandcholesterolbloodlevels,andpreventing obesity anddyslipidemia (Delgado-Andrade et al., 2016).NLL as an important source of proteins for humanconsumptionseemstobeparticularlypromisingasasourceofinnovativefoodingredientsduetoaverageproteincontentsimilartosoybeanandanadequatecompositionofessentialamino-acids.

Recently, it hasbeendemonstrated thatparticular seedproteins suchas conglutinproteinsβ1,β3, andβ6 fight type2diabetesthroughoutthemodulationoftheinsulinmolecularsignallingpathway(Lima-Cabelloetal.,2017).Thesepropertiesmaybeattributedtotheirparticularstructuralfeatures(Lima-Cabelloetal.,2016).Theseproteinsmightbepotentialnewbioactivemolecules with antioxidant, and anti-inflammatory activities (Lima-Cabello et al., 2018), opening the gate forpossibleplant-basedprevention,managementandtherapeuticapproachestotheworldmainhealthconcernandincreasingdiseasediabeticepidemics.

Theaimofthecurrentstudywastoevaluatetheabilityofb-conglutinproteinsfromNLLtoreverttheresponseatmolecularleveloftheinducedinsulinresistancestateofpancreaticPANC-1cells,checkinggenesinvolvedintheinsulinpathwayinT2DMpatients.

MATERIALANDMETHODS

Synthesisofgeneticconstructs

Beta-conglutinsexpressioninbacteriaE.coliwassetupusingthepET28a(+)vectorcontainingeachindividualb-conglutinisoformβ1,β2,β3,β4,andβ6,linkedto6xHis-Tag.

Proteinsexpressionandpurification

ProteinswereexpressedinRosetta™2(DE3)pLysSSingles™CompetentCells(Novagen),andpurifiedfollowingthemethoddevelopedbyJimenez-Lopezetal.(2016).

68

Cellcultureandtreatment

PANC-1cellswerepurchasedfromCIC(UniversityofGranada).PANC-1cellsweregrowninpoly-L-lysine-coatedflasks(∼2.0–2.5 × 106 cells/ml) inDulbecco’smodified Eagle’smedium (DMEM) supplementedwith 2 mMglutamine and 10%heat-inactivatedfetalbovineserumat37 °Candin5%CO2/95%air.Cellsweregrownasmonolayersanddetachedfromculturesbytrypsinization,andfrequentlysub-culturedtobeusedintheexponentialgrowthphaseforallexperiments.Cellswerewashedtwicewithphosphate-bufferedsolution(PBS,Sigma)andtreatedwith0.25%tryp-EDTA(Sigma)for10 minin5%CO2/95%airat37 °Chumidifiedatmosphere.Thetrypsinizationeffectwasneutralizedwithculturemedium.Thecellswerecollectedaftercentrifugationat1000×gfor5 minandwashedwithPBSforviabilityassays.CellcountingandviabilitywerecheckedusingaCountessIIFLAutomatedCellCounter(ThermoFisher)atthebeginningandendofeachexperimentusingrepresentativewells.Viabilitywasalways > 95%.

Cellswereculturedto80%confluenceandthentreatedwithβ1,β2,β3β4,orβ6purifiedproteinsfor24halone.Aliquotsofβ1,β2,β3β4,andβ6werestoredat–20°CinPBS.ThepurifiedconglutinswerethawedjustbeforeuseanddilutedinDMEMtodesiredconcentrations.20μgofeachpurifiedconglutinwasaddedtocultures.Afterthetreatments,cellswereharvestedforfurtheranalyses.

InsulinresistancePANC-1cellmodelandglucoseuptake

Panc-1cellswereseededin96-wellplatesinDMEMsupplementedwith10%(V/V)FBS,understandardcellcultureconditions(humidifiedatmosphere,5%CO2and37°C).Thedensityofthecellsineachplatewas2×104cells/mL.Thefollowingprotocolwasusedtodeterminetheoptimaldoseofinsulinandtreatmentdurationrequiredtoestablishinsulin-resistant(IR)cells:WeremovedtheoriginalmediumwhenthecellsbecameadherentandreplaceditwithDMEMcontaining3x10-53x10-63x10-73x10-83x10-9mol/Lof insulin (recombinanthuman insulin, Sigma), respectively, and checking for eachone theglucoseuptakebycells.Themodelofinsulinresistancewasestablishedafter24h.Westablishedtheinsulinconcentration(3x10-7mM)wherecellculturedidusetheloweramountofglucoseuptakeasthepointofinsulinresistanceforthePANC-1cellculture(IRPANC-1).Atthisstate,weconsiderthatcellsareinsulinsensitiveorinsensitivetodeterminewhetherβ-conglutinproteinscanimproveinsulin-dependentglucoseuptakecapacityofthecells.

Glucoseoxidasemethodwasusedtodetectglucoseconsumption(GC)levelsafter24h.Theglucoseoxidasemethodwasusedtodetecttheglucoseconcentrationineachgroup.Glucoseoxidasecatalysesthebreakdownofglucosetoglucoseacidandhydrogenperoxide. Peroxidase then catalyzes the formationof redquinone imide in a reaction involvinghydrogenperoxide,4-aminoantipyrine,andphenol.Theabsorbanceofredquinoneimideatawavelengthof490nmisproportionaltotheconcentrationofglucose.

Quantitativereal-timePCR

Real-timequantitativePCRtechnologywasusedtoassayGLUT-4mRNAexpressionfromeachexperimentalgroup.TotalRNAwasisolatedfromPANC-1andIRPANC-1cellsusingtheRNeasyTissueRNAisolationkit(Qiagen).First-strandcDNAwassynthesizedusingaHigh-CapacitycDNAArchiveKit(AppliedBiosystems).Forgeneexpressionassays,cDNAwasprepared,dilutedandsubjectedtoreal-timePCR,andamplifiedusingTaqMantechnology(LightCycler480quantitativePCRSystem,Roche). Primers and probes were used from the commercially available TaqMan Gene Expression Assays detection(Hs00168966_m1, Applied Biosystems;GenBank accession numberNM_001042.2). Relative changes in gene expressionlevelsweredeterminedusingthe2−ΔΔCtmethod.Thecyclenumberthatdetectedthetranscripts(CT)wasnormalizedtothecyclenumberforβ-Actin(Hs99999903_m1,AppliedBiosystems;GenBankaccessionnumberNM_001101.3)usedasthehousekeepinggene,referredtoasΔCT,wheretherelativemRNAlevelswerepresentedasunitvaluesof2∧[CT(β-Actin)–CT(geneofinterest)],havingCTasthethresholdcyclevaluedefinedasthefractionalcyclenumberatwhichthefluorescenttargetsignalpassesafixedthresholdabovethebaseline.PCRefficiencywasdeterminedbyTaqMananalysisonastandardcurvefortargetsandendogenouscontrolamplifications,whichwerehighlysimilar.

Statisticalanalysis

Valuesshownareexpressedasmeans±S.E.M.ofthethreeindividualexperiments.Statisticalsignificanceofthedatawasassessed using SPSS software by analysis of variance followed by Dunnett analysis. P-values ≤0.05 were consideredstatisticallysignificant.

RESULTS

PancreaticPANC-1insulinresistancecellmodel

PancreaticPANC-1cellsareusedforbiochemicalandnutritionalstudiesasacellculturemodelofhumanbetacellssincetheyretaintheirmorphologyandmostoftheirfunctioninculture.Thus,thiscelllinewouldbeusedtostudytheglucoseuptakeandthemodulationoftheinsulinpathwayinvitro.Insulinresistanceininsulinsensitiveorgansresultsinmetabolicdisorder such as hyperglycaemia, hyperinsulinaemia and hyper triglyceridaemia, which are common features of T2DM.Insulinresistanceinpancreaticcellsmainlycausesimpairedinsulinsynthesis.Inordertoestablishaninvitroinsulinresistantmodelofpancreaticcellsandevaluatetheeffectsofdifferentinsulinconcentrationsonglucosemetabolisminthecellmodel,


69

PANC-1cellswereincubatedwith3×10−5,3×10−6,3×10−7,3×10−8,and3×10−9mol/Linsulintobuildaninsulin-resistantcellmodel.Thecellswerestimulatedwithfreshinsulinfor24handtheglucoseuptakebythesecellswascarriedout.Theglucoseuptakebythecellswasdetectedbythemethodofglucoseoxidizes/peroxides(GOD-POD).Following3×10−7mol/LinsulinincubationofPANC-1cells,therewasthemostsignificantdecreaseintheconsumptionofextracellularglucose(P<0.05)comparedwithblankcontrolwithoutinsulinpre-treatment.TheadditionofinsulintoPANC-1cellsgrownin DMEM containing different amounts of glucose induced a statistically significant decrease (P<0.05) in the glucoseconsumptionafterincubationfor24h.

Conglutinproteinsb1,b3andb6increasedGLUT4mRNAexpression

Totestwhetherb-conglutinsreducedbloodglucose levelvia increasingtotalGLUT4proteinexpression,weanalysedm-GLUT4expressionlevelsinIRPANC-1byRT-qPCR.AsshowninFig.2,significantdifferencesinmRNAGLUT4expressionlevelswereobservedbetweencontrolc(-)(non-insulintreatedcells)andIRPANC-1c(+)(p<0.05),whereinsulinsignallingpathwaybiomarkersasINSR-1,Akt,PI3Kremainedlowlevelsunderinsulinresistancestate(Lima-Cabelloetal.,2017).However,b1,b3andb6conglutinproteinsupregulate(reverseback)theinsulinresistancestateinIRPANC-1cellmodelashigherinsulin-inducedupregulatingthemRNA-GLUT4expressioncomparedwiththec(+)control(p<0.05).Furthermore,nosignificantdifferenceswereobservedwhenIRPANC-1cellculturewaschallengedwithb2-ob4-conglutinproteins.

Interestingly,b1-,b3-,andb6-conglutinproteinsincreasetheglucoseuptakeinIRPANC-1cellculture.Thus,theseresultsindicatethatb-conglutinproteinsb1-,b3-andb6-mayalleviatehyperglycaemiaviafacilitatingGLUT4translocationtotheplasmamembraneratherthanviaincreasingthetranslocatorconcentration.

Figure1.EffectofNLLβ1-,β3-,β6-,β2-,andβ4-conglutinproteinsonmRNAexpressionofGLUT-4gene.PANC-1-pancreaticcellswereincubatedfor24 hwitheachpurifiedβ-conglutinproteins(β1,β3β6,β2,β4)alone.mRNAlevelsdeterminedbyreal-timeqRT-PCRofGLUT-4.ThebargraphshowsGLUT-4PANC-1-pancreaticcellsmRNArelative levels.Datarepresentthreeindependentexperiments.C(-):Untreatedcontrolpanc-1culturecells;C(+):insulin-treated(IR)controlPANC-1culturecells;β1,β3,β6,β2,andβ4+ insulin:β-conglutinchallenge IRPANC-1culturecells.p* < .05c(-) versusC(+);p** < .05β-conglutin+insulinversusC(+).

DISCUSSION

In thecurrentstudy,wedemonstrated that treatmentofpancreatic IRPANC-1 induced insulin resistantcell culturewithparticularNLLb-conglutinisoformsincreasedglucoseuptakeandincreaseGLUT-4mRNAexpressionlevels.Thesefindingssuggestthatb-conglutinswouldpotentiallybeabletosignificantlyreducebloodglucoselevelandimproveinsulinresistanceinT2DMinsulinresistantstate.

Finding an effective and natural alternative antidiabetic agent would be of enormous interest and importance for thepreventionandtreatmentofT2DMworldwide.Theseedsofsweetlupinspecies,particularlyNLL,havehighnutritionalvalueandbeneficialnutraceuticaleffects.Despitethesefeatures,NLLislargelyunderexploited,anditsconsumptionisatalongbase,but is increasing.Alternativeapplicationssuchasthe incorporationofNLLseeds innewfunctionalfoodsmayhelpfurther increase the use of NLL grain. In this regard, recent research pointed out the NLL major seed proteins, b-conglutin/vicilinproteinsfamily,asstrongpotentialcandidatefordiabetespreventionandtreatment(Lima-Cabelloetal.,2017;2018).

70

CONCLUSION

Thepresentstudyprovidedstrongevidencethatbeta-conglutinproteinsarecapableofalleviationinhyperglycaemiaandinsulinresistanceinpancreaticcells.NormalizationofGLUT-4mRNAexpressionlevelsoftransporterandincreasingglucoseuptakewastheunderlyingmechanismforthebeneficialeffectsofb-conglutinsinthestageofhyperglycaemiaandinsulinresistance,typicalofT2DM.Consequently,dietarysupplementationwithNLLseedproteinsprovidesfeasibilityforitsusageininsulinresistancetherapy,asanovelandsafefunctionalfood,withapotentialroleintheregulationofinflammatory-relatedprocesses.

ACKNOWLEDGEMENTSANDFUNDING

EU Marie Curie grant PIOF-GA-2011-301550; MINECO grants RYC-2014-16536 (Ramon y Cajal research program), andBFU2016-77243-P.

REFERENCES

Delgado-AndradeC.,OliasR.,Jimenez-LopezJ.C.,ClementeA.(2016).Nutritionalandbeneficialeffectsofgrainlegumesonhumanhealth.ARBOR172-779:a313,doi:10.3989/arbor.2016.779n3003.

Jimenez-LopezJC,MelserS,DeboerK,ThatcherLF,KamphuisLG,SinghKB.(2016).Narrow-leafedlupin(LupinusangustifoliusL.)b1-andb6-conglutin proteins antifungal activity, protecting plants against necrotrophic pathogen induced damage from SclerotiniasclerotiorumandPhytophthoranicotianae.FrontiersinPlantScience7:1856,doi:10.3389/fpls.2016.01856.

Lima-CabelloE.,Robles-BolivarP.,AlcheJ.D.,Jimenez-LopezJ.C.(2016).Narrow-leafedlupinb-conglutinproteinsepitopesidentificationandmolecularfeaturesanalysisinvolvedincross-allergenicitytopeanutandotherlegumes.GenomicsandComputationalBiology2(1):e29,doi:10.18547/gcb.2016.vol2.iss1.e29.

Lima-CabelloEetal.(2017).Narrow-leafedlupin(LupinusangustifoliusL.)β-conglutinproteinsmodulatetheinsulinsignallingpathwayaspotentialtype2diabetestreatmentandinflammatory-relateddiseaseamelioration.MolecularNutritionandFoodResearch61(5),doi:10.1002/mnfr.201600819.

Lima-CabelloE,Morales-SantanaS,FoleyRC,MelserS,AlchéV,SiddiqueKHM,SinghKB,AlchéJD,Jimenez-LopezJC.(2018).Exvivoandinvitroassessmentofanti-inflammatoryactivityofseedβ-conglutinproteinsfromLupinusangustifolius.JournalofFunctionalFoods,doi:10.1016/j.jff.2017.11.040.


71

FLAVONOIDS SYNTHETIC PATHWAY ENZYMES AS BIOMARKERS FOR THE STUDY OF BIOCHEMICAL AND PHYSIOLOGICAL CHANGES OCCURRING DURING NARROW-LEAFED LUPIN SEED GERMINATION AND SEEDLING

DEVELOPMENT

ELENALIMA-CABELLO1,JUAND.ALCHE1,JOSEC.JIMENEZ-LOPEZ1,2*

1Dept.Biochemistry,Cell&MolecularBiologyofPlants;EstacionExperimentaldelZaidin,SpanishNationalResearchCouncil(CSIC),Granada,Spain

2TheUWAInstituteofAgricultureandSchoolofAgricultureandEnvironment;TheUniversityofWesternAustralia,Perth,WA,Australia*Correspondingauthor:[email protected]

INTRODUCTION

TheLupinusgenuscomprisesover400species.Theirseedscontainapproximately36–52%proteins,30–40%fibers,and5–20%essentialoils,withvaluesdependingontheenvironmentalorgeneticconditions (Ref).OtherchemicalconstituentsdetectedinLupinussp.areoleicandlinoleicacids,flavonoidsincludingalkaloids,carotenoids,andpolysaccharides.Overall,thesweetlupinvarietiessuchasL.albus,L.angustifolius,orL.luteuspresentnutritionalandchemicalcharacteristicsmakingthemmoreedible.

Phenolicscompoundssuchasflavonesandisoflavonesarepresentinseveralpartsoflupin,includingtheseeds.However,germinationhasbeenwidelyusedforitsabilitytodecreaselevelsofanti-nutritionalfactorspresentinlegumeseeds,atthesame time improving the concentration and bioavailability of their nutrients. In this regard, germination of lupin seedsmodifies the compositionof theprotein fraction,andminimises thepresenceofphenolic (alkaloids) compounds,whichprotectionagainstdiseasemaybeassociatedwiththepowerfulantioxidantandfreeradicalscavengingpropertiesofthesecompounds,alsoactingasfungicides,bacteriostats,etc.Oneofthemostabundantalkaloidinlupinseeds,quinolizidine,isusedasanitrogensourceforseedlings,andtheyalsoplayadefensiveroleagainstpredatorsintheplant.

Alkaloid synthesis takes place in the stromaof leaf chloroplasts, following a light-regulatedbiosynthesis. Then they aretransportedviathephloemandstoredinvacuolesinalltheorgansoftheplant.Theseedsareespeciallyrichinalkaloids,containingupto5%(dryweight)whichrepresentsabout8–10%ofthetotalnitrogenstoredinlupinseeds.

Germination involves a great number of physiological changes, including synthesis, degradation and transformation ofdifferentcompounds(Zienkiewiczetal.,2011;Jimenez-Lopezetal.,2016a).Duringgermination,someoftheseedreservematerials,particularlyseedstorageproteins,e.g.lupinsvicilins,aredegradedandusedforrespirationandsynthesisofnewcellconstituentsofthedevelopingembryo,leadingtosignificantchangesinseedatthebiochemicalandphysiologicallevel.Furthermore,germinationprocessesfavourasignificantincreaseintheantioxidantcapacityoflupinseed(Jimenez-Lopezetal.,2016b),whichislinkedtochangesofphenoliccompoundsthatseemtobeassociatedwithanincreaseinthefreeradicalscavengingcapacity.Thisalsoincreasespotentialnutritivevalueandhealthqualitiesoftheseedsbyraisingthelevelsofbioactivecompoundsfornutraceuticalandfunctionalityoftheseeds(Lima-Cabelloetal.,2017;Lima-Cabelloetal.,2018).

Seedgerminationmayreducethetotalalkaloidcontent.However,thisdegradationcouldbeattributedtomobilizationofalkaloidalnitrogen,whichalsomaybeinfluencedbynitrogenmobilizationfromseedstorageproteinsalonggermination.ThepresentstudyaimstoidentifykeyplayersdrivingthefunctionalinterplaybetweenspecificmembersoftheNLLseedconglutinfamiliesandthealkaloidmetabolicpathwayduringseedgermination.

MATERIALANDMETHODS

Samplesandgerminationprocess

SeedsofL.angustifoliusL.wereusedinthisstudy.Severalassayswereperformedinordertoselectthebestconditionsforthe germination of the seeds such as different media (water, MS), different temperature conditions, and the optimalgerminationconditionswereperformedonwetfilterpaper,intoagerminationchamberunderenvironmentalcontrolledconditions:16hnight/25Cfor8hlightperdayexposure,andwateringoftheseedsduringgerminationtokeepthepaperalwayswet. L.angustifolius seedswerewashedwith sodiumhypochlorite solution (0.2gCl/l), thenwith steriledistilledwater,andscarifiedpriortogermination,inordertoavoidfungusproliferationandimprovethegerminationoftheseeds.Samplesweretakenat0(1-dayimbibition),2,3,4,5,7,9and11germinationdays.

GeneexpressionanalysesbyqPCR

Real-timequantitativePCRtechnologywasusedtoassaythedifferentgenes(mRNAexpression)inNLLofthefourconglutinfamilies(alpha,beta,gammaanddelta),andthekeyenzymesresponsibleofthealkaloidsmetabolic(synthetic)pathwaysuchaslysinedecarboxylase(L/ODC),lysineoxidase(LO),Twoacyltransferases(ATs):(–)-13α-hydroxymultiflorine/(+)-13α-

72

hydroxylupanine O-tigloyltransferase (HMT/HLT; EC 2.3.1.93), and p-coumaroyl-CoA/feruloyl-CoA: (+)-epilupinine/(–)-lupinine O-coumaroyl/feruloyltransferase (ECT/EFT-LCT/LFT). Total RNA was isolated from seed tissue at differentgerminationstages,using theRNeasyTissueRNA isolationkit (Qiagen).First-strandcDNAwassynthesizedusingaHigh-CapacitycDNAArchiveKit(AppliedBiosystems).Forgeneexpressionassays,cDNAwasprepared,dilutedandsubjectedtoreal-timePCR,andamplifiedusingTaqMantechnology(LightCycler480quantitativePCRSystem,Roche).SpecificFwdandRv primers were designed for each gene and Real-time PCR using QuantiTec SYBER green PCR master mix (AppliedBiosystems).Relativechangesingeneexpressionlevelsweredeterminedusingthe2−ΔΔCtmethod.Thecyclenumberthatdetectedthetranscripts(CT)wasnormalizedtothecyclenumberforubiquitin(usedasthehousekeepinggene)detection,referredtoasΔCT,wheretherelativemRNAlevelswerepresentedasunitvaluesof2∧[CT(β-Actin)–CT(geneofinterest)],havingCTasthethresholdcyclevaluedefinedasthefractionalcyclenumberatwhichthefluorescenttargetsignalpassesafixedthresholdabovethebaseline.Eachassayincludedatemplate-omittedcontrolandaRTnegativecontrol.

RESULTS

L.angustifoliusseedgermination

Wehavestudied the initial timecourseofNLLseedgerminationuntil the first leavesaredifferentiated,andcotyledonscontinue thedegradation.ThematureNLL seedconsistsofabrownseedcoat,absentendospermand largecotyledons(Figure1).Inthematureembryo,twocotyledonsandaradicleareclearlydistinguishable.Invitrogerminationwasfolloweduntiltheembryoturnedintoayoungsmallgreenplant.Thisgerminationperiodtakeplaceabout11days.Afterthisperiodofculture,seedlingsconsistedoftwocotyledons,stemandaroot,completelyturnedintoagreencolour.

Figure4.SeedgerminationstagesofL.angustifolius.Picturesincludefromimbibitionto11daysafterimbibition(DAI).

Wecanconsiderthisstageastheendoftheseedlingstageandstartoftheplantgrowth.Finally,after11days,theplantwasreadyforcontinuousgrowinginapot.

GeneexpressionanalysisofConglutinfamiliesandalkaloidenzymaticpathway

Geneexpressionanalysiswasperformedforallmembersofthedifferentconglutinfamilies.MostofthegenesexhibitedacharacteristicexpressionpatternwheremRNA%decreasedas thegerminationprocessprogresseduntil11days.Al thisstage,the%ofmRNAfortheseconglutingeneswasalmostabsent.However,Figure2showsacharacteristicpatternofexpressionforb3tob6,gamma1and2geneswhereinitialandmiddlestagesofgermination(3to5days)arecharacterizedbyabundanttranscriptsquantity,andwhereseedexhibitedanactivemetabolism.Interestingly,theexpressionanalysisoftheabovementionedgenes fromseedgerminated inwaterandMSmediumexhibiteda comparable levelof transcriptexpression,withfewexceptionsinsomegerminationsteps.

Interestingly,geneexpressionanalysiswasalsoperformedforkeyenzymesinvolvedinalkaloidsyntheticpathway,L/ODC,LO,HMT/HLTandECT/EFT-LCT/LFT,asdepictedinFigure3.

LOandECT/EFT-LCT/LFTexhibitedanincreasingexpressionpatternfrominhibitionstateto11daysofgermination.LODandHMT/HLTexhibitedamRNAhighexpressionpatternbetween3to5daysofgermination.

AlltheseenzymesexhibitedabundanttranscriptsinthesamestagesofgerminationcomparedtoNLLseedstorageproteins(betaandgammaconglutins).


73

Figure 2. Expression analysis of conglutin genes during seed germination. The housekeeping gene used as control wasubiquitin(UBQ).Relativegeneexpressionlevelsweredeterminedbyusingthe2−ΔΔCTmethod.ThecyclenumberatwhichthetranscriptsweredetectablewasnormalizedtothecyclenumberofUBQ,referredtoasΔCT.X-axisrepresentsDAI.Blackbars:IMBandgreybars(seedgerminatedwithoutMSmedia).Dotsbars:seedgerminatedinMSmedium.

Figure3.Expressionanalysisofalkaloidsyntheticpathwaygenesduringseedgermination.Thehousekeepinggeneusedascontrolwasubiquitin(UBQ).Relativegeneexpressionlevelsweredeterminedbyusingthe2−ΔΔCTmethod.ThecyclenumberatwhichthetranscriptsweredetectablewasnormalizedtothecyclenumberofUBQ,referredtoasΔCT.X-axisrepresentsDAI.Blackbars:IMBandgreybars(seedgerminatedwithoutMSmedia).Dotsbars:seedgerminatedinMSmedium.

74

DISCUSSION

Themainlimitingfactorofusinglupinasproteinsourcearealkaloidswhichintoxicdosescauseneuromuscularblockage,respiratorydepression,cyanosis,crampsandcardiacarrest.Forthatreason,manystudiesondecreaseofalkaloidcontentin lupin were carried out. Among many technological methods used for decrease of alkaloid content in lupin seedsgerminationhasbeenrecentlyconsideredasparticularlypromising.

The current study showedan increased transcripts expressionof alkaloids synthetic enzymes, and a general decreasingpatternofdifferentgenesrelatedtoconglutinfamilies.However,atthemiddleofthegerminationprocess(3to5days),bothtypeofgenesexhibitedabundanceoftranscripts.

PreviousstudiesshowedthatgerminationcausesanincreaseoftotalphenolsinsproutsofLupinusangustifolius.Anincreaseisalsoobservedatfourdaysandamorepronouncedoneatninedays.Ariseintotalphenolsofchickpeawasalsoreportedafterfivedaysofgermination.Alltheseresultsagreewithourstudyaboutthealkaloidenzymaticexpressionanalysis.

Thegerminationcausedacleardecreaseoftotalalkaloidscontentsinboththecases.However,thecontentsofthesamealkaloids during germination increased. For example, in the case of L. angustifolius, the increase of tigloyloxylupanine,3OHlupanine,angustifolineisnoted.ForL.luteustheincreaseofgraminecontentisobserved.Generally,thealkaloidcontentinsproutsisdependentonthelupinspecies,temperatureandtimeofgermination.

CONCLUSION

Severalvariationswereobservedingeneexpressionofconglutinandalkaloidssyntheticenzymesduringthedifferentdaysofgermination.However,comparablepatternofexpressionbetweenbothtypesofgeneswereobservedbetween3to6daysofgermination.ThisdatacouldinferthefunctionalinterplayinmetabolicprocessesduringseedsgerminationofNLLseedstorageproteins(conglutins)andthealkaloidmetabolicpathwaybeyondtheroleasnitrogensource.Thus,weproposethese gene families as molecular markers of the germination progressing, and particularly to dissect this physiologicalcomplicateprocess(germination)infunctionalsteps.

ACKNOWLEDGEMENTSANDFUNDING

EU Marie Curie grant PIOF-GA-2011-301550; MINECO grants RYC-2014-16536 (Ramon y Cajal research program), andBFU2016-77243-P.

REFERENCES

Jimenez-Lopez JC, Zienkiewicz A, Zienkiewicz K, Alche JD, Rodríguez-García MI. (2016a). Biogenesis of protein bodies during leguminaccumulationindevelopingolive(OleaeuropaeaL.)seed.Protoplasma253(2):517-530.doi:10.1007/s00709-015-0830-5.

Jimenez-LopezJC,MelserS,DeboerK,ThatcherLF,KamphuisLG,SinghKB.(2016b).Narrow-leafedlupin(LupinusangustifoliusL.)b1-andb6-conglutin proteins antifungal activity, protecting plants against necrotrophic pathogen induced damage from SclerotiniasclerotiorumandPhytophthoranicotianae.FrontiersinPlantScience7:1856,doi:10.3389/fpls.2016.01856.

Lima-CabelloEetal.(2017).Narrow-leafedlupin(LupinusangustifoliusL.)β-conglutinproteinsmodulatetheinsulinsignallingpathwayaspotentialtype2diabetestreatmentandinflammatory-relateddiseaseamelioration.MolecularNutritionandFoodResearch61(5),doi:10.1002/mnfr.201600819.

Lima-CabelloE,Morales-SantanaS,FoleyRC,MelserS,AlchéV,SiddiqueKHM,SinghKB,AlchéJD,Jimenez-LopezJC.(2018).Exvivoandinvitroassessmentofanti-inflammatoryactivityofseedβ-conglutinproteinsfromLupinusangustifolius.JournalofFunctionalFoods,doi:10.1016/j.jff.2017.11.040.

ZienkiewiczA,Jimenez-LopezJC,ZienkiewiczK,AlcheJD,Rodriguez-GarciaMI.(2011).Developmentofthecotyledoncellsduringolive(OleaeuropaeaL.)invitroseedgerminationandseedlinggrowth.Protoplasma248(4):751-765.doi:10.1007/s00709-010-0242-5.


75

TOCOPHEROL CONTENT OF ALMOND OILS PRODUCED IN EASTERN MOROCCO

ABMELHAOUIR,BFAUCONNIERM-L,CSINDICM,AADDIM;AABIDM,

AMIHAMOUA.,ASERGHINI-CAIDH,AELAMRANIA

ALaboratoiredeBiologiedesplantesetdesmicro-organismes,FacultédesSciences,UniversitéMohamedIer,Oujda;Maroc.

BLaboratoiredeQualitéetSécuritédesProduitsAlimentaires,GemblouxAgro-BioTech,UniversitédeLiège;Belgique.CLaboratoiredeChimieGénéraleetOrganique,GemblouxAgroBio-Tech,UniversitédeLiège;Belgique

INTRODUCTION

AlmondisthemostimportanttreenutcropinMoroccointermsofacreageandproductionvalue.Almondplantationscoveratotalareaof151,000hawithanestimatedaverageannualproductionof99.000tonsofshelledproducts.9%ofthisareawhichprovidesupto14%ofMoroccanproductionofalmondsislocatedineasternMorocco(MAPM,2014).RecentlyeasternMoroccoregionwassupportedbytheBelgiandevelopmentagency"BTC"throughthe"PROFAO*"projectforplanting6000haofalmondtreesandfortheimprovementofalmondvaluechainatthepost-harvestlevel,particularlytheevaluationofalmondsanditsderivedproducts(oilandotherco-products).Almondsareagooddietarysourceoftocopherols(vitaminE),sterols,andflavonoids,suggestingplayingaroleinhealthpromotion.Furthermoreconsumptionoftocopherolshavebeenassociated with the natural antioxidant benefits and health outcomes (Maguire et al., 2004). The concentrations oftocopherols in fully ripenedalmondkernelshavebeenstudied indifferent regionsofMorocco (Kodadetal.,2014),buttocopherolcontentsineasternMoroccohasnotyetbeenstudied.Thegoalofthisstudyconcernstocopherolanalysis inalmondoilsproduced ineasternMorocco.Thevarietiesused inthisstudyare:Ferragnes,Feraduel (F/F)andFournatdeBrezenaudoriginatedfromFrance;MarconafromSpainandBeldilocalecotypefromeasternMorocco.PROFAO*:ProjetFilièreAmandesdeL’Oriental,“ThealmondvaluechainineasternMorocco,PillarIIoftheMoroccanGreenPlan,supportedbyBelgiandevelopmentagency(BTC2011-2017)

MATERIALANDMETHODSPlantmaterialTodeterminetocopherolcontentsasaqualityparameterofalmondoils(AO)producedineasternMorocco,AOoffivemainvarietiesgrowninthisregion(Fournat,Marcona,Ferragnes/FerraduelcoupleandlocalecotypeBeldi)weretested.Almondsweretrituratedusinganoilscrewpress(KOMETModèleDD85G).Thealmondsvarieties(Figure1)arecollectedinapilotareaintheeasternMorocco(SIDIBOUHRIA:34°44’13.6’’N,002°20’15.0’’W),

FournatdeBrezenaud Marcona Ferragnes/Feraduel BeldiecotypeFigure1:ThemostcommonalmondvarietiesgrownineasternmoroccoTocopherolcontentsanalysisThedifferenttocopherolisoforms(α-,β-,γ-andδ-tocopherols)wereevaluatedfollowingtheAOCSmethodCe8-89(AOCS1989).AnoilhexanesolutionwasanalyzedbyHPLCwithfluorescencedetectorHPLC-FLD(AgilentTechnologiesseries1200system,AgilentTechnologies),equippedwithanautomaticinjector,onanUptisphere120A°NH2column(150mm*3mm,3µm) Interchim (Montluçon, France) andmaintained at 30 °C. The injection volumewas 10µL. Themobile phasewashexane/2-propanol(99:1,v/v)elutedataflowrateof1mLmin-1.Thetocopherolswereidentifiedandquantifiedbyexternalstandardization(Mixtureoftocopherols:α-tocopherol,β-tocopherols,γ-tocopherols,δ-tocopherols)obtainedfromSigma-Aldrich(Steinheim,Germany).

76

StatisticalanalysisTocopherol analyseswere carried out on triplicate for each sample of almondoil varieties. All statistical analyseswereperformedwiththeSPSSsoftwareforWindows(SPSS.21,USA).Valuesofdifferentparameterswereexpressedasthemean(X¥±standarddeviation).ThenormaldistributionwasverifiedaccordingtoShapiroWilktest.Duncan'smethodwasusedformeancomparison.Onlyvariableswithaconfidencelevelsuperiorto95%(P<0.05)wereconsideredassignificant.

RESULTS

AlmondOilsextractedfromlocalBeldiecotypeandthemainintroducedvarietiesFerragnes/Ferraduel(F/F),MarconaandFournatwereanalyzedfortheirquantitativeandqualitativetocopherolcontents.Theaverageoftotaltocopherolquantitiesobservedis528.98mg/kgAO,withtheα-tocopherolasadominantcompoundinalmondsoil.Totaltocopherolscontentandproportionofdifferenttocopherolshomologuescontent(α-,β-,γandδtocopherols)ofanalyzedalmondoilsbyHPLC-FLDaresummarizedinTable1.

Table1:Totalcontentandproportionofmaintocopherolhomologuesinfivescrew-pressedalmondoils(mg/kgAO)AlmondoilTocopherolsmg/kgAO

Fournat Marcona Ferragnes/Feraduel Beldi

α-tocopherol 483.98ab±7.83 456.44bc±2.87 425.03c±7.59 517.02a±8.56β-tocopherol 3.17a±0.78 1.77c±0,01 2.28bb±0.25 2.95a±0.33Ɣ-tocopherol 14.27a±0.82 2.75b±0.01 5,80c±0.14 9.01d±1.01δ-tocopherols ND ND ND NDTotaltocopherols 501.43ab±8.05 460.96bc±2.88 433.11c±7.88 528.98a±9.71Significantdifferencesareshownbydifferentletters(a-d).ND:notdetected

DISCUSSION

Tocopherolanalysisshowedthepresenceof3tocopherolisoforms(α-,β-and,γ–tocopherol).SimilarresultswerefoundbyKornsteineretal.,(2006),Zhuetal.,(2015a,b)andZhuetal.,(2017).Thehigherα-tocopherolcontent(517mg/kgoil)wasfound inAOBeldi ecotype.While the lower contentswere registered in the introduced varietieswhich range between484mg/kgoilforFournatand425mg/kgoilforthecoupleF/F.Regardingtoβandγtocopherols,ourresultsshowthatβ-tocopherolcontentsrangefrom1.77to3.17mg/kg,andγ-tocopherolfrom2,75to14,27mg/kgoilrespectivelyforMarconaand Fournat, while intermediate values are recorded for F/F. The total tocopherol content of the introduced varietiescultivatedineasternMoroccoismuchhigher(Table1)comparedtothesamevarietiescultivatedinSpain(Marcona:374.1,Fournat:402.4,F/F:385.2mg/kgoil)(Kodadetal.,2011)Thisisanimportantparameterforthevalorizationoftheseproductsfortheirusesasfoodstarsandincosmeticsaswell.AlthoughtheBeldiecotypeischaracterizedbyhighdiversityrelatedtonaturalhybridizationbetween thenativesalmond trees (Melhaouietal., 2017)andbya strongpresenceofdoubles itstocopherolrichnesscanbeconsideredasanaddvalueforthelocalproductionandcouldbeexclusivelyorientatedtowardvirginalmondoilextraction.Sincealmondoilextractionbyscrewpresscanpreservenaturalantioxidantsandfat-solublebioactivecompoundswhichmakethisoilwithinterestingnutritionalandcosmeticproperties.

CONCLUSION

Thisstudyshowsa largevariabilityofcontents fordifferent tocopherolhomologues in theanalyzedalmondoilswithα-tocopherolasadominantcompound.Thisvariationisrelatedtothegenotypeaswellastothegeographicallocalization.Moreover,tocopherolrichnessinBeldiecotypecouldbeusedasanaddvalueforvirginalmondoilextractionwithinterestingnutritionalandcosmeticproperties.

REFERENCES

Jambazian,P.R.,Haddad,E.,Rajaram,S.,Tanzman,J.,Sabaté,J.,2005.Almondsinthedietsimultaneouslyimproveplasmaalpha-tocopherolconcentrationsandreduceplasmalipids.J.Am.Diet.Assoc.105,449–454.https://doi.org/10.1016/j.jada.2004.12.002Kodad,O.,Estopanan,G.,Juan,T.,Company,R.S.i,2014.TocopherolconcentrationinalmondoilfromMoroccanseedlings:Geographicaloriginandpost-harvestimplications.J.FoodCompos.Anal.33,161–165.https://doi.org/10.1016/j.jfca.2013.12.010Kodad, O., Estopanan, G., Teresa Juan, AliMamouni, Rafel Socias i Company, 2011. Tocopherol Concentration in AlmondOil: GeneticVariationandEnvironmentalEffectsunderWarmConditions.JAgricFoodChem6137–6141.Kornsteiner,M.,Wagner, K.H., Elmadfa, I., 2006. Tocopherols and total phenolics in 10 different nut types. Food Chem. 98, 381–387.https://doi.org/10.1016/j.foodchem.2005.07.033


77

Maguire, L.S.,O’Sullivan, S.M.,Galvin, K.,O’Connor, T.P.,O’Brien,N.M., 2004. Fatty acid profile, tocopherol, squalene andphytosterolcontent of walnuts, almonds, peanuts, hazelnuts and the macadamia nut. Int. J. Food Sci. Nutr. 55, 171–178.https://doi.org/10.1080/09637480410001725175MAPM,2014.Ministèredel’agricultureetdelapechemaritimeVeille-économique-secteur-amandier-Notestrategique,N°99.Availableat:http://www.agriculture.gov.ma/pages/veille/veille-economique-secteur-amandier-juillet-2014,.Melhaoui,R.,Abid,M.,Mihamou,A.,Sindic,M.,Caid,H.S.,Elamrani,A.,2017.Flowering,aCriticalPhenologicalStageasaLimitingFactorforAlmondNativeEcotypesCultivationinEasternMorocco.Appl.Microsc.47,157–159.Zhu,Y.,Taylor,C.,Sommer,K.,Wilkinson,K.,Wirthensohn,M.,2015a.Influenceofdeficitirrigationstrategiesonfattyacidandtocopherolconcentrationofalmond(Prunusdulcis).FoodChem.173,821–826.https://doi.org/10.1016/j.foodchem.2014.10.108Zhu, Y.,Wilkinson, K.L.,Wirthensohn,M.,Al, E., 2017. Changes in fatty acid and tocopherol content during almond (Prunusdulcis, cv.Nonpareil)kerneldevelopment.Sci.Hortic.225,150–155.https://doi.org/10.1016/j.scienta.2017.07.008

78

IMPROVEMENT OF SPRAY RETENTION ON BARLEY LEAVES

H.H.BOUKHALFA,M.BELHAMRA

DPAZAlaboratory,UniversityMohamedKhiderBiskra,FacultyofSciencesandsciencesofnatureandlifeDept.Agronomy,BP145RP07000Biskra,Algeria

CorrespondingauthorE-mail:[email protected]

SUMMARYThe droplet size distribution of agricultural sprays is a key parameter during the plant protection product applications.Phenomena governing spray retention on plants are investigated for a very long time in order to optimise pesticideapplication.Thesecanbedividedinphysicalandchemicalpropertiesofeitherthetargetorthedrop.Leafmorphologymayplay an important role in spray deposition. Different leaf structures such aswax, hairs, edges and veins are importantimpingement and retention variables. Surfactants are nowadays very useful additives to improve the effectiveness ofphytosanitarytreatments.Theycontributetochangetheimpacttypesandthustheamountofsprayretainedbytheleavesofthetreatedplant.

WeperformedtestsofretentiononwholebarleyplantsonBBCH-scale12andsmallpiecesofbarleyleavesatthesamestageofgrowth.Sprayingwasdoneinthreeways:waterwithoutsurfactant,waterwithBreak-Thru®S240andwaterwithLi700®.Thethreeslurriesoffluoresceincontainedinanamountof0.2g/l.Fluoresceinretainedbytheleavesinbothcasesisthenmeasuredbyaspectrofluorometer.Theretentiontestsonwholeplantsshowthatit istripledbythefirstsurfactantanddoubledbythesecond.Byconsonsmallpiecesofbarleyleaves,theamountwasincreasedbytheuseofsurfactantsbutnottothesamescale.Thisstudyconcludedthattheuseofsurfactantsinspraypesticidesmayincreasetheamountofretentionasafunctionofleafareaandthesurfactantused.

INTRODUCTIONRetentionismainlyassociatedwithdropletprimaryadhesion,whilebouncingandsplashingareseenasdetrimental.Impactoutcomesdependonleafsurfaceandspraymixtureproperties.Onsuperhydrophobicspecies,surfactantsareoftenusedtoenhancesprayformulationperformancesbyaffectingthephysicochemicalpropertiesofdroplets.

Theaimofthestudyistotesttwosurfactantsusingsimultaneousobservationofdropletimpactsbyhighspeedimagingandfluorescenttraceranalysisofdeposits.WorksofHollowayetal.(2000)confirmedalsothat,tank-mixadjuvantscanhaveaconsiderable influenceon theefficiencyof deliveryofwater spays. Theyaffect thephysicochemical propertiesof spraydropletsintermsoftheirultimateimpactionandspreadingbehaviour.Surfactantsarethemostwidelyusedadjuvanttypeinagrochemicalapplicationsandthemostefficientinenhancingtheirefficacy(Wangetal.,2007).

MATERIALSANDMETHODSTheexperimentalmethodologyexplained inBoukhalfaetal. (2014)wasassociatedtoanexperimentonwholeplantsofbarley.Foreachformulationtensprayswererealizedonfivebarleyplantsatthetwoleavesgrowthstage(BBCHstage12)grown indoor incontrolledconditions.Theamountof sprayactually retainedbybarleyplantswasassessedbydosingafluorescenttraceraddedtothemixtures.Barleyplantswereplacedlinearlybelowthecenterofamovingnozzleatthespeedof2m/susingapressureof2bars.Thelaststepwasmeasuringleafareaforeachsprayandcalculatingtheamountofspraysretainedbybarleyplants.RESULTSANDDISCUSSION

Dropletsimpactbehaviour:Results obtained by image analysis for the three spraymixtures are represented by volumetric distribution of differentimpactbehaviourofdropletsrelatingtotheirdiametersandvelocities.Table1:Volumetricdistributionofdropletsimpactbehaviour


79

Resultsofwatershowthehigheramountofbouncing.SplashingoccursinbothCassie-BaxterandWenzelwettingregimesaswellasadhesion.

For the mixture containing Break Thru S240, bouncing is not observed at the same scale of water. This behaviour ischaracteristicofthe lowdynamicsurfacetensionofthissuper-spreaderorganosiliconeadjuvant.CassieBaxterregime isobservedveryfewtimes.Break-thruincreasegreatlyadhesionanddecreasehighlyreboundcomparingtowater.

TheresultsforthewettingagentLi700,showthereforethatsplashingoccursonlyinWenzelwettingregime.Decreaseofadhesion is compensated by the increase of shattering inWenzel regime related to the higher VMD (volume mediandiameter).

Retentionrelatedtothemixture:Sprayretentionforeachmixturewerecomparedonfigure1.

Figure1.Comparisionretentionforthreemixtures

Figure 1 represents volumes of spray retained by hole plants of barleymeasured by spectrofluorometer. The diagramconfirmstheeffectofadjuvantsonthespraycharacteristics.Inhisstudies,Stock(1997)confirmedthatthesuperspreadingeffectoforganisiliconematerialscanhaveasignificantimpactuponretentionefficiency.Buthethoughtthatthesubsequentsuperspreadingmay,dependingonsprayvolume, leadtoexcessiverunoffandareducednetretentionwhilsttheinitialeffectonretentionmaybebeneficial.Ourresultsconfirmthattheuseoftankmixsurfactantscanimprovesprayretentiononbarleyleaves.CONCLUSIONTank-mixadjuvantsaffectpositivelyretentionandimproveapplicationefficiency.Sprayretentionisdifferentfromaplanttoanotherone,sothenumberoftrialsmustbeincreasedinordertoobtainasignificantresult.Dependingonthespraymixture,dropletsfragmentedinWenzelregimeaccountedfor28-46%ofretentionatfirstimpact,withaclearrankingasafunctionofDST.LITERATUREBoukhalfa, H. H., Massinon, M., Belhamra, M., & Lebeau, F., (2014). Contribution of Spray Droplet Pinning Fragmentationto CanopyRetention.CropProtection,56,91-97.

80

Boukhalfa,H.H.,Massinon,M.,&Lebeau,F.,(2013).High-speedimagingusetopredictsprayretentiononbarleyleaves.CommunicationsinAgriculturalandAppliedBiologicalSciences,Vol78(2)(1-386(2013)),31-36.Massinon,M.,BoukhalfaH.H.,Lebeau,F.,(2014).Theeffectofsurfaceorientationonsprayretention.PrecisionAgriculture15:241–254.Massinon,M.,Boukhalfa,H.H.,&Lebeau,F., (2014).Sprayretentionassmentcombininghigh-speedshadowimageryandfluorescencetechniques.InternationalAdvancesinPesticideApplication:AspectsofAppliedBiology122,2014(pp.219-225).Warwick,UK:AssociationofAppliedBiologists.Massinon,M.,Boukhalfa,H.H.,Marechal,P.,&Lebeau,F.(2012,Juillet).Theeffectofleaforientationonsprayretentiononblackgrass.Paperpresentedat11thinternationalconferenceonprecisionAgriculture,Indianapolis,USA.SmithD.B.,AskewS.D.,MorrisW.H.,ShawD.R.&BoyetteM.(2000).Dropletsizeandleafmorphologyeffectsonpesticidespraydeposition.TransactionsoftheASAE.43(2):255-259.StockD. (1997).Doweneedadjuvants?Mechanisticstudiesand implicationsfor futuredevelopments.Proc.50thN.Z.PlantprotectionConf.:185-190.WangC.J.&LiuZ.Q.(2007).Foliaruptakeofpesticides.Presentstatusandfuturechallenge.PesticideBiochemistryandPhysiology.87:1-8.


81

EVAUATION OF SPRAY RETENTION VARIABILITY ON HYDROPHOBIC PLANT LEAVES

H.H.BOUKHALFA,M.BELHAMRA

LaboratoryDEDSPAZA,DepartmentofAgronmicalSciences,MohamedKhiderUniversity

BiskraAlgeria,BP145RPBiskra,Algeria

INTRODUCTIONSpraying iswidespreadinagriculturalpracticetoprotectcropsfromavarietyofpests. Inpracticeanamountofproductalwaysgreaterthanrequiredisappliedtoprovidethelevelofcropprotectionneeded.SprayretentionismainlyassociatedwithdropletadhesionandsplashinginWenzelwettingstate.Impactoutcomesdependonleafsurfaceandspraymixtureproperties.Barleyfoliageisorientedmainlyverticallyandtheleavesaredifficulttowetbecauseoftheirdensecoveringofmicrocrystalline epicuticularwax. And that affect spray retention. Drop behaviour during impact affects also retention.ObservationofdropimpactsusinghighspeedimagingshowsthatfragmentationinWenzelwettingregimecancontributetoretention.

MATERIALANDMETHODSMeasurements of retention were done for two surfactants and compared to water on barley leaves (BBCH12) usingspectrofluorometry.Break-Thru®S240(Organosiliconesurfactant)attheconcentrationof0,1%andLi700®(Phospholipidsurfactant)attheconcentrationof0,25%wereappliedtofoliageinaqueoussprays.Thethreetankmixturesweremarkedbyfluoresceintracerataconcentrationof1g/l.

Thesprayswereproducedbyaflat-fannozzleTeejet11003andapressureof2bars,mounted50cmheightabovethetargetonarampmovingataspeedof2m/s.Sprayingswereperformedinlaboratoryatatemperatureof18°Candrelativehumidityof40%.

Foreachformulationtensprayswererealizedonfivebarleyplantsatthetwoleavesgrowthstage(BBCHstage12)grownindoorincontrolledconditionsandtenspraysrealizedonindividualbarleyplantsatthesamegrowthstage.Theamountofsprayactuallyretainedbybarleyplantswasassessedbydosingfluorescenttracer.Barleyplantswereplacedlinearlybelowthecenterofthemovingnozzle.Thelaststepwasmeasuringleafareaforeachsprayandcalculatingtheamountofspraysretainedbybarleyplantstodeterminethevariabilityofsprayretention.

RESULTSSprayingon5barleyplantsSprayretentionmeasuredon5linearbarleyplantsarerepresentedin(Figure1).Thereisavisiblevariabilitybetweenthetentrials forall tankmixturestested.Theuseofadjuvantmay increasesprayretentionbutthere isno influenceonthevariability.

Figure1.Retentionvariabilitymeasuredon5barleyplantsfromthethreemixtures.

0.0000

0.0200

0.0400

0.0600

0.0800

0.1000

0.1200

0.1400

1 2 3 4 5 6 7 8 9 10

Retention from spraying onthe5plants

Retentiontapwater(µl/cm²) Retentiontapwater_BT(µl/cm²)Retentiontapwater_Li700(µl/cm²)

82

OnewaysprayingonindividualbarleyplantsSprayretentionmeasuredonindividualbarleyplantsfromonewayspryingarerepresentedin(Figure2).Thediagrammeshowsthesameresultsobtainedwith5linearbarleyplants.Thereisgreatsprayretentionvariabilitybetweeneachplant.

Figure2.Retentionvariabilitymeasuredforonewaysprayingonindividualbarleyplantsfromthethreemixtures.DoublesprayingonindividualbarleyplantsSprayretentionmeasuredon5linearbarleyplantsarerepresentedin(Figure3).Thesamevariabilityobtainedbyone-waysprayingisobtainedwithedabblesprays.Weperformedthistrialinordertotesttheinfluenceofintersectionofsprayjetsapplied in field applications. Spray retention variability between each plant is the same in all cases tested and for allformulationsusedinthisstudy.

Figure3.Retentionvariabilitymeasuredfordabblesprayingonindividualbarleyplantsfromthethreemixtures.DISCUSSIONA 3D virtual spraying model predicting spray droplet interception and retention by single plant architecture has beendevelopedandusedforfocusingonthevariabilitybyMassinonetal.(2015).It investigatedasingle3Dplantmodelasafunctionofthesprayquality,thevolumeperhectareappliedandtheplantsizefortwocontrastedformulationscenariosrepresentativeoflowandhighsprayliquidwettingproperties.Resultsobtainedbythismodelshowedthatretentionrangedfrom6.8%to96.6%ofa fulladhesiondependingonsprayqualityandformulationscenaro.Theseresultscooborateourresults.Massinonetal.(2015)explanedthatthevariabilityofdepositsincreaseswithdecreasingsprayfineness,appliedvolumeperhectareandplantsizebecauseofthereducednumberofdropletcontributingtoretention. InourexperimentthesameresultisobservedwitnformulationcontainingBreaktruewitchiscaracterizedbyfinenessofdropletsandthoseobtanedwithformulationcontainingLi700caracterizedbylownumberofdroplets.

-0.0200

0.0000

0.0200

0.0400

0.0600

0.0800

0.1000

0.1200

1 2 3 4 5 6 7 8 9 10

Retentionfromsimplesprayingonindividualplants

Retentiontapwater(µl/cm²)

Retentiontapwater_BT(µl/cm²)

Retentiontapwater_Li700(µl/cm²)

-0.0500

0.0000

0.0500

0.1000

0.1500

0.2000

1 2 3 4 5 6 7 8 9 10

Sprayretention from dabblespraying onindividual plants

Retentiontapwater(µl/cm²)

Retentiontapwater_BT(µl/cm²)

Retentiontapwater_Li700(µl/cm²)


83

CONCLUSIONTheaimofthisworkistoobservetheretentionvariabilitythatcanoccurduringatreatmentdependingontheequipment,tankmixformulationandcropproperties.Tank-mixadjuvantsaffectpositivelyretentionandimproveapplicationefficiency,buttheyhaven’tanyeffectonsprayretentionvariability.Sprayretentionisdifferentfromaplanttoanotherone,sothenumberoftrialsmustbeincreasedinordertoobtainasignificantresult.REFERENCESBoukhalfa, H. H., Massinon, M., Belhamra, M., & Lebeau, F., (2014). Contribution of Spray Droplet Pinning Fragmentationto Canopy

Retention.CropProtection,56,91-97.Boukhalfa,H.H.,Massinon,M.,&Lebeau,F.,(2013).High-speedimagingusetopredictsprayretentiononbarleyleaves.Communications

inAgriculturalandAppliedBiologicalSciences,Vol78(2)(1-386(2013)),31-36.Massinon,M.,Dumont,B.,DeCock,N.,OuledTalebSalah,S.,&Lebeau,F.,(2015).Studyofretentionvariabilityonanearlygrowthstage

herbaceousplantusinga3Dvirtualsprayingmodel.CropProtection,78,63-71.Massinon,M.,BoukhalfaH.H.,Lebeau,F.,(2014).Theeffectofsurfaceorientationonsprayretention.PrecisionAgriculture15:241–254.Massinon,M.,Boukhalfa,H.H.,&Lebeau,F., (2014).Sprayretentionassmentcombininghigh-speedshadowimageryandfluorescence

techniques. International Advances in Pesticide Application: Aspects of Applied Biology 122, 2014(pp.219-225).Warwick, UK:AssociationofAppliedBiologists.

Massinon,M., Boukhalfa, H. H,Marechal, P, Lebeau, F. (2012). The effect of leaf orientation on spray retention on blackgrass. 11 thinternationalconferenceonprecisionAgriculture,Indianapolis,USA.

SmithD.B.,AskewS.D.,MorrisW.H.,ShawD.R.&BoyetteM.(2000).Dropletsizeandleafmorphologyeffectsonpesticidespraydeposition.TransactionsoftheASAE.43(2):255-259.

StockD.(1997).Doweneedadjuvants?Mechanisticstudiesandimplicationsforfuturedevelopments.Proc.50thN.Z.PlantprotectionConf.:185-190.

84

EVALUATION OF GROWTH PERFORMANCE OF OULED DJELLAL LAMBS BY SEX AND LITTER SIZE IN A SEMI-ARID REGION OF ALGERIA

KDEGHNOUCHE,BNACERCHERIF,HHBOUKHALFA

LABORATORYDEDSPAZA.MOHAMEDKHIDERUNIVERSITY.BISKRA.07000.ALGERIA

INTRODUCTION RuminantlivestockisoneofthekeysectorsofAlgerianagricultureinwhichthecomponent(smallruminant)predominates.AccordingtothestatisticsofthespecializedservicesoftheMinistryofAgricultureandRuralDevelopmentin2015,sheepfarming accounts for nearly 80%of the total numberof livestock in the country andAlgeria is ranked5th in theworldproductionofsheepmeat.Despitetheexceptionalgrowthofthissector,sheepmeatremainsinaccessibleforalargepartofAlgerianhouseholdswithmediumandlowincomes.ThelowproductivityofdomesticherdsisattributedaccordingtoBencherif(2011)toamisbehaviorofbreedingandfeedingofherdswhichisoftenofextensivetype.Theobjectiveofthisworkistostudyandanalyzetheimpactsexofthelamb,andthesizeofthelitteronmeasurablecriteria(birthweight,weightattypicalages,averagedailygainsatagestypes)intheOuledDjellallambsfrombirthtoweaninginordertoprovidethebreederwithassistanceinmanagingtheherdonthebasisofreproductionandgrowthcriteriaontheonehandandtoallowgeneticevaluationewesbasedonprolificacyanddairyvalue.MATERIALSANDMETHODSAnimalsThestudyconcernedanumberof200lambs(maleandfemale)ofOuledDjellalbreedfrommultiparousandprimiparousewesthatdidnotundergoheatsynchronization.TheywerebornbetweenOctober10thandDecember9th,2016.Theywereraisedinsemi-intensive,inadditiontobreastmilk,theyhadbarleyhayattheirdisposalandtheywerepreviouslyidentifiedwithnumberedearrings,theywerehousedinsheepfoldwithnaturallight.Weighingthe lambswasdoneusingascalewithamaximumcapacityof50kg±100g,andall informationfrombirthtoweaningisrecordedonindividualgrowthrecordsforeachlamb.Thestudiedvariables:Controloftheevolutionoflambgrowthfrombirthtoweaningrepresentedbyliveweight(WV)andmeandailygain(ADG)wasperformedasfollows:liveweightin(kg)atd0(atbirth),d10,d20,d30andd90(weaning).-theADGin(g)between:d0-d10;d10-d20;d20-J30andd30-d90.Thefactorsofvariation:-Thesexoftheproduct(male,female)-Thesizeofthelitter(single,double)Statisticalanalysis:Descriptivestatisticsandone-wayanalysisofvariance(ANOVA)wereperformedwiththeSPSSsoftware(2013version20)forPVandGMQanalysis.-ThecorrelationresultswerecalculatedbythePearsontestfortheevaluationoftherelationshipbetweenliveweightatdifferentgrowthphasesoflambs.Alltheaveragesoftheresultswerecalculatedwiththeirmeanstandarderrors(mean±E.S.M).Thestatisticaldifferencewasreportedat(P<0.05).-Thegeneralizedlinearmodel(GLM)wasusedtotesttheeffectsofthefactorsonthevariables,bytheapplicationoftheTtestforindependentsamplestoestimatethemeaningorthehomogeneitybetweenthedifferentsetsofdata(comparisontestbetweentheaverages).-ThefrequenciesofeachquantitativevariableweregraphicallyrepresentedusingExcel(2007).RESULTS:Growthperformancefrombirthtoweaning:I-Dependingonthesizeofthelitter:1-Weightatbirth:Themeanliveweightofthelambsatbirthis4.19±0.94kg.Singlelambstendtohavehigherbirthweightscomparedtodoubles(4.70±0.54kgvs.3.88±0.30kg)respectively(Figure1).2-Weightattypicalages(10,20,30and90):Singlelambstendtohavegreaterweightsthantwinsthroughouttheexperiment(Figure2)


85

Meanweightsobservedat10,20,30and90jcorrelateverysignificantlywithbirthweight(r=0.56P=0.009;r=0.57P=0.008;r=0.54P=0.013andr=0.63P=0.003respectively).Birthweightandmeanweightsat4typicalagesdiffersignificantlywithlittersize(P<0.05)

3-AverageDailygainsItappearsthatsinglestendtohavehigherADGscomparedtodoubles(Figure3).Thecorrelationsaresignificantbetweentheaveragedailygainsatthedifferenttypicalages,(the30dayADGiscorrelatedwiththe20-dayADGwithacorrelationcoefficientr=0.68andP=0.001,itisalsocorrelatedwiththeADGat90dayswithacorrelationcoefficientr=0.64andP=0.002,alsothe90dayADGiscorrelatedwiththe20dayADGwithacorrelationcoefficientr=0.57andP=0.008).Thereisnosignificantcorrelationbetweenbirthweightandaveragedailygainat30daysafterbirth(P>0.05/P=0.30),whereasitisamilkproductionindex.4-Evolutionoftheweight:"Growthcurve"Theliveweightinsingleanddoublelambsevolvesaccordingtoafirstorderpolynomialregressioncurve,theequationsarerespectivelyy=4.108x-1.163andy=2.478x-0.362ofwhich(xisthetimeandyistheweight).Singlebirthlambsalwaysgrowlargerthandoubles.Theytendtohavehigherliveweightsthandoublesfrombirthtoweaning.

II- Accordingtosex:

1-Birthweight:Theaverageliveweightof lambsatbirth is4.19±0.94kg.Male lambsshowedaslightdifferenceinbirthweightcomparedtolambs(4.37±0.5vs4±0.42kgrespectively)Birthweightdidnotdiffersignificantlywithlambsex(P>0.05)/P=0.39)2-Weightattypicalages(10,20,30and90days):Malelambstendtohavehigherweightsthanewelambsforthedurationoftheexperiment(Figure5).Theweightsatthedifferenttypicalagesdonotdiffersignificantlywiththesexofthelamb(P>0.05/P=0.23,P=0.16,P=0.29,P=0.86respectivelyatthetypicalages10,20,30and90days).

Figure 1: Difference in birth weightbetweensingleanddoublelambs

Figure 2: Variations in weights at differenttypical ages (kg) by litter size

Figure3:Variationsinaveragedailygains(g)bylittersizeinOuledDjellallambs.

Figure4:EvolutionofweightrelativetolittersizeinOuledDjellallambs

86

Figure5:Variationsinweight(kg)bysexinOuledDjellallambs.3-Theaveragedailygains:ItappearsthatmaleshavehigherADGsthanewesfrombirthupto20days,andthentheopposite(Figure6).Alsoatthetypicalages,ADGsdonotdiffersignificantlywiththelambsex(p>0.05/P=0.40,P=0.34,P=0.62andP=0.59respectivelyattheADGsat10,20,30and90days).(Figure6)4-Evolutionoftheweight«Growthcurve»Liveweight inmaleand female lambsevolvesaccording toa firstorderpolynomial regression curve, theequationsarerespectivelyy=3.092x+0.027andy=3.031x-0.699ofwhich(xisthetimeandyistheweight).Malelambsarestillgrowingfasterthanewelambs(Figure7).Theytendtohavehigherliveweightsthanfemalesfrombirthtoweaning(Figure7).

DISCUSSIONThemeanliveweightoflambsatbirth(4.19kg)issimilartothemeanbirthweightreportedinlambsbornfromcrossesbetweenOuledDjellalfemalesandD'menmales(3.99kg)(Lebiedetal.2015)TheweightresultsforthedifferenttypicalagesfoundinthisstudyareverysimilartothosereportedbyBendiabandDekhili(2012)forlambsofthesamebreed.ThesuperiorityinweightgaininfavorofsimplebirthtypehasalsobeenreportedbyKerfaletal,(2005).CONCLUSIONTheresultsobtainedattheendofthisstudyallowedustoshowthat:-LiveweightandaveragedailygainsofOuledDjellallambsfrombirthtoweaningarecloselyrelatedtolittersize.-Similarly,averysignificantcorrelationwasobservedbetweenmeanweightsattypicalagesandbirthweight.-Thegrowthperformanceofpre-weanedOuledDjellallambsisnotrelatedtothesexofthelamb.-TheseweightandgrowthperformancestestifytothesatisfactorygrowthpotentialoflambsoftheOuledDjellalbreed.Attheendoftheseresults,itappearsthatthe"sizeoflitter"factorhasasignificantinfluenceongrowthperformance,whichmustbetakenintoaccount.Finally,thisworkshouldbesupplementedbytheidentificationandstudyoftheeffectsofotherfactorswhichcaninducethegrowthoflambs,futurebreeders,inotherwordsthefutureoftheOuledDjellalbreed.LITERATUREBENDIABNetDEKHILIM.,(2012).FacteursinfluençantlacroissancedesdansleNordestAlgérien.RevueAgriculture.3-4p.BENCHERIF S., (2011). L'élevage pastoral et la céréaliculture dans la steppe algérienne Evolution et possibilités de développement.Agriculture,economyandpolitics.AgroParisTech.French.269p.

Figure6:Variations inaveragedailygains(g)bysexforOuledDjellallambs.

Figure 7: Evolution of weight by sex inlambsOuledDjellal.


87

LEBIEDM.,ADAOURIM.,TEMMARR.,SEBBAGHL.,DJOUADIS.,BALOULIN.,(2015).Contrôledesperformanceszootechniqueschezdesovins(femellesOuledDjellalcroiséesavecdesmâlesD’menetdeleursdescendantsF1).UniversitéSaDahlabConsultéle12/05/2017.KERFALM.,CHIKHIA.,BOULANOUARB.,(2005).PotentialitésproductivesdelaraceD'ManauDomaineExpérimentaldel'INRAàErrachidia.ActesduSymposiumInternationalsurleDéveloppementDurabledesSystèmesOasiens.INRAMaroc.9p.

88

MAP


89

NOTES

90

NOTES


91

NOTES

92

NOTES

VOL 83(1) (2018) COMMUNICATIONS IN AGRICULTURAL AND … · 2018. 12. 11. · VOL 83(1) (2018)...

Documents

Transcript of VOL 83(1) (2018) COMMUNICATIONS IN AGRICULTURAL AND … · 2018. 12. 11. · VOL 83(1) (2018)...