3rd bioproscale symposium inhomogeneities in large-scale ......gaseous biofuels from agroindustrial...

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www.bioproscale-conference.org 3 rd BioProScale Symposium 2 to 4 April 2014 Berlin – Germany Institute for Biotechnology and Fermentation in Berlin (IfGB) Seestrasse 13, 13353 Berlin, Germany In cooperation with Institute for Biotechnology and Fermentation in Berlin Organisers Technische Universität Berlin – Chair of Bioprocess Engineering & Institute for Biotechnology and Fermentation in Berlin (IfGB) Location Technische Universität Berlin, Institute for Chemistry, Lecture hall C130 Strasse des 17. Juni 115, 10623 Berlin (Charlottenburg), Germany Three-day symposium about development and application of bioprocesses in industrial scale Inhomogeneities in large-scale bioprocesses System biology and process dynamics Quality by design in bioprocess development High throughput applications, in-line process analytics, data handling and analy- sis, multivatiate data processing, scale down methods, evolutionary strategies for strains and processes Scale down approaches and process analytical tech- nologies for advanced process design Comprehensive process monitoring, non-invasive sensors for all process scales, mul- tiparameter sensors, addressing inhomogeneities by sensor applications and mul- tiposition sampling, modelling and control approaches, influence of early process steps on later down-stream operations Novel reactors, automation and control concepts Process design including feed-back and feed-forward strategies, process control by data based and mechanistic models, model-based experimental design strategies Chair of Bioprocess Engineering

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    3rd BioProScale Symposium

    2 to 4 April 2014Berlin – Germany

    Institute for Biotechnology and Fermentation in Berlin (IfGB) Seestrasse 13, 13353 Berlin, Germany

    In cooperation with

    Institute for Biotechnology and Fermentation in Berlin

    Organisers Technische Universität Berlin – Chair of Bioprocess Engineering & Institute for Biotechnology and Fermentation in Berlin (IfGB)

    Location Technische Universität Berlin, Institute for Chemistry, Lecture hall C130 Strasse des 17. Juni 115, 10623 Berlin (Charlottenburg), Germany

    Three-day symposium about development and application of bioprocesses in industrial scale

    Inhomogeneities in large-scale bioprocessesSystem biology and process dynamics

    Quality by design in bioprocess development High throughput applications, in-line process analytics, data handling and analy-

    sis, multivatiate data processing, scale down methods, evolutionary strategies for strains and processes

    Scale down approaches and process analytical tech-nologies for advanced process designComprehensive process monitoring, non-invasive sensors for all process scales, mul-tiparameter sensors, addressing inhomogeneities by sensor applications and mul-tiposition sampling, modelling and control approaches, influence of early process steps on later down-stream operations

    Novel reactors, automation and control concepts Process design including feed-back and feed-forward strategies, process control by

    data based and mechanistic models, model-based experimental design strategies

    Chair of Bioprocess Engineering

  • Welcome addressDearColleagues,LadiesandGentlemenDearGuestsandStudents

    Myco-workersandIwouldliketowarmlywelcomeyoutoBerlintothe3rdBioProScaleSymposium.Theaimofthissymposiumistocontinuetheintensivediscussionswhichstartedduringtheprevioussymposiain2009and2012.Thisyearweextendthefocusoflarge-scaleconsiderationstothewholeprocessdevelopmentlineandwillalsotakeintoconsiderationhowtoimplementmoreconsistencyintothedevelopmentofnewprocesses.Weareespeciallyexcitedaboutthefascinatingopportunitieswhichbecomepossiblethroughautomationtechnologiesandnewanalyticalprinciplesanddevices.ThesystematicimplementationofQualitybydesign(QbD)andProcessAnalyticalandControlTechnologiesincreasetheefficiencyofbioprocessdevelopmentandtherobustnessoftheprocessesthemselves.

    AnimportantaspectofthisSymposiumistheparticipationofscientistsandengineersfromdifferentbiotechnologicaldisciplineswhichareallconnectedbythesimilarityoftheprincipleswhicharerelevantfortheindustrialscale.Consequently,alsotherequirementtostartbioprocessdevelopmentfromtheperspectiveofthelargescaleprocessisthesameforallareasofappliedbiosciencesandabasicneedtosavetimeandcosts,anddecreasetheinvestmentrisks.

    Weareverydelightedtohaveawealthofpromisinglecturesinourprogrammeandwouldliketoextendourgratitudeespeciallytothespeakerswhofollowedourinvitationandwillshareanddiscusstheirex-pertisewithus,aswellastoourexhibitorsandsponsors,whoprovidedasubstantialbasisforapleasantatmosphere.

    IwishyouallaveryinterestingsymposiumandagreatstayinBerlin!

    Exhibitors & Sponsors

    Introduction

    IfGB is a brand of VLB Berlin e.V.www.vlb-berlin.org

    Scientific advisory boardDr.HenkNoorman(DSM,TheNetherlands)

    Prof.Dr.MatthiasReuss(UniversitätStuttgart,Germany)

    Prof.Dr.NicoOosterhuis(CellutionBiotech,TheNetherlands)

    Dr.ClausLattemann(Sanofi,Frankfurt,Germany)

    Dr.FrankDelvigne(UniversityofLiege,Belgium)

    Prof.Dr.KristV.Gernaey(DTUChemicalEngineering,Lyngby,Denmark)

    Dr.StefanJune(TUBerlin,Germany)

    Prof.Dr.PeterNeubauer(TUBerlin,Germany)

    Professor Dr. Peter NeubauerTechnische Universität Berlin – Chair of Bioprocess Engineering

    About the organisers

    Technische Universität Berlin: Department of Biotechnology – Chair of Bioprocess Engineering

    TheDepartmentofBiotechnologyhassixchairs:BioprocessEngineering,MedicalBiotechnology,Bioanalysis,MicrobiologyandGenetics,BrewingTechnologyandAppliedBiochemistry.

    TheChairofBioprocessEngineering,whichwasnewlyestablishedin2008,hasthreefocusareas:

    (i) Large-scalebioprocessingincludingcellphysiologyandfluxanaly-sisofmicrobialprocessesininhomogenousbioreactorsystems,scale-downsimulators,mobilesensorsandsamplingdevicesformeasurementsinthebulkliquidoflarge-scalebioreactors

    (ii) Biocatalysis–developmentofnewbiocatalystsandbiocatalyticproductsonthebasisofstructurebasedevolutionandmetabolicengineering

    (iii) Highthroughputbioprocessing,robotsinbioprocessdevelopment,especiallyforrecombinantproteins

     www.bioprocess.tu-berlin.de

    IfGB – Institut für Gärungsgewerbe und Biotechnologie zu Berlin

    Foundedin1874,undertheumbrellaoftheInstituteofFermentationandBiotechnologyinBerlin(IfGB)fermentationorientedresearchandeducationhasbeenconductedformorethen140years–alwaysinclosecooperationwiththeTechnischeUniversitätBerlin(resp.itspredesessorinstitutions).Since2003theVersuchs-undLehranstaltfürBrauereiinBerlin(VLB)e.V.isthesouleholderofIfGB.

    Since2003,underthebrandnameIfGBservicesandtrainingforthespiritsindustryanddistillershavebeenoffered.Startingin2009ourserviceandtrainingprogrammeswillbeexpandedintothefieldofbio-technology–againinclosecooperationwiththeInstituteofBiotechnol-ogyofTUBerlin.

     www.ifgb.de

    Compliance note: For the placement of the company logo on our website and in the conference documentation we received from Sanofi-Aventis a financial support of 1000 €.

     www.bioproscale-conference.org

  • 3rd BioProScale Symposium 2014

    Programm at a glance, Wednesday, 2 April 2014

    13:00 Welcome address and introductionPeterNeubauer(DepartmentofBioprocessEngineering,TUBerlin,Germany)

    13:30 Plenary Lecture: Exploring the heterologous genomic space for building, stepwise, complex, microbial strains for large-scale bioprocessing (L01)EleftheriosTerryPapoutsakis(DelawareBiotechnologyInstituteandDepartmentofChemicalandBiomolecularEngineering,UniversityofDelaware,Newark,USA)

    Quality by design in bioprocess development

    Chair AndreasKnepper(TUBerlin,Dep.ofBioprocessEngineering)

    14:15 Key note lecture: Approaching the microbial growth space for systematic bioprocess development (L02) RaivoVilu(TechnicalUniversityofTallinnandCCFFT,Tallinn,Estonia)

    14:45 Coffeebreakwithpostersessionandexhibition

    15:20 Reviving QbD – A mechanistic approach (L03)PatrickSagmeister(TUWien,Austria)

    15:50 Key note lecture: Plasmid DNA production by E. coli: Cell engi-neering, cultivation techniques and process monitoring (L04)AlvaroR.Lara(UniversidadAutónomaMetropolitana-Cuajimalpa,UAM,Mexico)

    16:20 From shake flasks to bioreactor in the production of a recombi-nant glycoprotein in a filamentous bacterial culture (L05)MauricioA.Trujillo-Roldán(InstitutodeInvestigacionesBiomédicas,UniversidadNacionalAutonomadeMexico,MexicoCity,Mexico)

    16:40 Sponsor talk: Navigator software for process development and optimisationTomiAho(BioptimaOy,Tampere,Finland)

    16:45 Coffeebreakwithpostersessionandexhibition

    Chair NicolasCruz-Bournazou(TUBerlin,Dep.ofBioprocessEngineering)

    17:15 Optimisation of a single-use bioreactor for the expansion of human mesenchymal stem cells at bench-top scale by means of CFD: A QbD approach (L06) StephanKaiser(ZürcherHochschulefürAngewandteWissen-schaften,Switzerland)

    17:35 Consistent high throughput bioprocess development under process relevant conditions (L07) FlorianGlauche(Dep.ofBioprocessEngineering,TUBerlin,Germany)

    18:05 Mini Pilot Plant (MPP) for fast and reliable upstream development (L08)SimonUnthan(ForschungszentrumJülich,Jülich,Germany)

    18:25 Low-cost devices and technologies for bioprocess, monitoring, control, modeling and optimisation (L09)GueguimKanaEvaristeBosco(UniversityofKwazuluNatal,Pietermaritzburg,SouthAfrica)

    18:45 Single-cell cultivation in picoliter scale: Opportunity for bioprocess development (L10)AlexanderGrünberger(ForschungszentrumJülichGmbH,IBG-1:Biotechnology,Jülich,Germany)

    19:05 From extracellular environment to functional phenotype: Quantitative comparison of single cell physiology in static and steady environments (L11)ChristianDusny(LaboratoryofChemicalBiotechnology,TUDort-mundUniversity,Dortmund,Germany)

    19:30 Poster session, Exhibition & Welcome Reception

    21:30 End

    © 2012 Hamilton Robotics GmbH. All rights reserved. All trademarks are owned and/or registered by Hamilton Bonaduz AG in Switzerland and/or other countries.

    For more information visit: www.hamiltonrobotics.com

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    DYNAMIC LIQUID CLASSIFICATION Classifi cation of liquids according to their physical parameters determined from a pressure curve test during pipetting. Enables a fl exible determination of liquid classes and improves precision of pipetting for unknown liquids, mixtures of liquids, and liquids of variable quality.

  • Novel reactors, automation and control concepts

    Chair MirjaKrause(TUBerlin,Dep.ofBioprocessEngineering)

    9:00 Plenary lecture: Bioprocess development for the production of gaseous biofuels from agroindustrial wastes (L26) GerasimosLyberatos(SchoolofChemicalEngineering,NationalTechnicalUniversityofAthens,Athens,Greece)

    9:45 Single-use bioreactors for microbial application: Feasibility and recent advances (L27)NicoOosterhuis(CELLutionBiotechB.V.,Nieuwendijk,TheNetherlands)

    10:05 Design space definition for a stirred single-use bioreactor family (L28)ThomasDreher(SartoriusStedimBiotechGmbH,Göttingen,Germany)

    10:25 Solutions for the application of disposable technologies in a bio-production plant (L29)BenjaminMinow(RentschlerBiotechnologieGmbH,Laupheim,Germany)

    10:45 Coffeebreakwithpostersessionandexhibition

    Chair ErichKielhorn(TUBerlin,Dep.ofBioprocessEngineering)

    Programm at a glance, Friday, 4 April 2014

    11:15 Key note lecture: From High throughput process development to PAT applications in downstream processing (L30)JürgenHubbuch(KIT,Karlsruhe,Germany)

    11:45 Hybrid cybernetic modeling of Escherichia coli metabolic adaptations to carbon source availability: A step forward in the modeling of microorganism behavior within industrial bioreactors (L31)NathalieGorret(INRA,Toulouse,France)

    12:05 infoteam iLAB: A software platform for optimised bioprocess development (L32)IngridSchmid(infoteamSoftwareAG,Bubenreuth,Germany)

    12:25 Key note lecture: Modelling across bioreactor scales: methods, challenges and limitations (L33)KristV.Gernaey(DTUChemicalEngineering,Lyngby,Denmark)

    12:55 Concluding remarks PeterNeubauer(TUBerlin,Germany)

    13:00 Endofsymposium

    Programme

    Programm at a glance, Thursday, 3 April 2014

    Scale down approaches and process analytical technologies for advanced process design

    Chair StefanJunne(TUBerlin,Dep.ofBioprocessEngineering)

    9:00 Key note lecture: Investigating the impact of different CO2/HCO3-levels on metabolism and regulation – Example: Corynebacterium glutamicum (L12)RalfTakors(UniversityofStuttgart,Stuttgart,Germany)

    9:30 Response of Corynebacterium glutamicum to oscillations in a three-compartment scale-down reactor concept (L13)AnjaLemoine(DepartmentofBioprocessEngineering,TUBerlin,Germany)

    9:50 Scale-down meets Omics: Challenging metabolism of Coryne-bacterium glutamicum by oxygen inhomogeneity (L14)MarcoOldiges(ForschungszentrumJülich/InstituteofBio-andGeosciences-IBG-1:Biotechnology,Jülich,Germany)

    10:20 Simulating large scale conditions in a scale-down bioreactor: Impacts on the cell physiology of Escherichia coli (L15)ChristianReitz(DepartmentofBioprocessEngineering,TUBerlin,Germany)

    10:40 Sponsor talk: Capacitance based measurement of online viable cell density and monitoring of changes in physiological statesJensRupprecht(Hamilton-Messtechnik,Bonadouz,Switzerland)

    10:45 Sponsor talk: EloTrace – Analysis of the cell polarizability as process analytical toolStefanJunne(TUBerlin,Dep.ofBioprocessEngineering)

    10:55 Coffeebreakandpostersessionandexhibition

    Chair JuliaGlazyrina(TUBerlin,Dep.ofBioprocessEngineering)

    11:25 Towards a single empirical correlation to predict kLa across scales and processes (L16)DanielaQuintanilla-Hernandez(TechnicalUniversityofDenmark,Lyngby,Denmark)

    11:45 Redesigning yeast processes to circumvent heterogeneities issues in large scale bioreactors (L17)StephaneGuillouet(INSA-LISBP,Toulouse,France)

    12:05 Key note lecture: Microbial heterogeneity affects bioprocess robustness: future of dynamic single-cell analysis for large-scale bioprocess control (L18)FrankDelvigne(UniversityofLiege,Belgium)

    12:35 Lunchbreak,postersessionandexhibition

    Chair MarioBirkholz(TUBerlin,Dep.ofBioprocessEngineering)

    14:00 Monitoring functions in managed microbial systems by cytometric bar coding (L19)SusannMüller(UFZ–HelmholtzCentreforEnvironmentalResearch,DepartmentofEnvironmentalMicrobiology,Leipzig,Germany)

    14:30 Use of on-line flow cytometry for the characterisation of microbial stress dynamics during the bioprocess (L20)AlisonBrognaux(GemblouxAgroBio-Tech,Gembloux,Belgium)

    14:50 Mobile multi-sensor systems for the 3-D characterisation of in-dustrial scale processes and the investigation of gradients (L21)AnikaBockisch(DepartmentofBioprocessEngineering,TUBerlin,Germany)

    15:10 Application of wireless sensors for the detection of inhomo-geneities in stirred tanks (L22)JoachimVenus(Leibniz-InstituteforAgriculturalEngineering,Potsdam-Bornim,Dept.Bioengineering,Potsdam,Germany)

    15:40 Sponsor talk: In situ analysis of cell count and size distributionFriedelH.Schwartz(SequipS+EGmbH,Düsseldorf,Germany)

    15:50 Sponsor talk: GRETA – High throughput multifermenter systemGyorgyRajkai(BelachBioteknikAB,Skogås,Sweden)

    15:55 Coffeebreakandpostersessionandexhibition

    Chair PeterNeubauer(TUBerlin,Dep.ofBioprocessEngineering)

    16:25 Key note lecture: Engineering biology: Enabling a knowledge-based bioeconomy (L23)JürgenEck(BRAINAG,Zwingenberg,Germany)

    16:55 Key note lecture: Fermentation scale-down – A view from industrial practice (L24)HenkNoorman(DSMBiotechnologyCenter,Delft,TheNetherlands)

    17:25 Distinguished lecture: Concepts for scale down studies based on CFD agent based modelling approaches (L25)MatthiasReuss(UniversityofStuttgart,Germany)

    19:30 Conference dinnerRestaurantNolle,S-Bahnbogen203,Georgenstraße,10117Berlin(BahnhofFriedrichstrassse)

  • Opening session

    13:00 Welcome address and introductionPeter Neubauer Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering [email protected]

    13:30 Plenary Lecture: Exploring the heterologous genomic space for building, stepwise, complex, microbial strains for large-scale bioprocessing (L01) Eleftherios Terry Papoutsakis, Stefan M. Gaida, Sergios A. Nicolaou, Nicholas R. Sandoval, Kyle A. Zingaro, Yongbo YuanDelaware Biotechnology Institute and Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, USA. [email protected]

    Abstract:Therepertoireofmetabolitesthatcanbeproducedbybio-basedprocessesascommodityorspecialtychemi-calsiseverexpanding,andincludesorganicacids,alkanes,long-chainalcoholsandaldehydes.Severalmoleculesarealsoconsideredsuitableasbiofuels,fromtheestablishedethanoltohigheralcohols,hydrocarbon-likemolecules,andprecursorssuchasbutyricacid.Mostindustriallyrelevantchemicalsaretoxictomicrobes,anissuethatlimitsthedevel-opmentofeconomicallyattractivefermentationandbioremediationprocesses.Howdoesonegeneratethenmicrobialstrainsthatareconsiderablymoreresistanttotheintendedmetaboliteunderrealbioprocessingconditions?Wewillarguethattheheterologousgenomicspaceandthemetagenomicspaceremainlargelyunexploredforthisandothermetabolic-engineeringapplications.Akeylimitationinusingheterologousgenomicormetagenomiclibrariesinfunc-tionalgenomicsandgenomeengineeringisthelow-levelexpressionofheterologousgenesinscreeninghosts,suchasEscherichiacoli.Wewilldiscussanovelstrategy(basedonflow-cytometryscreeningofGFP-traplibraries)toovercomethislimitationanddemonstratetheapplicationofthisstrategyinbuildingcomplextolerancecapabilities.UsingE.coli,wedevelopedfirstseparateandthenacompositephenotypeofimprovedgrowthandsurvivalunderethanolstressbyutilizingtheLactobacillusplantarumgenomicspace.WewillshowthatthisstraincansignificantlyimproveethanolproductivityinapracticallysignificantMelle-Boinot-likefermentationprocess.

    Wednesday, 2 April 2014

    3rd BioProScale Symposium 2014

  • Wednesday, 2 April 2014

    Quality by design in bioprocess developmentChair AndreasKnepper(TUBerlin,ChairofBioprocessEngineering)

    14:15 Key note lecture: Approaching the microbial growth space for systematic bioprocess development (L02) Raivo ViluTechnical University of Tallinn and CCFFT, Tallinn, Estonia – [email protected]

    Abstract:Recentdevelopmentsofsystemsbiologyandsyntheticbiologyhavecreatednewchallenges-needstodevelopandintroducehighthroughputomics-methodstogetherwiththehighthroughputdatahandlingandmodellingmethods,whichallowtocarryoutcomprehensivequantitativediagnosticsofphysiologyofbacterialcellsnotonlyinonepointofpossiblegrowthconditionsbutintheregionofgrowthspaceinterestingfortheresearchersand/orprocessdevelopers.Anapproachincludingchangestatcultivationmethods(A-stat,D-statetc.),absolutequantitativeomics-methods(proteomics,metabolomicsetc.)andmodellingmethods(FBA,SCMetc.)willbedescribedanditsuseinstudiesofevolutionofgeneticvariabilityduringthecontinuouscultivationofE.coli,acetateoverflowmetabolisminE.coliandgrowthratedepend-entmetabolisminL.lactiswillbeillustrated.ItwasshownusingtheA-statcultivationtogetherwiththenextgenerationsequencingthatgeneticvariabilityofbacteriaintheculturecollectionsandduringthecontinuouscultivationsshouldbetakenintoaccountiffullyreliablequantitativedataneedtobegenerated.Theresultsofthestudiesoflevelsofgrowthratedependentregulationmechanismsshowedthatinthebothbacteriainvestigatedthepost-translationalregulationisplayingamoreimpor-tantroleincomparisonwithtranscriptionalandtranslationalcontrols.ItwaspossibletoformulateandproveanovelregulationmechanismofacetateoverflowinE.coliusingthesystemsbiologyplatformdevelopedandstudyingdifferentknock-outmutantsofthebacteria.Futurechallengesofgrowthspacestudieswillbediscussed.

    14:45 Coffeebreakandexhibition

    15:20 Reviving QbD – A mechanistic approach (L03) Patrick Sagmeister, Christoph Herwig Vienna University of Technology, Institute of Chemical Engineering, Research Area Biochemical Engineering, Vienna, Austria, [email protected]

    Abstract:Followingthepharmaceuticalqualitybydesign(QbD)initiative,thedevelopmentanddemonstrationofprocessunderstandingemergedasaprimarydemandfromtheregulatoryauthorities.Thisshouldenablethepharmaceuticalindustrytoactivelyhandlewantedorunwantedprocessvariabilityandhencetoguaranteeaqualityproduct“bydesign”.Inthelastdecade,statisticalmethods(statisticalexperimentaldesign,data-drivenmodels,statisticalprocesscontrol)emergedastheprimarytoolstoputQbDintopractice.Althoughstatisticalmethodsaresuitabletocapturetherelation-shipsbetweenprocessparametersandqualityattributes,theyallowlittleinsightandunderstandingintheunderlyingtechnicalorbiologicalmechanistic.Furthermore,scale-effectscannotberuledoutandthetransferabilityofknowledgetootherprocessesisdifficult.Here,wesketchtherevivingofQbDusingmechanisticmethods.Weshowhowmechanisticmethods(kineticmodels,firstprinciplerelationships)canbeusedforthebioprocesslifecycleandinterlinkedwithstatisticalmethods.Thisincludesi)mechanisticmodelsassistedefficientandpreciseexperimentationtoextractthesameknowledgefromlessexperiments,ii)sounddemonstra-tionofscalableprocessunderstandingbyrevealingtrueunderlyingrelationshipsiii)clearidentificationoftrueprocessoptimaandiv)consist-entuseofmodelsfromtheuseinthescreeningphasetomodel-basedcontrolstrategieswithinmanufacturing.

    15:50 Key note lecture: Plasmid DNA production by Escherichia coli – Cell engineering, cultivation techniques and process monitoring (L04)Alvaro R. LaraUniversidad Autónoma Metropolitana-Cuajimalpa (UAM), México – [email protected]

    Abstract: Escherichiacolicultivationsareaccompaniedbyacetateexcretion(overflowmetabolism),whichhasharmfuleffectonthegrowthandwastescarbontoundesiredproducts.Growthsubstrates(glucose,aminoacids)andspecificgrowthrate(μ)areimportantparametersinfluencingcellphysiologyandacetatemetabolism.Inthisstudysystemsbi-ologyapproachwasused–advancedcontinuouscultivationmethods(A-statandD-stat)withtranscriptome,proteomeandmetabolicfluxanalysiswereusedtomonitorregulationpatternsofmRNAvsproteinandproteinvsfluxatdifferentspecificgrowthrates.Itwasshownthatacetateoverflowwasstartedatμ=0.27±0.02h-1inparallelwithexcretionofpyrimidinepathwayintermediatescarbamoylphosphate,dihydroorotateandorotate.Almost11%carbonwaswastedatspecificgrowthrate0.5h-1.Overflowofacetatewascausedbythecarboncataboliterepressionofacetyl-CoAsyn-thethase(Acs)anddisruptionofthePTA-ACSnodeindicatingimbalancebetweencarbonconsumptionandbiosynthet-icpathways.Itwasobservedthatonthelevelofproteinabundanceinreproductionpathways(synthesisofbiopolymerbuildingblocks)coverslargepartofproteomesynthesis(25%ofATPcostoftotalproteome)andishigherthanthatforenergygeneration(10%).Onthecontrary,carbonoverflowwasobservedinparallelwiththereductionofATPspilling(36%),TCAcycleandglycolysisfluxesindicatingmoreeffectiveenergymetabolism.Itcanbeexplainedbyincreasedapparentcatalyticactivitiesofenzymes(almost3.5timesatspecificgrowthrate0.1h-1vs0.5 h-1,especiallyATPgeneratingenzymes)andbythefactthatlessproteinswasneededtobesynthesizedforbiomassproduction.ToimproveE.coligrowthseveralmutantsweredesignedwhichhadAcsrepressionoractivationmutations.Repressionreleasedmutantshadincreasedbiomassyieldandpostponedacetateproduction.Itwasalsoshownthatbalancedgrowthmedia(additionofaminobasesoraminoacids)caneffectivelyreducecarbonwastingtounwantedbyproductsindicatingbigpotentialtodesignpropermediaforbioprocesses.

    Programme

  • 16:20 From shake flasks to bioreactor in the production of a recombinant glycoprotein in a filamentous bacterial culture (L05)Mauricio A. Trujillo-Roldán Instituto de Investigaciones Biomédicas, Universidad Nacional Autonoma de Mexico, Mexico City, Mexico – [email protected]

    Abstract: CultureconditionsinshakeflasksaffectfilamentousStreptomyceslividansmorphology,aswelltheproductiv-ityandO-mannosylationofrecombinantAla-Pro-richO-glycoprotein(knownasthe45/47kDaorAPAantigen)fromMycobacteriumtuberculosis.Threedifferentshakeflaskgeometrieswereusedtoprovidedifferentshearandoxygena-tionconditions;andtheimpactofthoseconditionsonthemorphologyofS.lividansandtheproductionofrAPAwascharacterizedandevaluated.SmallanddispersedmycelialaggregatesobtainedinbaffledandstainlesssteelcoiledflasksimprovetheproductionandincreasethedegreeofO-mannosylationoftherecombinantprotein,incomparisonwithlargeaggregatesobtainedinconventionalshakeflasks.Inordertoscale-upfromshakeflaskstobioreactor,theeffectofagitationonmorphologyofStreptomycesstrainswereusedtoobtainvolumetricpowerinput(P/V)valuesthatcanbeusedtoobtainamorphologyofS.lividansinbioreactorsimilartothemorphologyreportedincoiled/baffledshakeflasks.MorphologyofS.lividanswassuccessfullyscaled-up,obtainingsimilarmyceliasizesinbothscales(baffledandcoiledshakeflasksandbioreactor).Moreinterestingly,thequalityoftherecombinantglycoproteinmeasuredastheamountofmannosesattachedtotheC-terminalofAPAwasalsoscaled-up;withuptofivemannosesresiduesinculturescarriedoutinshakeflasks;andsixinbioreactor.However,finalbiomassconcentrationwasnotsimilar,indicatingthatalthoughtheprocesscanbescaled-upusingthepowerinput,othersfac-torslikeoxygentransferrate,tipspeedorenergydissipation/circulationfunctioncanbeaninfluenceonbacterialmetabolism.RecentstudiesonpowerinputandoxygentransferratesinshakeflasksallowustoconcludethatP/Vtakenasaglobalparameter,isnotadefinitiveparameterthatcandeterminefilamentousbacteriagrowthandmorphology,notevenrecombinantglycoproteinproduction.ButitcanbeproposedthatthatoxygentransferinthecenterofthepelletsandhydromechanicalstressmightbethemorerelevantparametersthanP/V.

    16:40 Sponsor Talk: Navigator software for process development and optimisationTomi Aho, Bioptima Oy, Tampere, Finland

    16:45 Coffeebreak,postersessionandexhibition

    Chair NicolasCruz-Bournazou(TUBerlin,ChairofBioprocessEngineering)

    17:15 Optimisation of a single-use bioreactor for the expansion of human mesenchymal stem cells at bench-top scale by means of CFD: a QbD approach (L06)Stephan C. Kaiser1, Valentin Jossen1, Carmen Schirmaier1, Ann Siehoff2, Silke Brill2, Gerhard Greller3, Alexander Tappe3, Dieter Eibl1, Regine Eibl1 1Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Grüental, 8820 Wädenswil, Switzerland, [email protected]. 2Lonza Cologne GmbH, Nattermannallee 1, 50829 Köln, Germany, 3Sartorius Stedim Biotech, Landstrasse 94-108, Göttingen, Germany.

    Abstract: Thein-vitroexpansionofhumanmesenchymalstemcells(hMSCs)isofgrowinginterest,becauseoftheirlimitedavailabilityandtheincreasingdemandsformedicalapplications,wheretheprimarycellshaveshownagreatpotentialfortreatmentofawiderangeofdiseases.Currently,thecellsareexpandedin2-Dculturesbecauseoftheirad-herence.However,thosesystemsaremostlynon-instrumentedandlimitedinscalebecauseofthelackofmixing.Thisresultsinconcentrationgradientsand,therefore,supplyofhighcellamountswithconsistentlyhighqualityisdifficultorimpossibletoensure.ApromisingalternativemaybeprovidedbysuspensionculturesofhMSCscultivatedonmicro-carriers(MCs)instirredSUbioreactors,whichofferthebenefitofhigherprocesssafetyduetothelowercontaminationrisk.However,thesebioreactorswereoriginallydesignedforthecultivationofanimalcellculturesthatarecharacter-izedbyhigherrobustnessthantheprimaryhMSCs.Thus,theyareequippedwithimpellers,whicharenotoptimizedforthecultivationofhMSCsattypicaloperationconditions.Thisstudypresentsanoptimizationapproachofacommerciallyavailable,stirredSUbioreactorbymeansofComputationalfluiddynam-ics(CFD).BasedonDesignofExperiments(DoE),differentgeometriesoftheimpellerswereinvestigatedintermsoffluidflow,powerinputandshearstress.Insubsequentsuspensioninvestigations,therequiredimpellerspeedsforMCsuspensionwereidentified,whichprovidessufficientmixingwhilepreventingexcessiveshearstresses.Consequently,adesignspaceforthesuccessfulin-vitroexpansionofhMSCswasdefined.

    17:35 Consistent high throughput bioprocess development under process relevant conditions (L07)F. Glauche1, A. Knepper1, L. Theuer1, M. Heiser1, F. Wollny1, S. Bigesse1,2, A. Neubauer2, S. Arain3, G. John3, J. Aschoff4, B. Stehlik4, I. Schmidt4, N. Violet5, R. King5, D. Gölling6, A. Raab6, G. Kiesewetter1,6, R. Nolte1,6, P. Neubauer1 1Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, [email protected], 2BioSilta Europe GmbH, Berlin, Ger-many, www.biosilta.com, 3PreSens PreSens Precision Sensing GmbH, Regensburg, Germany, www.presens.de, 4Infoteam Software AG, Bubenreuth, Germany, www.infoteam.de, 5Technische Universität Berlin, Chair of Measurement and Control, Berlin. Germany, www.mrt.tu-berlin.de, 6Organo-balance GmbH, Berlin, Germany, www.organobalance.de

    Abstract: Inthefieldofbiotechnology,thedevelopmentofcost-effectiveproductionprocessesisatime-andlabor-intensivetaskduetothevastdesignspacetobescreened.Quiteoften,processparametersresultfromtrialanderrorbasedexperimentation.Throughtheeyesofanengineer,mostscreeningsystemslackfromrelevantinformationandtheconsistencyinscaling-upislimited.Althoughthereisageneraltrendtowardsminiaturizedandparallelizeddevelopmentofbioprocesses,thedegreeofautomationismostlylimitedtosinglesteps.Withacombinationofhighthroughputcultivationsystems,sensortechnology,datahandling,mathematicalmodelingandexperimentaldesignstrategies,theAUTOBIOconsortiumisworkingonplatformtechnologiesinordertosignificantlyshortenprocessdevel-opmenttimeandcosts.TheNeubauerlabhasset-uptworobotstations,whichallowautomatedmicrobialcultivationsinmultiwellplateformatsandina48-stirredtankbioreactorsystem.

    3rd BioProScale Symposium 2014

  • 18:05 Mini Pilot Plant (MPP) for fast and reliable Upstream Development (L08)Simon Unthan, A. Radek, M. Oldiges, W. Wiechert, S. NoackForschungszentrum Jülich, Jülich, Germany, [email protected]

    Abstract: Forindustrialbiotechnology,itishighlyimportanttoselectthemostpromisingproductionhostsandme-diumcompositionsinearlystagesofupstreamdevelopment.However,theopposingdemandsofdetailedprocessin-sightduringhighthroughputaregenerallynotfulfilledbyclassicalcultivationsystems.Today,thisgapisthereforeoftenbridgedbyuseofhighqualityMTP-cultivationdevices,enablingthenon-invasivemonitoringofpH,pO2andbiomasswhileprovidingsufficientoxygentransfer.However,thesedevicesstilldependonmanualworktoinoculate,sampleorinducecultivations,whichlimitstheoverallspeedandthroughputofupstreamdevelopment.Toaddressthisissue,weconstructedaminipilotplant(MPP)byembeddingaBioLectorinaroboticenvironmenttoautomatecompleteworkflowsforupstreamdevelopment.Theconnectedliquidhandlingplatformenablestriggeredadditionofsubstances(i.e.inductors,precursorsorfedsubstances)toindividualcultivationexperiments.Moreover,dif-ferentgrowthmediaarepreparedandinoculatedinasterileenvironmentestablishedbylaminarflow.Togainadeeperprocessunderstanding,cultivationsamplesareharvestedandcentrifugedautomaticallytoprovidesupernatantsforsubsequentquantita-tiveanalysiswithvariousfullyautomatedassaysinMTPscale.WiththisMPPathandweevaluated22novelL-LysineproducingCorynebacteriumglutamicumstrainsindifferentmediaforgrowthandglucoseuptakerateaswellasL-Lysineyieldandproductivitywithintwoweeks.ConcentrationsofglucoseandL-Lysineincultivationsam-pleswereautomaticallymeasuredontheMPPbyanenzymaticorbiochemicalassay,respectively.AsaresultthenovelstrainC.glutamicumD67wasidentifiedwhoshowedabove20%increasedL-LysinetiterscomparedtothereferenceproducerDM1933.TheincreasedL-LysineproductionbyD67wassubsequentlyalsoobservedinlabscale(1L),basicallyconfirmingourresultsobtainedontheMPP.ThisexemplaryfindingshowsthepotentialofourMPPapproachandmoregenerallyspeaking,howfutureupstreamdevelopmentwillbenefitfromroboticautomation.

    18:25 Low-cost devices and technologies for bioprocess, monitoring, control, modeling and optimisation (L09)Gueguim Kana Evariste BoscoUniversity of Kwazulu Natal, Pietermaritzburg, South Africa, [email protected]

    Abstract: Bioreactorsarecentraltobioprocessdevelopmentandproduction.Atthepresentstate,theirhighcostim-pedesparallelmultivariateexperimentationstogeneratehighthroughputdataforprocessdevelopment.Inthiswork,thepotentialityofusingalow-costopen-sourcemicrocontrollersuchastheArduinoboardforbioprocessmonitoringandcontrolispresented.Then,wereviewedmorethanthirtylow-costactuatorsandsensors,whichcaninterfacethebioreactionandthecontroller.Thesesensorsgenerateastreamofdataonbioprocessbehavior,requiredformodelingandoptimisation.

    18:45 Single-cell cultivation in picoliter scale: opportunity for bioprocess development (L10)Alexander Grünberger, Christopher Probst, Simon Unthan, Nurije Mustafi, Julia Frunzke, Stephan Noack, Wolfgang Wiechert and Dietrich KohlheyerForschungszentrum Jülich GmbH, IBG-1: Biotechnology, Jülich, Germany, [email protected]

    Abstract: Novelanalyticaltechniqueshaveprovidedmoredetailedinformationoncellularheterogeneityinbiotechno-logicalprocessesinthelastyears.Nevertheless,thecomplexinterplaybetweenenvironmentalchangesandcellularre-sponseisfarfrombeingcompletelyunderstood[1].Totacklesomeofthesechallenges,microfluidicsingle-cellanalysisofferspromisingtechnicalconceptstoperformindepthcell-to-cellheterogeneityinvestigations.Inthiscontribution,wewillpresentasystematicandcomprehensivestudyutilizingmicrofluidicbioreactorstoanalyzebacterialprocessesonsingle-celllevel[2].Incomparisontodropletbasedsingle-cellcultivationandsimpleagarpadcultivationmethods,thepresenteddeviceareoperatedunderwell-definedandconstantenvironmentalconditions.Corynebacteriumglutamicum,whichisanindustrialworkhorseforaminoacidproduction,wasanalyzedatsingle-cellleveltogainnovelinsidesintoindustrialbioprocesses.ThegrowthrateofC.glutamicumwildtypeundervariousenvi-ronmentalconditionswasinvestigatedsystematically.Surprisingly,single-cellcultivationsinacontinuouslyperfusedmicrofluidicbioreactorsystemrevealedelevatedgrowthratesµ=0.62±0.02h-1)incontrasttolarge-scalebatchcultivations(µ=0.42±0.02h-1)[3].Basedonthesefindings,potentialeffectorswereinvestigated.Itwasfoundthatprotocatechuate-anironchelator-,significantlyinfluencesgrowthratesofC.glutamicum[4].Furthermore,theL-valineproducerC.glutamicumaceEwasinvestigatedbymeansofourrecentlydevelopedgeneticallyencodedfluores-cencereportersystemsfortheintracellulardetectionofamino-acidsatsingle-cellresolution[5].Interestingly,thestrainexhibitedstrongpopulationheterogeneitywithrespecttogrowthaswellasproductivitywithindividualcellsexpressingdifferentfluorescencelevels.Latestresultswillbediscussedinthepresentation.Ourresultsdemonstratethatmicrofluidicpicoliterbioreactorsarepowerfultoolstogainsingle-cellknowledgeofbiotechnologicalproc-essesessentialforupcomingprocessoptimizationandresearch.Nevertheless,single-cellbioreactorsarestillinanearlyphaseofdevelop-ment,makingacriticaldiscussionofadvantages,disadvantages,existingchallengesandnecessaryimprovementsnecessary.

    References:[1]Grünbergeretal.CurrOpinBiotechnol,2014,inrevision.[2]Grünbergeretal.LabChip,2012.12(11):p.2060-2068.[3]Grün-bergeretal.BiotechnolBioeng,2013.110(1):p.220-228.[4]Unthanetal.BiotechnolBioeng,2014:111:359-371.[5]Mustafietal.PlosOne,2014.14(4):9:e85731.

    Programme

  • 3rd BioProScale Symposium 2014

    19:05 From extracellular environment to functional phenotype: Quantitative comparison of single cell physiology in static and steady environments (L11)Christian Dusny1, Alexander Grünberger2, Oliver Frick1, Dietrich Kohlheyer2, Wolfgang Wiechert2, and Andreas Schmid11Laboratory of Chemical Biotechnology, TU Dortmund University, Germany – [email protected] 2Institute of Bio- and Geosciences, IBG-1, Forschungszentrum Jülich GmbH, Germany

    Abstract: Naturehasequippedmicroorganismswithanextensiverepertoireofsophisticatedcellularmechanismstoadapttophysicochemicalchangesintheextracellularenvironment.Thesemechanismsincludestochasticalterationsofregulatorycircuitsaswellasspecificadaptationstoexternalstimuli,whichstronglyvaryfromcelltocell.1Consequent-ly,cellsofamicrobialpopulationexhibitsignificantphysiologicaldiversitydespiteofclonality,whichcanbeaccessedexclusivelybysinglecellanalysis.Inordertounambiguouslyassigntheoriginofcell-to-celldifferencestoeitherenvironmentalfactorsorintrinsicsto-chasticity,thecontroloftheextracellular(micro-)environmentisafundamentalrequirement.Todate,onlymicrofluidiccultivationsystemsofferthepossibilitytopreciselycontroltheextracellularenvironmentandeliminatetheunavoid-ableenvironmentalinhomogeneitiesoccurringduringbulkcultivations.2Weherepresentasystematiccomparisonofinherentlydifferentmicrocultivationtechnologiesinordertoquantifytheresponseofcellularphysiologytotheextracellularenvironmentatasinglecelllevel.Wecomparedmicrofluidiccontactlesstrappingwiththenegativedielectrophoresis(nDEP/Envirostat)andcontact-basedhydrodynamicsingle-cellcultivationsystems(MGC),bothallowingforconstantenvironmentalconditionsbycontinuouscellperfusion,withsolidagarose-basedcultivationpadsonthebasisofphysiologicalandmorphologicaldata.3,4Inordertoallowaquantitativecomparisonofthethreesystems,unifiedanalyticalmethodsfortheprecisequantificationofspecificgrowthrates,cellmorphologyanddivisioncharacteristicsofsinglemicroorganismsweredeveloped.CorynebacteriumglutamicumATCC13032servedasamodelorganismduringthisstudyandwasinvestigatedunderotherwiseidenticalcultivationconditions.Independentoftheemployedcultivationsystem,exceptionallyhighspecificgrowthratesof0.6h-1couldbeobservedonbothsinglecellandcolonylevel.Thesefindingsleadtotheconclusionthatanoptimalnutrientandoxygensupplywaspresentinallemployedsystemsandmaximalgrowthwasratherlimitedbythephysiologicalcapacityofthecellsthanbytheenvironment.Itcanalsobededucedthatallcellsexhibitahighlysimilarmeta-boliccapacity.Incontrasttogrowth,thestaticenvironmentofagarosepadsmanifesteditselfinconsiderabledifferencesinthesnappingdivisionangleandcelllengthdistributionbeforeandafterdivision,whilecellscultivatedwithnDEPandMGCshowedhighlysimilarlengthanddivisionangledistributions.Tothebestofourknowledge,thisstudyrepresentsthefirstsystematicanalysisofphysiologicalresponsestosteadyandstaticextracellularenvironmentsatsinglecellresolution.Moreover,theresultsnicelyshowthepotentialofobservingphysiologicalphenomenaatasinglecelllevelunbiasedbypopulationactivityanduncontrollableenvironmentalinfluences.

    References: 1.Fritzschetal.AnnuRevChemBiomolEng(2012).2.Dusnyetal.Appl.Environ.Microbiol.78,7132-7136(2012).3.Grünbergeretal.Biotechnol.Bioeng.110,220-228(2013).4.Youngetal.NatProtoc7,80-88(2012).

    19:30 Poster session & Exhibition & Welcome Reception Foyer of the Institute for Chemistry

    21:30 End

    ChairofBioprocessEngineering

  • Scale down approaches and process analytical technologies for advanced process design

    Chair StefanJunne(TUBerlin,ChairofBioprocessEngineering)

    9:00 Key note lecture: Investigating the impact of different CO2/HCO3-levels on metabolism and regulation – Example: Corynebacterium glutamicum (L12)J. Buchholz, B. Blombach, Ralf TakorsInstitute of Bioprocess Engineering (IBVT), University of Stuttgart, Germany – [email protected]

    Abstract:Itisawell-knownfactthatlargescalebioreactorsshowmixingheterogeneitiesmirroredbygradientsofsub-strates,dissolvedgasesorpH.Whileimpactsofvaryingsubstrateordissolvedoxygenlevelswereinvestigatedmani-fold,ourcontributionfocusesonthedistinctinteractionofchangingCO2/HCO3-levelsoncellularperformance–atopicwhichhasbeendiscussedrarelyformicrobialcells.StudyingtheindustrialworkhorseCorynebacteriumglutamicumaseriesoflab-scalefermentationswasperformedanalyzingthemetabolicandtranscriptionalresponseondifferentCO2/HCO3-stimuli.Besidesanovelscale-downdevicewasdevelopedenablingthein–depthanalysisofintracellulardynam-icscausedbyextracellularheterogeneities.Aftercharacterizationofthisdevicesub-cellulardynamicswereelucidatedasafunctionofexternalCO2/HCO3-heterogeneitiesmirroringtypicalscenariosoflargescaleconditions.

    9:30 Response of Corynebacterium glutamicum to oscillations in a three-compartment scale-down reactor concept (L13)Anja Lemoine, Nina Maya, Robert Spann, Stefan Junne, Peter Neubauer Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany – [email protected]

    Abstract: Corynebacteriumglutamicumiswidelyappliedfortheindustrialproductionoftheaminoacidlysine.Theselarge-scalenutrientlimitedfed-batchprocessesarecharacterizedbygradientsofsubstrateandoxygen.Scale-downex-perimentsareusedforthesystematicinvestigationoftheimpactofheterogeneousconditionsonthephysiologicandmorphologicstateofthecells.Therefore,twoscale-downconceptsarecomparedinthisstudy:(i)atwo-compartmentreactorcomprisingofastirredtankreactorandaplugflowreactor,inwhichthefeedissuppliedtotheculture(highsubstrateavailabilityandoxygenlimitation),and(ii)athreecompartmentreactor,wherethetwocompartmentreactorisextendedbyasecondplugflowmodule(lowsubstrateavailabilityandoxygenlimitation).Thesezonesareoccurringinindustrial-scalelysineproduction.Resultsshowareducedlysineproductionandaconcomitantaccumulationofthefreeaminoacidsalanine,aspartate,glutamineandglutamateunderheterogeneousconditions.Theintracellularlactateconcentrationshowedanincreaseonlyinthethreecompartmentreactor,underliningthenecessitytobroadentheconceptofatwo-compartmentscale-downreactortoareactorconceptreflectingmultiplezones.

    9:50 Scale-down meets Omics: Challenging metabolism of Corynebacterium glutamicum by oxygen inhomogeneity (L14)Friedrich Käß1, Ioanna Hariskos1, Andrea Michel1, Robert Spann1,2, Peter Neubauer2, Stefan Junne2, Wolfgang Wiechert1, Marco Oldiges1 1Forschungszentrum Jülich; Institute of Bio- and Geosciences IBG-1: Biotechnologie, Jülich, Germany – [email protected], 2Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany

    Abstract: C.glutamicumhasprovenitspotentialasplatformorganismforIndustrialBiotechnologywithannualpro-ductionofmillionsoftonsofaminoacids,organicacidsandderivativesatbioreactorscalesofseveralhundredcubicmeters.Inthiswork,atwo-compartmentreactorsystem(aerobicSTR/nonaeratedPFR)havebeenusedtogeneratedefinedoscillationsofoxygenandsubstratesupplyduringcultivationofC.glutamicumwtandlysineproducingstrainC.glutamicumDM1933.Withresidencetimesinnon-aeratedPFRfrom40secondsuptominutes,robustnessandperformanceofmicrobialmetabolismischallengedinbatchaswellasfed-batchexperiments.Glucoseconsumption,respiratoryactivityandsideproductconcentrationsshowfundamentaldifferenceswhencomparinghomogeneousandoscillatingconditions.Moststrikingly,omics-datafromthemetabolome,proteomeandtranscriptomeshowonlymarginaleffectswithrespecttooscillation.ThisindicatesthattheregulonofC.glutamicumisveryrobustagainstoscil-latoryconditions,whichconstitutesanimportantmechanismforrobustnessinindustrialprocesses.

    10:20 Simulating large scale conditions in a scale-down bioreactor: Impacts on the cell physiology of Escherichia coli (L15)Christian Reitz, Eva Brand, Ping Lu, Robert Spann, Sergej Trippel, Stefan Junne, Peter NeubauerTechnische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany – [email protected]

    Abstract:Escherichiacoliisanimportantproductionstrainforrecombinantproteinproductioninlarge-scalefed-batchbioprocesses.Duetolimitedmixingcapacities,gradientsexistconcerningthenutrientandoxygenavailabilitywhenacertainscaleandcelldensityisreached.Inordertoinvestigatetheimpactoftheseheterogeneousconditions

    Thursday, 3 April 2014

    Programme

  • onthephysiologicstateofthecells,experimentsinascale-downtwo-compartmentreactorareperformed.Itconsistsofastirredtankreactorconnectedtoaplug-flowreactor,whichrepresentsconditionsatthefeedzonebyacombinationofoxygenlimitationandahighconcentrationofsubstrate.MetabolomeanalysisatcultivationsofE.coliK12W3110showanaccumulationofmetabolitesderivingfrompyruvate(acetate,formateandlactate)aswellasanincreasedproductionof(non-canonical)branched-chainaminoacids.AsrevealedbyexperimentswiththerapidsamplingunitBioScope,theshorttimeresponsetoaglucosepulseisdependentwhethercellshadbeenexposedtooscillatingconditionsbefore.Asnon-canonicalaminoacidsareintroducedintorecombinantproteins,theincorporationintorecombinantproteinsunderoscillat-ingconditionshastobeinvestigated.

    10:40 Sponsor talk: Capacitance based measurement of online viable cell density and monitor-ing of changes in physiological statesDr. Jens Rupprecht, Hamilton-Messtechnik, Bonadouz, Switzerland – [email protected]

    Abstract: WithrespecttotheFDA’sProcessAnalyticalTechnology(PAT)frameworkforInnovativePharmaceuticalDevelopment,Manufac-turing,andQualityAssurance,furtheranalyticaltoolsarerequestedtobeimplementedforthesupervisionandcontrollingoffermentationprocesses.Goalisanimprovementofproductqualityandreductionofproductioncostsandtime.Thiscanbeachievedbyanincreaseinprocessautomation,amoreefficientenergyandmaterialuseandcontinuousprocesssupervisionandqualityassurance.Howevertodaysestablishedpoolofreliableandautoclavablein-linetoolsisrestrictedtopH,dissolvedoxygen,conductivity,redoxpotential,temperatureandcellmassasopticaldensity.Inparticularopticaldensityprovidesonlyinformationviatheturbidityofaculturewithoutbeingabletodistinctbetweenlivinganddeadcells,gasbubblesandcellsaswellasmicro-carrierandcellsalsobeingsensitivetoexternallightsources:Resultinginarestrictedquantityandqualityofinformationonthefermentationbatch.Intherecentyearscapacitancebasedmeasurementshavebeenemployedinanincreasingamountoffermentationscoveringagrowingrangeofsinglecellorganisms.Besideitsabilitytomeasuretheviablecellvolumeinabioreactor,itwasshownthatthistechniqueisabletoprovideusefulinformationonfermentationeventslikenutrientdepletion,virusinfectionoroptimalinductiontime.Supportedbyotheranalyticaltoolslikegasanalysis(e.g.respirationorphotosyntheticoxygenevolution)andoff-lineinvestigationsofnutrientavailability,precisetimepointsofthoseeventscanbedeterminedandusedtosuperviseandcontrolfermentationruns.InJanuary2014HamiltonincorporatedtheBioTechdivisionofFogaleNanoTech.WiththisacquisitionHamiltonisnowtheonlycompanyofferingthemainparametersofin-lineprocessanalyticsunderoneroof,promisingcomprehensiveandintensivesupportandservice.

    10:45 Sponsor talk: EloTrace – Analysis of the cell polarizability as process analytical toolAlexander Angersbach1, Viktor Bunin1, Anja Lemoine2, Peter Neubauer2, Stefan Junne21EloSystemsGbR,Berlin,Germany,[email protected]ätBerlin,ChairofBioprocessEngineering,Berlin,Germany.

    Abstract: ThecellpolarizabilitycanbemonitoredatlineapplyingelectroopticalmethodsandautomatedsamplepreparationwithEloSystems’monitoringdeviceEloTrace.Inseveralexperiments,weshowedthatthemonitoringoftheanisotropyofpolarizabilityofrod-shapedbacteriainbatchculturessupportedabetterinsightintothecell’sphysiologicstagesduringtheprocess.TheapplicationofthemethodologytoEscherichiacolibatchcultivationsofferedthepossibil-itytodividethegrowthphaseinseveralpartswithrespecttothespecificaceticacidsynthesisrate.Furthermore,acor-relationbetweentheanisotropyandbiomassyieldinE.coliK12fed-batchandchemostatculturesisfound.Currently,thesystemisappliedforthequantificationofimpactsofoscillatingnutrientandoxygensupplyinC.glutamicumcul-turesinscale-downexperiments.Sincethepolarizabilityseemstoberelatedstronglytotheoverallmetabolicactivityofthecell,theimpactofstressduringcultivationcanbemonitoredforthepurposeofprocessoptimizationandcontrol.

    10:55 Coffeebreak,postersessionandexhibition

    Chair JuliaGlazyrina(TUBerlin,ChairofBioprocessEngineering)

    11:25 Towards a single empirical correlation to predict kLa across scales and processes (L16)Daniela Quintanilla-Hernandez1, Krist V. Gernaey1, Mads O. Albæk2, Stuart M. Stocks21Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark – [email protected] A/S, Fermentation Pilot Plant, Bagsværd, Denmark

    Abstract: Mathematicalmodelsareincreasinglyusedinfermentation.Nevertheless,oneofthemajorlimitationsofthesemodelsisthattheparameterstheyincludeareprocessspecific,e.g.thevolumetricmasstransfercoefficient(kLa).OxygentransferwasstudiedinordertoestablishasingleequationtopredictkLa,anddatafromarangeofprocesses–pilotandproductionscale–wereextracted.On-lineviscositywasmeasuredforallprocesses(56batches).Off-linerheologicalmeasurementswereperformedforthepilotscaleprocesses(26batches).Theapparentviscositywasevalu-atedwith5differentcalculationsoftheaverageshearrate.TheexperimentalkLavaluewasdeterminedwiththedirectmethod;however,eightvariationsofitscalculationwereevaluated.Severalsimplecorrelationswerefittedtothemeas-uredkLadata.ThestandardempiricalequationwasfoundtobebestforpredictingkLainallprocessesatpilotscaleus-ingoff-lineviscositymeasurements,andusingtheequationfromHenzlerandKauling(1985)toevaluatetheshearrate.Inaddition,aparametersetofthestandardempiricalequationwasfoundthatcanpredictoxygentransferinBacillusprocessesatallscalesusingon-lineviscositymeasurements.Asinglecorrelationforallprocessesandallscalescouldnotbeestablished.

    3rd BioProScale Symposium 2014

  • Thursday, 3 April 2014

    11:45 Redesigning yeast processes to circumvent heterogeneities issues in large scale bioreactors (L17)Stephane GuillouetINSA-LISBP, Toulouse, France – [email protected]

    Abstract: TheyeastSaccharomycescerevisiaebelongstosuchyeastspecieswhichswitchfromapureoxidativemetabolismtoarespiro-fermentativemetabolismevenunderfullyaerobicconditionsassoonastheglucoseexceedsaconcentrationofabout0.5gl-1.ThisspecificcharacteristicofS.cerevisiaehasbeensupportiveforallfermentativeprocessessuchasalcoholicbeverageorethanolproductionbutrepresentsadisadvantageforproductionofyeastbiomass(baker’syeastproduction)aswellasbiomass-relatedyeastproducts(suchasheterologousproteins).Therefore,yeastbiomassproductionbioprocesseshavebeenusuallycarriedoutunderaerobicfed-batchmodewheretheglucosefeedingisfinelycontrolledtokeepaverylowresidualconcentration(

  • 14:30 Use of on-line flow cytometry for the characterization of microbial stress dynamics during the bioprocess (L20)Alison BrognauxGembloux Agro Bio-Tech, Gembloux, Belgium – [email protected]

    Abstract:Microbialcellpopulationheterogeneityisnowrecognizedasamajorsourceofissuesforthedevelopmentandoptimizationofbioprocesses.Flowcytometryisaverypowerfultoolforthefollowupofphysiologicalpropertiesofmicrobialcellsinprocess-relatedconditionsatthesinglecelllevel,andcanbeusedtostudythedynamicsofsegrega-tiondirectlyinbioreactors.Inthiscontext,specificinterfaceshavebeendevelopedinordertoconnectflowcytometer(FC)directlyonbioreactorforautomatedanalyses.Inthiswork,weproposeasimplifiedversionofsuchinterfaceanddemonstrateditsusefulnessformultiplexedexperiments.ThisautomatedFCsystemhasbeentestedforthefollowupofthedynamicsofanE.colipfis::gfpAAVfluorescentbio-reporteranditsPIuptake,correlatedwithmembranepermeability.Thisbioreporteriscomposedofafispromoter,agrowthdependentpromoter-indicatorofthenutrientstatusofcells,fusedtoageneexpressinganunstablevariantofGFP.TheresultsobtainedshowedthatthedynamicsoftheGFPsynthesisiscomplexandcanbeattributedtoacomplexsetofbiologicalparameters.Segregationinthemembranepermeabilityhasbeennoticed.Thisworkdemonstratesthatasimplifiedversionofon-lineFCcanbeusedattheprocesslevelfortheinvestigationofthedynamicsofcomplexphysiologicalmechanisms.

    14:50 Mobile multi-sensor systems for the 3-D characterisation of industrial scale processes and the investigation of gradients (L21)Anika Bockisch, S. Junne, P. NeubauerTechnische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

    Abstract:Inlargescalefermentationprocesses,thereisalimitationinthepowerofmixing.Consequently,theoccur-renceofgradientsislikely.However,theavailablesensor-andsampling-technologyisnotsufficienttoquantifythesegradientsandcharacterizelargescaleprocesses.Inordertoincreasetheknowledgeaboutgradientsandtheirimpactontheprocessperformance,multi-positionsensorshavebeendevelopedforinsitumonitoringatdifferentzonesintheliquidcoreofindustrialbioreactors.Theseanalysesarecombinedwithofflinestudiesonthecell’smetabolism,morphology,andphysiology.IthasbeenshownthattheonlinemonitoringisachievableforthepH-value,redoxpotential,DO-concentration,conductivity,pressure,andtemperature.Thedatafromlargescaleissuitableforthedesignofexperimentsinatwo-compartmentscale-downreactor,mimickingtheconditionsofthelargescale.ResultsofS.cerevisiaecultivationexperi-mentsindicatetheimpactofoscillatingconditionsonthemetabolicactivity,cellsize,andrespiratoryactivity.Thecombinationofmulti-positionsensorsandphysiologicstudiesallowstheidentificationofcriticalreactorzones.Onlinemonitoringofthesezoneswillallowafastdetectionofdisturbances,whiletargetsforprocessoptimizationareidentified.

    15:10 Application of wireless sensors for the detection of inhomogeneities in stirred tanks (L22)Joachim Venus1, Thormann, S.1, Hälsig, C.2 1Leibniz-Institute for Agricultural Engineering Potsdam-Bornim, Dept. Bioengineering, Potsdam, Germany – [email protected] GmbH, Teltow, Germany

    Abstract:Renewablefeedstockscanbeutilizeddirectly,e.g.asenergycarriers,aspackagingmaterials,asfibres,fortheproductionofcolouringagentsoraslubricants.However,theycanalsobeconvertedbiotechnologicallybyenzymesandmicroorganisms,givingusaccesstoamultitudeofnew,biocompatibleproductsandpossibleuses.Oftentheeconomyofbioprocessesisstilltheproblembecauseinthecaseofhugeproductquantitiesthepriceisaffectedmainlybyrawmaterialcostsandtheoverallyield,respectively.Fortheperformanceimprovementoftheentirefermentation,whichisstilloftencarriedoutinbigstirredtanks,anewapproachhasbeeninvestigatedbasedonwirelesssensors.Theaimofthestudywastoidentifyanyzonesofinhomo-geneitiesforthebulkliquidinordertooptimizetheprocessconditions.Besidesthesensors,aspecificexperimentalset-up(Fig.1)hasbeendevelopedtoavoiddisturbancesofthehydrodynamicbehaviourofthesystem.Forthespecificcaseoflacticacidfermentation,firstresultswillbepresentedupto1m³bioreactorvolume.ThankstotheanalyticaldevicesforparameterslikepHvalueandconductivityabetterunderstandingoftheturbulenceshasbeenachieved.Thear-rangementofseveralsensorsallowsanindividualpositioningforfurthermeasurementsconcerningtheinfluencesofprocess-determiningparameters.

    15:40 Sponsor Talk: In situ analysis of cell count and size distributionFriedel H. Schwartz (Sequip S+E GmbH, Düsseldorf, Germany)

    15:50 Sponsor talk: GRETA – High throughput multifermenter systemGyorgy Rajkai (Belach Bioteknik AB, Skogås, Sweden)

    15:55 Coffeebreak,postersessionandexhibition

    3rd BioProScale Symposium 2014

  • Programme

    Chair PeterNeubauer(TUBerlin,ChairofBioprocessEngineering)

    16:25 Key note lecture: Engineering biology: Enabling a knowledge-based bioeconomy (L23)Juergen EckBRAIN AG, Zwingenberg, Germany – [email protected]

    Abstract:Heavilyengineeredwhole-cellbiocatalystsandhighlyeffectiveenzymespromiseimprovementforexist-ingprocessesorcouldenablenovelproductideas,pavingthewaytoaknowledge-basedBioeconomy[1].Both,thereplacementoftraditionalprocessesfortheproductionofindustrialchemicalsbymulti-stepbiosyntheticprocessesandtheestablishmentofnovelproductsandcorrespondingsustainableprocessesarebasedonfairlyrecenttechnologicalinnovationsinfieldssuchasmicrobialgenomics,metagenomics,producerstrainengineeringandcoordinatedprocessdevelopment[2;3;4].Thedramaticincreaseofinformationaboutthetruebiosyntheticpotentialofmicroorganismsandtheirenzymesmakesitpossibletodiscoverandoptimizeapplicableenzymesaccordingtheprocessrequirementsandtodesignbiologicalroutesandprocessesforthesynthesisofindustrialchemicals.

    References:[1]EuropeanCommission.2012.InnovatingforSustainableGrowth:ABioeconomyforEurope.http://ec.europa.eu/research/bioeconomy/pdf/201202_innovating_sustainable_growth_en.pdf.[2]Lorenz&Eck.2005.NatRevMicrobiol3:510-516.[3]Gaboretal.2012.JMolBiol418:16-20.[4]Mampeletal.2013.TrendsBiotechnol31:52-60

    16:55 Key note lecture: Fermentation scale-down – A view from industrial practice (L24)Henk NoormanDSM Biotechnology Center, Delft, The Netherlands – [email protected]

    Abstract:Innovationsin(molecular)biotechnologyandchemistryfortheconversionofbiomassintobio-basedproducts,nowarerapidlyadvancingtobioprocessscale-upandimplementationinindustry.There,themanufacturingvaluechainconnectsbiomassasnovelfeedstock,biomasspretreatmentandhydrolysis,(bio)conversionofsugarsintomonomericintermediates,andnovelpurificationschemestomeetfinalproductspecifications.Allneedtobeproveneconomicandrobustinindustry.Inparticular,theshiftfromsugarstobiomassasfeedstockpresentsthebioprocesstechnologyfieldwithaformidableadditionalchallenge,andnewsolutionsarerequired.Processmodeling,high-throughputscreening,scale-downsimulation,processintensificationandpilot/demo-scalecampaignsareimportantforde-riskingandfasterimplementation.Ontopofthis,sustainabilitymetricssuchaslessgreenhousegasemissions,energyinputandwasteneedtobeclearlylinkedtotheprocessmassandenergybalances.Aleadingprincipleistotaketheindustrialfermentationasreferencepoint,andfromtherescale-downtothelabswheretheoptimizationresearchisbeingdone.Foraproperscale-down,experimentaldatafromtheindustrialreactorprovideguidancetodesignandoperationofscale-downsimulators,butalsocomputationstogetsufficienthigh-resolutioninformation.Itisimportanttouseasthoroughlyaspossiblethescarcedatafromindustrialfermentationstovalidatecomputationalresults.Inrecentyears,goodprogressinthisareahasbeendemonstratedasshowninthispresentationviaexamplesfromDSM,appliedtoviablebusinesscasesfore.g.antibiotics,vitamins,lignocellulosicbio-ethanol,andbio-plastics.

    17:25 Distinguished lecture: New concepts for designing scale down operations based on CFD coupled agent-based modelling and simulation (L25)Matthias Reuss, Alexei LapinStuttgart Research Centre Systems Biology and Institute of Biochemical Engineering, University Stuttgart, Nobelstr. 15, 70569 Stuttgart – [email protected]

    Abstract:Thelectureaimsatdiscussinganewstrategyforamodelbaseddesignofscale-downexperimentstooptimal-lymimicthelifelinesofindividualcellsinlargebioreactors.Thedesignconceptisbasedontheagent-basedsegregatedmodellingapproach,inwhichtheliquidistreatedasacontinuum(Euler)andthedispersedbiophaseistrackedwiththeaidofaLagrangianrepresentation(Lapinetal.,2004,2006,2010).ApplicationofthismodellingstrategyisexemplifiedwiththesugaruptakerateofE.coli(Lapinetal.,2006).Animportantoutcomeofthesimulationisaquantitativeportrayoftheadventuresofindividualcellsintermsofvariationofsugaruptakerateduringthespatio-temporaltrackingoftheagents.ThenewapproachsuggestedinthelecturefurtheranalysesthisinformationwiththeaidofaFourier-transfor-mation.Applyingthesameapproachtothescaledownexperiment–wellmixedvesselcoupledwithaplugflowintherecyclingflow–allowsanadaptationoftheparametersofthescale-downoperationtooptimallyportraythesituationinthelargescalereactor.

    References: Lapinetal.2004.Ind.Eng,Chem.Res.43,4647-4656.Lapinetal.2006.Chem.Eng.Sci.61,4783-4797.Lapinetal.2010.Adv.Biochem.Eng./Biotechnol121,23-43

    19:30 Conference dinner

    Restaurant Nolle, S-Bahnbogen 203, Georgenstraße, 10117 Berlin

    (directly located at Bahnhof/Station Friedrichstrassse, individual journey)

  • Novel reactors, automation and control conceptsChair MirjaKrause(TUBerlin,ChairofBioprocessEngineering)

    9:00 Plenary lecture: Periodic bioprocesses and their use for nutrient removal and for the production of gaseous biofuels from agroindustrial wastes (L26)Gerasimos LyberatosSchool of Chemical Engineering, National Technical University of Athens, Athens, Greece – [email protected]

    Abstract: Periodicbioreactoroperationprovidesseveralpossibilitiesthatarenotpossiblewitheitherbatchorcon-tinuousstirredtankbioreactorsoperatedatsteady-state.Theseincludeflexibilityofoperation,enhancedbioreactorperformance,prevalenceofselectedmicrobialpopulationsandindefiniteretentionofrecombinantstrains.ApartfromperiodicoperationofCSTR-typereactors,sequencingbatchreactorsareperiodicallyoperatedreactorsbynature.Itisdemonstrated,asanexamplethatpartialnitrification/denitrificationmaybeeffectivelycarriedoutthroughappropriateoperationofanSBR.AnotherbioreactortypethatisinherentlyperiodicbynatureisthePeriodicAnaerobicBaffledReac-tor.Itisahighrateanaerobicreactorthatismaintainedinaperiodicstateatalltimes,despitereceivingconstantfeed,withdistinctiveadvantages.Theoptimizationoftheprocessoperationisgreatlyfacilitatedbyappropriatedynamicmodeling.Examplesoftheuseofthisreactorfortheproductionofgaseousbiofuels,suchashydrogenandmethane,fromagroindustrialwastesarepresented.

    9:45 Single-use bioreactors for microbial application: feasibility and recent advances (L27)Nico M.G. Oosterhuis1, S. Junne2, P. Neubauer21 CELLution Biotech BV, Assen, The Netherlands – [email protected] Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany

    Abstract: Nowadayssingle-usebioreactorsarefullyacceptedinthebiopharmaceuticalindustry.Reactorsupto2000Lworkingvolumearecommonlyused.However,thesebioreactorsarelimitedintermsofmass-transferandmixingcapabilitiesandthereforeonlysuitedforapplicationinmammaliancellculture.Single-useprocessingoffersthesameadvantagesformicrobialprocessesasformammalianprocesses,sothereisaneedforsingle-usebioreactorsapplicableformicrobialprocessesaswell.TheCELL-tainer®technology,basedona2-dimensionalrockingmotionisavailablenowwithaworkingvolumefrom0.15–25Linoneandthesamebagaswellasfrom10–150Linonebagsize.KLavaluesof300h-1andabovehavebeenreportedforbothsizesofreactors.CulturedataofE.coliandaRhodutorulayeastshowthattheCELL-tainer®single-usebioreactoriscomparabletostirredfermentersandthussuitableformicrobialcultiva-tions.ApplicationinboththeseedtrainandasfinalproductionsystemforsmallbatchesinGMPispossiblenow.

    10:05 Design space definition for a stirred single-use bioreactor family (L28)Thomas Dreher, Ute Husemann, Christian Zahnow, Gerhard GrellerSartorius Stedim Biotech GmbH, Göttingen, Germany – [email protected]

    Abstract: Single-usebioreactorscontinuetogainlargeinterestinthebiopharmaceuticalindustry.Theyareexces-sivelyusedformammalianandinsectcellcultivations,e.g.productionofmonoclonalantibodiesandvaccines.Thisismotivatedbyseveraladvantagesofthesebioreactorslikereducedriskofcrosscontaminationsorshortleadtimes.Single-usebioreactorsdifferintermsofshape,agitationprincipleandgassingstrategy.Hence,adirectprocesstransferorscale-upcanbeachallenge.Conventionalstirredstainlesssteelorglassbioreactorsarethereforestillconsideredasthegoldstandardduetotheirwell-definedandcharacterizedproperties.Basedonthisknowledgeastirredsingle-usebioreactorfamilywasdevelopedwithgeometricalratiossimilartoconven-tionalreusablesystems.TofollowaQualitybyDesign(QbD)approachthesingle-usebioreactorfamilyevaluatedherewascharacterizedbyusingprocessengineeringmethods.Fordefinitionofadesignspacethepowerinputpervolume(P/VL),mixingtimeandkLa-valuesweredeterminedforthedifferentscales.Basedontheresultsconclusionsaboutthesuitabilityandfieldsofapplicationforthesingle-usebioreactorsarepossible.

    10:25 Solutions for the application of disposable technologies in a bioproduction plant (L29)Benjamin MinowRentschler Biotechnologie GmbH, Laupheim, Germany – [email protected]

    Abstract: Inbiopharmaceuticalindustry,characterizedofcostpressureandincreasedcompetition,disposabletechnol-ogyisawelcomemeasuretoreducetimelinesandcostforthedevelopmentandproductionofdrugs.RentschlerisoneofthepioneersintermsofemploymentofdisposableequipmentandasaCMOtheneedtoensureacertaindegreeofequipmentdiversitythatcanbeofferedisobvious.ThisinturncomplicatestheincorporationofnewDisposableTechnologiesintoalreadyexistingfacilitieswhetherstainlesssteelor“plastic”.InordertoreachasatisfyingdegreeofharmonizationtailormadesolutionsforcertainDisposableequipmentwasdeveloped.Thispresentationaimsforreviewingthelatestimprovementsandtheimpactonprocessperformance.

    10:45 Coffeebreak,postersessionandexhibition

    Friday, 4 April 2014

    3rd BioProScale Symposium 2014

  • Chair ErichKielhorn(TUBerlin,ChairofBioprocessEngineering)

    11:15 Key note lecture: High throughput process development for the purification of biopharmaceuticals (L30)Jürgen HubbuchInstitute of Process Engineering in Life Science, Section IV: Biomolecular Separation Engineering, Karlsruhe Institute of Technology (KIT), Germany, [email protected]

    Abstract: ModernarchitectureofbiopharmaceuticaldevelopmentandproductionhasseenarapidchangeoverthepastdecadeandiscurrentlycomposedbyinitiativessuchasQualitybyDesign,ProcessAnalyticalTechnologyandearlyproductassessmenttomitigaterisksduringdevelopmentandmanufacture.Inthedownstreamareaofbiophar-maceuticalproduction,thedemandforfastprocessdevelopmentwithlimitedmaterialhasbecomeeverydaylife.Tomeetthisdemand,highthroughputprocessdevelopment(HTPD)strategiesforapplicationindownstreamprocessdevelopmenthavebeendeveloped.Inordertogainmaximalbenefitfromthisexperimentalapproach,theanalyticalmethodsappliedtoevaluatetheexperimentsperformedrequireautomatedperformanceandathroughputmatch-ingtheexperimentalspeed.Inaddition,eventhoughHTSapplieslowvolumeset-ups,experimentalspaceshouldbeminimizedintermsoffeedstockvolumeneededwhileatthesametimemaximisingthelevelofinformationgained.Whilethemethodologyiscurrentlybasedonstatisticaldataevaluationonthelongrunafundamentalmechanisticunderstandingisrequired.ThepresentationwillgiveashortoverviewonandcasestudiesfromtheapplicationoftheHTPDmethodologyandexamplesshowingitslinkagewithinthecurrentdevelopmentframeworkofthebiopharmaceuticalindustry.

    11:45 Hybrid cybernetic modeling of Escherichia coli metabolic adaptations to carbon source availability: A step forward in the modeling of microorganism behavior within industrial bioreactor (L31)Nathalie GorretINRA, Toulouse, France – [email protected]

    Abstract: Knowledgeofthemicrobialkineticsincomplexdynamicenvironmentisrequiredinordertounderstandanddescribethebehaviorofcellscultivatedinlarge-scalebioreactorwhereconcentrationgradientstakeplace[1,4].Growthofmicroorganismsonsubstratemixturesdisplaydiversegrowthresponsescharacterizedbysimultaneousorsequential/preferentialuptakeofcarbonsources.Withtheobjectivetosimulatethemetabolicbehaviorofmicroor-ganismsfacinglocalconcentrationgradients,ahybridmetabolicandhydrodynamicmodelhastobeintended;asaconsequencethedegreeofcomplexityofbothbuildingblockshastobeconsidered.Inthisidea,theaimofthisworkistocomparedifferentdynamicalmetabolicframeworksandidentifytheoptimalmodelcomplexityi.e.thetrade-offbetweencomplexity(numberofparameters,variables)andrealism(predictionrangeandaccuracy)inordertomodelsystematicallyE.colimetabolicadaptationstocarbonsourceavailability.Threedifferentmetabolicmodelingapproach-eswerecompared:apublishedKineticmodelwithenzymaticandtranscriptionalregulation[2],whichtheoreticallypresentsahigherdegreeofcomplexity,aHybridCyberneticModelandaLumpedHybridCyberneticmodelwithlowerdegreeofcomplex-ity.HybridCyberneticmodeling(HCM)waspreviouslyusedtopredictsuccessfullydiauxicgrowthpatternsand/orsimultaneousconsump-tionofsubstrates[3].Thehybridcyberneticframeworkfocusesonadynamicdescriptionofmetabolismbasedonasetofsub-networksoftheglobalmetabolicpathwayscalledelementarymodes.Theregulatoryprocessesareinterpretedasoptimalallocationofresourcesrequiredforenzymesynthesisamongdifferentmodesinordertomaximizeaglobalobjectivefunctionsuchascarbonuptake[5].Inthiswork,aHybridCybernetic(HCM)andaLumpedHybridCybernetic(L-HCM)modelsweredevelopedandoptimizedindependentlyusingaglobalestimationalgorithmtofindspecificparametersbasedonexperimentaldata.Numericalsimulationsusingthe3formalismshavebeencarriedoutfromvarioussetofexperimentaldatainordertoevaluatethepredictabilityandgenericityofthemodels.BothdevelopedmodelscanpredictthebehaviorofE.coliinbatchenvironmentswithverygoodcoefficientofdeterminationR2rangingfrom0.87to0.98.ThisstudyshowsthatHCMandespeciallyL-HCMhaveagoodtrade-offbetweennumberofparametersandaccuracycontrarytothelargescalemetabolicmodel.L-HCMshowsverygoodaccuracytosimulatebiomass,glucoseandacetateconcentrations;consequentlyitcouldbeanefficienttooltosimulatedynamicalmetabolicbehaviorofE.colifacingfluctuatingenvironmentwhenintegratedwithahydrody-namicmodel.

    References: [1]Enforsetal.2001.JBiotechnol85,175–185.[2]Kotteetal.2010.MolecSystBiol6.[3]Kimetal.2008.BiotechnolProg24,993–1006.[4]Laraetal.2006.MolecBiotechnol34,355-381.[5]Patnaik.2000.BiotechnolAdv18,267–288

    12:05 Infoteam iLAB: A Software Platform for Optimized Bioprocess Development (L32)Ingrid SchmidInfoteam Software AG, Bubenreuth, Germany, [email protected]

    Abstract: Biotechnologybeingoneofthekeytechnologiesofthe21stcenturyisthebasisfortheproductionofmanypharmaceuticals,industrialbiocatalystsandfinechemicals.Itisexpectedthatin2030onethirdoftheworldwideindus-trialproductionwilloriginatefrombiotechnologicalprocesses.Thebiggestchallengetoachievethedemandingaimsisthereductionofthedevelopmenttimeandcostsfornewbioprocesses.infoteamisdevelopinganewlabautomationprocessdatamanagementandprocesscontrolsoftwareplatformcallediLAB.iLABisdevelopedaspartoftheresearchprojectAutoBIO,sponsoredbytheGermanfederalMinistryofEducationandResearch.

    Programme

    Friday, 4 April 2014

  • 3rd BioProScale Symposium 2014

    ThebasicideaofAUTOBIOistoapplyautomationtechnologiesfromthehigh-throughputapplicationdomainonthedevelopmentofbioprocesses.Bioprocessdevelopmentisminiaturizedandparallelized.Methodsandtechnologiesusuallyonlyestablishedforbigscalefermentationareadaptedtothesmallscreeningandlabscale.Therefore,thequalityoftheprocessitselfandofdatageneratedduringthedevelopmentisenhanced,productyieldsareincreasedanddevelopmenttimesarereducedsignificantly.iLABisaflexibleanddevice-independentsoftwareplatformthatprovidesaneasy-to-useinterfaceforthemonitoringandvisualizingofbiotechnologicalprocesses.Basedonexistinglaboratoryautomationstandards,thecoreofiLABisawell-structureddatabase.TheintegrationofDoEanddynamicprocessmodelingtoolsarepossible.Thefeedbackofresultingactuatingvariablesrealizesawell-docu-mentedonlineprocessoptimizationinaclosedloop.iLABisdevelopedaccordingtotheIEC62304softwaredevelopmentlifecycleprocessandtheEU-GMPGuidelineandincludeskeyfeaturesliketrackingandtracing,audittrail,userandrolemanagementandelectronicsignatures.InthispresentationweprovideanoverviewonthebasicfeaturesofiLABanditsopenarchitecturethatisdesignedtobeflexiblyadaptedtospecificapplicationrequirements.

    12:25 Key note lecture: Modelling across bioreactor scales: methods, challenges and limitations (L33)Krist V. GernaeyDepartment of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark (DTU), DK-2800 Kgs. Lyngby, Denmark. [email protected]

    Abstract:Scale-upandscale-downofbioreactorsareveryimportantinindustrialbiotechnology,especiallywiththecurrentlyavailableknowledgeontheoccurrenceofgradientsinindustrial-scalebioreactors.Moreover,itbecomesincreasinglyappealingtomodelsuchindustrialscalesystems,consideringthatitischallengingandexpensivetoacquireexperimentaldataofgoodqualitythatcanbeusedforcharacterizinggradientsoccurringinsidealargeindustrialscalebioreactor.Butwhichmodelbuildingmethodsareavailable?Andhowcanoneensurethattheparametersinsuchamodelareproperlyestimated?Andwhatarethelimitationsofdifferenttypesofmod-els?Thispaperwillprovideexamplesofmodelsthathavebeenpublishedintheliteratureforuseacrossbioreactorscales,includingcomputationalfluiddynamics(CFD)andpopulationbalancemodels.Furthermore,theimportanceofgoodmodelingpracticewillbehighlightedaswell.

    12:55 Concluding remarksPeter Neubauer Technische Universität Berlin, Institute for Biotechnology, Chair of Bioprocess Engineering – [email protected]

    13:00 Endofsymposium

    ChairofBioprocessEngineering

  • Poster abstracts

    Determination of cultivation conditions for auto-mated bioprocess development (P01)M. Heiser1 , A. Knepper1, F. Glauche1, L. Theuer1 , F. Wollny1, S. Bigesse1,2,A. Neubauer2, P. Neubauer11 Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, www.bioprocess.tu-berlin.de 2 BioSilta Europe GmbH, Berlin, Germany, www.biosilta.com

    With„QualitybyDesign“(QbD)astheleitmotif,consistentbioprocessdevelopmentcanstartatmicroliterscale.Settingupaprocessatthatstagemustthereforetakethesameconsidera-tionsintoaccount,asonewouldapplytolarge-scalebioproc-esses.Here,theimplementationofauto-induction,automatedprocessmonitoringandscale-uponamultiwellplateandminibioreactorplatformareshown.Importantfeaturesthatwereincludedaremonitoringofkeyparameters(opticaldensity,pH,evaporation,glucose-andacetatelevelinthemedium).Theywereappliedtomicrolitrescalecultivationsinshakenmultiwellplatesandscaleduptoamillilitrescalestirredtankminibioreac-torsystem.

    Sensor equipment for quantification of spatial heterogeneity in large bioreactors (P02)Anders Nørregaard1, Luca R. Formenti1, Stuart M. Stocks2, Brian Madsen3, John M. Woodley1, Krist V. Gernaey1 1 Department of Chemical and Bioche-mical Engineering, Technical University of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark 2 Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsværd, Denmark3 Novo Nordisk A/S, Hallas Allé, DK-4400 Kalundborg, Denmark

    Suspensioncultivationinlargestirredtankreactorssuffersfromimperfectmixingandpressuregradientsduetothelargesizeoftheliquidcolumninthebioreactors.Thisleadstogradientsofsubstrateconcentrationsandinturncellpopulationheteroge-neity.Theprocessesinlargescalecannotbedirectlycomparedtolaboratoryscaleexperimentsduetothesereasons,andthus,inordertounderstandthelargescaleprocesses,experimentaldatahastobecollectedatlargescale.Thecostofacquiringdataatlargescaleishigh.Thebioreactorsareusuallyrunwithalimitedarrayofsensorsandinordertoapplymoresensorequipmentthebioreactorhastobemodifiedwhichisbothcostlyandresultsinproductiondowntime.Thepresenceofthreephases(gas,liquid,andsolid),andtheopaquenatureofthefermentationbrothtogetherwiththenecessityofheatsterilizationfurtherincreasestherequirementstothesen-sorequipment.Inordertoaddresstheseissuesthisstudyaimstomakeaninvestigationintofreelyfloating,batterydrivensen-sorparticlesthatcanfollowtheliquidmovementinthereactorandmakemeasurementswhilebeingdistributedinthewholevolumeofthebioreactor.Themethodleavesaminimalfootprintandcanbeappliedtorunningproductiontogatherlargescalefermentationdata,withouttheneedofdedicatedexperimentalcultivations.Ultimately,datadescribingthespatialheterogeneitycanbeusedtoenhanceexistingprocessmodelsandtocreatebet-terscale-downstrategiesforlab-scaleexperiments.Accurateprocessmodelsandlab-scaleexperimentscouldinturnleadtoamorescientificapproachtoscalingofbiotechnologicalproc-esses.

    Investigation of the scale-up related CO2/HCO3-stimulus in Corynebacterium glutamicum (P03)Jens Buchholz, Bastian Blombach, Tobias Busche, Jörn Kalinowski, Ralf Ta-kors, Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany, [email protected]

    C.glutamicumisafacultativeanaerobic,Gram-positiveorgan-ismthatgrowsonavarietyofsugarsandorganicacidsandistheworkhorseforthelargescaleproductionofanumberofaminoacids,suchasL-glutamateandL-lysine.Inlargescaleproductionprocessestypicalbioreactorvolumesreachupto750m³tokeepproductioncostslow,resultinginmixingtimesupto240s[1,2].Consequently,cellsareexposedtogradientsoftemperature,substrateconcentrations,pH-valuesorpartialpressuresofthevariousdilutedgasesase.g.O2andCO2[3].Thelatterisadditionallyenforcedduetothehydrostaticpressure,causedbythereactorheightandtheexistenceofpoorlyven-tilatedregionsduetolargervolumes[4].Here,weinvestigatedthescale-uprelatedCO2/HCO3-stimulusonthemetabolismofC.glutamicum.Batchfermentationswithglucoseassolecarbonandenergysourcewithelevated(quasi-stationary)pCO2levelsofupto(300–400)mbarintheliquidphaseshowednodifferenceinthegrowthratebutslightlyhigherbiomassyieldcomparedtostandardconditionsindicatingthatC.glutamicumcantolerateconsiderablehighCO2/HCO3-concentrations.Incontrast,experi-mentswithsignificantlyreducedCO2concentrationsobtainedbystrippingconditions(3vvm)withairledtothreephasicgrowth,whichcanbecomplementedbyincreasingtheproportionofCO2intheinletgasflow.Transcriptomeprofilesobtainedbymicroar-rayanalysesincombinationwiththedeterminationofenzymeactivitiesledtoacomprehensivepictureoftheadaptationtoCO2/HCO3-limitingconditions,indicatinganindirectactivationofdecarboxylationreactionsbytriggeringthethiaminbiosynthe-sis.UnderhighCO2/HCO3-conditions,microarrayexperiments,revealedacomplextranscriptionalresponsewithmostgenesassignedtothecompletedtxR/ripAreguloncontrollingtheironhomeostasisinC.glutamicum.

    References: [1]R.Kelle.L-Lysineproduction,in:Eggeling&Bott(Eds.)Handbookof

    Corynebacteriumglutamicum,CRCPress,BocaRaton,2005[2]Junker.J.Biosci.Bioeng.,97(2004)347-364.[3]Laraetal.Mol.Bio-

    technol.,34(2006)355-381.[4]Baezetal.Ramírez.Biotechnol.J.,6(2011)959-967

    Development and application of a bioreactor scale-down simulator for fermentations of Coryne-bacterium glutamicum under dynamic CO2/HCO3- gradients (P04)Jens Buchholz, Michaela Graf, Bastian Blombach, Andreas Freund, Tobias Busche, Jörn Kalinowski, Ralf Takors Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany, [email protected]

    Theuseofscale-downsystemstoimitateindustrial-relevantreactorinhomogeneitiesandtodetermineconcludingmetabolicandphysiologicalimpactshasbeenrecentlyreviewed[1,2].Inprincipal,mostconceptsfocuson(i)thesimulationofthereducedmixingqualityoccurringatproductionscaleincombinationwith(ii)oscillatingnutrientorgassupplyduringfermentationproc-esses.Eventhough,thegeneralapplicationofC.glutamicumasplatformorganisminscale-up/downstudieswasrecentlyintensi-fied[3,4],themetabolicandtranscriptionalresponsetolargescale-relevantdissolvedCO2levelshasbeenrarelyinvestigated[5,6].

    Poster abstracts

  • Independenceonourresultsobtainedunder(quasi-)station-aryCO2/HCO3-levels[6],wepresentthedevelopmentandapplicationofacascadebioreactorsystem(CBS)comprisingofthreestirredtankreactors(STRs)putinseries:mainreactor(MR),cascadereactor1(CR1)andCR2.Intotalcirculation,theapplicationofflowratesbetweenF=(0.5–4)Lmin–1facilitatedtheimitationofresidencetimesintherangeofaboutτ=(0.6–4)min(CR1;CR2).Aftersuccessfulprocesstechnicalcharacteriza-tionofthesystem,batchfermentationsusingC.glutamicumwild-type(WT)wereperformed.IndustrialrelevantCO2levelsofaboutpCO2=150mbar(CR1)and300mbar(CR2)wereinstalledbyvariationofthegasinletcompositionand/orgasflowrates.Asaresult,asimilargrowthphenotypeasobtainedinrefer-encebatchfermentationsperformedintheMRunderstandardCO2/HCO3-levelswasobserved,insummaryleadingtogrowthratesofµ=(0.42±0.03)h–1,glucoseconsumptionratesofqS=(0.87±0.06)gg–1h–1,andbiomassyieldsofYXS=(0.48±0.01)gg–1.Additionally,thetranscriptionalresponseobservedbyDNAmicroarrayswasrecorded.SampleswerethereforewithdrawnsimultaneouslyfromtheCBS(MRasreference)revealingafasttranscriptionalresponse,despitetheshortresidencetimesintheindividualCRsof>CR1vs.MR.

    References: [1]Neubauer&Junne.21(2010)CurrOpinionBiotechnol21:114-121[2]Takors.J.Biotechnol.,160(2012)3-9[3]Käßetal.BioprBiosystEng.(2013)1-12[4]Käßetal.MicrobCellFact,13(2014)6[5]Bäumchenetal.J.Biotechnol.,128(2007)868-874.[6]Blombachetal.

    J.Biotechnol.,(2013)

    Scale-down model of manufacturing bacterial cultivation: A case study (P05)V. Mangiafridda1, C. Boucher1, M. Allagadda1, S.K. Guddeti2, G. De Conciliis2, W. Vassillo2, A. D’Avino2 1Novartis Vaccines & Diagnostics, Technical Development Bacterial Drug Substance Development, 53100 Siena, Via Fiorentina 1, Italy, [email protected]. 2Novartis Vaccines & Diagnostics, Manufacturing Science & Technology Primary, 53100 Siena, Via Fiorentina 1, Italy

    Adown-scalemodelofamanufacturingprocessisrequiredinordertosupportmanufacturingtroubleshootingandimprove-mentsandtoexecuteprocesscharacterizationactivities.Inthisposter,theactivitiesperformedtoestablishalabscalemodelofa50xscalemanufacturingprocessaredescribed,withparticularfocusonthedown-scalingstrategyappliedtothefermentationstep.Inordertoreproducethefermentermanufacturingmixingandmasstransferproperties,severaladjustmentsofthelabscalebioreactorconfigurationwererequired,includingoptimi-zationoftheimpellerposition,dissolvedoxygencascadecontrolparametersandworkingvolume,throughaKLastudy.Finally,twofeasibilityrunswerecarriedouttodemonstratethesuit-abilityofthedown-scaledprocess.Growthprofiles,productivityoffermentationbatches,andotherproductqualityindicatorsdemonstratedthecomparabilitybetweenthelab-scaleandlarge-scalematerial.

    Integration of rapid sampling experiments into a scale-down two-compartment bioreactor study of Escherichia coli Sergej Trippel, Eva Brand, Robert Spann, Dennis Runge, Ping Lu, Basant El Kady, Stefan Junne, Peter Neubauer Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 71, ACK 24, D-13355 Berlin, Germany

    SincetheresidencetimeofsampleportsofthePFRpartinscaledownreactorsisintherangeof>10seconds,shorttermresponsesareobservedbycombiningthescale-downreactorandtherapidsamplingunitBioscope.Theresponsetoapulseofglucoseunderaerobicandanaerobicconditionsisinvestigated.Theresponseofcells,whichhadbeengrownunderoscillatoryconditions,iscomparedtotheresponseofcells,whichhadbeengrownunderheterogeneousconditions.Itisobservedthatthepreferredroutetowardsthesynthesisofbranched-chainaminoacidsafterapulsedependsonthehistoryofgrowthconditions.Thepresentedmethodologyshouldbeunderstoodasabridgebetweensystemsbiologyfortheinvestigationoftheregulatorynetworkunderindustriallyrelevantconditions.

    Process development for Rhodotorula glutinis: From shake flasks to pilot scale (P06)Eric Lorenz1, Runge D.1, Schmidt L.2, Stahl U.1, Bader J.31 University for Technology Berlin, Germany, [email protected] 2 University of Applied Sciences Jena, Germany, 3 Beuth University of Applied Sciences Berlin, Germany

    Regardingfurtherincreaseofhumanpopulation,conscioususeofnaturalresourcesbecomesmoreandmoreimportant.Inthenextyears,oneofthemainproblemsisthesufficientsupplyofprotein-richfood.Fishisanefficientwaytomeetthisdemand.Anotheradvantageoffishisitshighcontentofvitamins,miner-alsandpolyunsaturatedfattyacids(PUFAs),(FAOreport2012).Duringthelastthreedecades,consumptionoffishhasmorethandoubled.In2010,148mmtoffishasanutritionsourcewereharvested.However,yieldoffishfromindustrialfishingisnotsufficienttocovertheworldwidedemand.Nowadays,45%ofseafoodisalreadyproducedbyaquaculturing.Fishmeal(FM)andfishoil(FO)areessentialingredientsoffishfeedtoensuresuitablefishqualityandquantity.Duetooverfish-ing,thequantityofFMandFOdecreased,simultaneouslylead-ingtohigherprices.TheobjectiveofthecooperativeprojectFENA,supportedbyBLE,wastofindastrategyforthesubstitutionoffishmealandfishoilbyalgaeandyeast.Therefore,wesuccessfullydevelopedahigh-cell-densityprocessforoleaginousyeastRhodotorulaglutinis.Thisstrainwasselectedafterascreeningof32oleagi-nousyeaststrains.Crucialselectioncriteriawereproteinandlipidcontent,aswellasaminoandfattyacidprofiles.Forinitialshakingflaskexperimentsandthesubsequentprocessoptimiza-tion,designofexperiments(DoE)wasconducted.Hence,variousfactorssuchastheC/N-ratio,temperature,andnitrogenandcarbonsourceswereexamined.Theobtainedresultsweretrans-ferredtoa5Lbioreactorcultivationresultinginexcellentgrowthperformance(80gL-1)andlipidaccumulation(30%).Finally,thisvalidoptimizedprocesswassuccessfullyscaled-upto150Lpilotscalebioreactor.Achievedbiomasswillbeusedtosupplementfishfeedinanimaltrials.Inconclusion,theproductionprocessenablesapartialsubstitu-tionoffishmealandfishoilforsustainableandhigh-valuefishnutrition.

    3rd BioProScale Symposium 2014

  • Poster abstracts

    Modeling regulation of carbon and nitrogen me-tabolism in large-scale processes with Escherichia coli (P07)Michael Löffler, Joana Simen, Alexander Broicher, Andreas Freund, Ralf Takors Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany, [email protected]

    Developmentandoptimizationofproductionstrainsandproc-essesiscommonlyperformedinlab-scalefermenters(1–100L).Consequently,bioprocessesneedtobescaled-uptoindustrialscalefollowingtheconstraintthatperformancedataofthelabareequallyachievedinlargeproductionreactors.However,cellsinproductionscalefermentersexperiencesubstrateandoxygengradientswhiletravellingthroughthedifferentzonesofthereactorcausingalargenumberofunderperformances.Itisthegoaloftheprojecttoaddresstheurgentscale-upproblembyprofoundsystemsbiologystudiesusingE.coliasarelevantmodelstrain.Multi-scalesystemsbiologyanalysiswillbeperformedfocusingontheinterplayofsubstrate-gradientbasedstimuliandthedy-namicmetabolicandtranscriptionalresponseincells.Metabo-lomeandtranscriptomedatawillbemeasuredinscaled-downexperiments(Plug-Flow-Reactor,Cascade-Bioreactor)simulatingthegradientsofthesubstantialsubstratescarbon(C-,glucose)andnitrogen(N-,ammonia).Quantitativetranscriptdata,basedonnextgenerationsequencingwillbegatheredusingdiversemutantandreporterstrainsofE.coliforsetting-updata-drivenmetabolicandregulatorymodels.

    Kinetic modelling of the central carbon metabo-lism of Escherichia coli considering flux estima-tions (P08)Sebastian Hans, Anja Lemoine, Nicolas Cruz, Peter Neubauer, Stefan Junne Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstraße 76 ACK24, D-13355 Berlin, Germany, [email protected]

    Kineticmodelsareincreasinglyusedformodelbasedprocess-developmentand–control.Thequalitybasedprocessdesigncanbeenhancedbystatisticallyrelevantparametersandmeasurements.However,thereisalackofsuitablemodels,whichareabletodescribedynamicalfed-batchprocesses.Inthepresentedwork,amodelofthecentralcarbonmetabolismofEscherichiacoliK12W3110isdeveloped,whichrepresentsafusionofamodeloftheTCAcycle[1]withmodelsdescribingtheaminoacidsynthesesandacetateformation.Adynamicmetabolicfluxanalysisbasedonamassbalanceapproach[2]wasappliedforthekineticparameterestimation,whenreactionrateswhereadjustedtothefluxes.Thisallowsimprovedparam-eterestimation,whennotallintermediatescanbemeasuredaccurately.Usingthiscombinatorialmethod,thekineticmodelcouldbeadjustedsuccessfullyintheTCApathway.

    References:[1]Singh&Ghosh.Theor.Biol.MedModel,2006.3-27[2]Desaietal.Biotechnology.1999.71,191-205

    Screening of Klyveromyces marxianus strains iso-lated from traditional dairy products for ethanol production from lactose (P09)Fatemeh Nejati1, Ralf Greiner21 Islamic Azad University-Shahrekord Branch, Shahrekord, Iran2 Department of Food Technology and Bioprocess Engineering, Max Rubner Institut, Federal Research Institute of Nutrition and Food, Karlsruhe, Germany

    Inthisstudy11yeaststrainspreviouslyisolatedfromdairyproductsandidentifiedasKlyveromycesmarxianuswereevalu-

    atedforethanoltoleranceandethanolproduction.Theresultsshowedthatethanolconcentrationfrom6%canhaveareduc-tionongrowthoftheyeasts.Inamediumcontaining25g/llactose,ethanolconcentrationsweredeterminedtobe0.61to1.70g/lafter24hrsoffermentation.Although,ethanolproduc-tionwasnothigh,itseemsthatchangingandmodifyingsomeparametersinfermentationsuchascarbonsourceandnutrientingredientamount,temperature,incubationtimeandinocula-tionsizewouldresultsinhigheryieldsofethanol.

    Docosahexaenoic acid production with marine or-ganism Crypthecodinium cohnii in the wave-mixed single-use bioreactor CELL-tainer (P10)Friederike Hillig, Nadine Porscha, Stefan Junne, Peter Neubauer Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Ger-many, [email protected]

    Cultivationofmarineorganismsexhibitsthebigchallengetoavoidcorrosioninconventionalstainlesssteelbioreactorsduetothehighchlorideionconcentrationofmarinemedia.Theapplicationofsingle-usebioreactors(SUB)offersapossibilitytocircumventthisproblem.However,mostoftheorganismshavealsoahighoxygendemandand,inthecaseofCrypthecodiniumcohnii,theyaresensitivetoshearforces.Therefore,thechoiceoftheappropriatebioreactorsystemiscrucial.TheCELL-tainer(CELLutionBiotechBV,Assen,Netherlands)provideshighoxygentransferratesandwasthereforeappliedforthecultiva-tionofC.cohnii.AcomparisonofthisSUBwiththeconventionalstirredtankreactorwasperformedandthegrowthandproduc-tionperformanceinbothdevicesispresented.Additionally,spe-cialattentionwasaddressedtothecellphysiologydeterminedbytheapplicationofflowcytometry.Resultsprovethefeasibilityoftheconceptasanalternativetostainlesssteelreactorsinearlystagesofmarineprocessdevelopment.

    Effect of phosphate concentration and oxygen oscillation conditions on clavulanic acid produc-tion in Streptomyces clavuligerus (P11)Howard Ramirez1,2, Juan Arzate2, Nicolas Cruz-Bournazou2, Stefan Junne2, Peter Neubauer2, Rigoberto Rios11Bioprossess Research Group, University of Antioquia, Colombia2Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, [email protected]

    Clavulanicacid(CA)isproducedbyfermentativeprocesseswithStreptomycesclavuligerus(Sc).Despiteitsweakantibacterialactivity,itisapowerfulinhibitorofβ-lactamaseenzymeswithsomeimportantapplicationsalreadyavailableinthemarket.Large-scaleproductionofantibioticsisperformedatgrowth-limitingconcentrationsofinorganicphosphate.ThisplaysanimportantroleinCAsynthesis,becauseitisrelatedwiththeavailabilityofglyceraldehyde-3-phosphate,whichisthefirstprecursorfortheclavulanicacidproduction.Inthiswork,twocultivationswerecarriedoutusingeither0.58g/LK2HPO4or0.8g/LK2HPO4in250mLUltraYield

    ©shakeflask.Thecultureswereexposedtooxygenoscillationconditionsat30hand48hseparatelybytherepeatedinterruptionofshakingandtheac-cumulationofCAwasinvestigated.Aprocessmodelofthebatchfermentationwasdeveloped,appliedinaMatlab®environmentandcomparedwithexperimentaldata.Resultsindicateagoodsimulationperformanceatthebiomassandsubstrateconcentra-tion.