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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

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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 peter.neubauer@tu-berlin.de

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. papoutsakis@dbi.udel.edu

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 – raivo@kbfi.ee

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, patrick.sagmeister@tuwien.ac.at

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 – alara@correo.cua.uam.mx

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 – maurotru@gmail.com

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, stephan.kaiser@zhaw.ch. 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, florian.glauche@tu-berlin.de, 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, s.unthan@fz-juelich.de

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, kanag@ukzn.ac.za

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, a.gruenberger@fz-juelich.de

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 Schmid1

1Laboratory of Chemical Biotechnology, TU Dortmund University, Germany – christian.dusny@bci.tu-dortmund.de 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 – takors@ibvt.uni-stuttgart.de

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 – anja.lemoine@tu-berlin.de

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 – m.oldiges@fz-juelich.de, 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 – christian.reitz@tu-berlin.de

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 – j.rupprecht@hamilton-messtechnik.de

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 Junne2

1EloSystemsGbR,Berlin,Germany,angersbach@biotronix.de.2TechnischeUniversitätBerlin,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. Stocks2

1Department of Chemical and Biochemical Engineering, Technical University of Denmark, Lyngby, Denmark – danaquh@kt.dtu.dk2Novozymes 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 – stephane.guillouet@insa-toulouse.fr

Abstract: TheyeastSaccharomycescerevisiaebelongstosuchyeastspecieswhichswitchfromapureoxidativemetabolismtoarespiro-fermentativemetabolismevenunderfullyaerobicconditionsassoonastheglucoseexceedsaconcentrationofabout0.5gl-1.ThisspecificcharacteristicofS.cerevisiaehasbeensupportiveforallfermentativeprocessessuchasalcoholicbeverageorethanolproductionbutrepresentsadisadvantageforproductionofyeastbiomass(baker’syeastproduction)aswellasbiomass-relatedyeastproducts(suchasheterologousproteins).Therefore,yeastbiomassproductionbioprocesseshavebeenusuallycarriedoutunderaerobicfed-batchmodewheretheglucosefeedingisfinelycontrolledtokeepaverylowresidualconcentration(<0.5gl-1)andtomaintainthespecificgrowthratebelowacriticalvalue,calledcriticaldilutionrate(Dc).Suchstrenuousmonitoringconditionsbecomechallengingwhentheproductioniscarriedoutinlargescalebioreactorswheremicroorganisms,duetomixingissues,aresubmittedtofluctuationsinconcentrationsofe.g.substrateandoxygenwithanintensityandafrequencydependingontheoperat-ingscale.Analternativetocontrolledfeedingofsugarbasedfeedstockwouldbetheuseofadditivesabletodelaythemetabolicswitch.WewillpresenthereanexampleofstrategyappliedtotheproductionofS.c.underpureoxidativemetabolism.WedemonstratedthatadditionofoleicacidalloweddelayingandmodulatingthetransitionfromrespiratorytofermentativemetabolisminS.cerevisiae.Theresponseofyeasttooleicacicwasstudiedthroughsystemicanalysiscouplingmetabolicsandtranscriptomics.Wefurtherinvestigatedthepotentialofoleicacidtocircumventheterogeneitiesissuesinlargescalebioreactorsbymimickingtheseeffectsinscale-downbioreactorcoupledwithcellpopulationanalysisandcomputersimulation.

12:05 Key note lecture: Microbial heterogeneity affects bioprocess robustness: Future of dynamic single-cell analysis for large-scale bioprocess control (L18)Frank DelvigneUniversity of Liege, Belgium – f.delvigne@ulg.ac.be

Abstract: Microbialphenotypicheterogeneitystronglyimpactsbioprocesses,asdemonstratedforseveralcellfacto-ries.Phenotypicheterogeneityinbioprocessingconditionsisofteninvestigatedbyusingfluorescenttranscriptionalreportersincombinationwithsinglecelldetectiontechnique,suchasflowcytometry.However,therearenowsomeevidencesthatgeneinductionisnottheonlymechanismsubjectedtostochasticity.Indeed,variabilityatthelevelofmetabolicnetworkcanalsoleadtodifferentcorrespondingphenotype,suchasthespecializationofafractionofthecellpopulationtotheuptakeofaspecificsubstrateoraspecificby-product.Theseconsiderationsareofprimaryimpor-tancefortheunderstandingofphenotypicheterogeneitysinceclassicaldetectiontechniquesarenotdirectedtowardsmetabolicprocesses.Basedonthesetheoreticalconsiderations,ascientificstrategyinvolvingon-lineflowcytometrycoupledtodifferentbioreactorconfigurationswillbepresentedandtheimportancefortheselectionofanappropriatefluorescentreportersystemwillbedemonstrated.E.coliwillbeusedasarepresentativecellfactoryexhibitingsignifi-cantphenotypicheterogeneitywithsevereconsequencesonbioprocesses.

12:35 Lunchbreak,postersessionandexhibition

Chair MarioBirkholz(TUBerlin,ChairofBioprocessEngineering)

14:00 Monitoring functions in managed microbial systems by cytometric bar coding (L19)Susann Müller1,Christin Koch2, Ingo Fetzer1, Susanne Günther1, Hauke Harms1

1UFZ – Helmholtz Centre for Environmental Research, Department of Environmental Microbiology, Leipzig, Germany – susann.mueller@ufz.de2UFZ - Helmholtz Centre for Environmental Research, Department of Bioenergy, Leipzig, Germany

Abstract: Thestillpoorlyexploredworldofmicrobialfunctioningisabouttobeuncoveredbyacombinedapplianceofoldandnewtechnologies.Especiallybacteriaarestillinthedarkbothinviewoftheirphylogeneticaffiliationaswellastheirmetaboliccapabilitiesandfunctions.However,withtheadventofsophisticatedflowcytometricandcellsortingtechnologiesinmicrobiologicallabsthereisnowthepossibilitytogainthisknowledgewithoutcumbersomecultivationapproaches.Asingle-cellbasedapproachcalledcytometricbarcoding(CyBar)forfastidentificationofreactivesub-communitieswasused.Functionalityofspecificsub-communitieswasuncoveredbyprocessingCyBardatawithabioticreactorparametersusingSpearman’scorrelationcoefficient.ThedynamicsofcellularDNA-contentsandcellsizesasgrowthin-dicatorsweredeterminedtodetectthemostactiveindividuals/sub-communitiesandtodeterminetheirphylogeneticaffiliationuponcellsortingandcorrelatetheirabundancetocertainabioticfactors.Newbiostatistictoolsaredevelopedtoevaluateandinterprettheresultinghugedatasets.Theprocedureiseasytoperform,quickandhasthecapacitytodesignandcontrolbiotechnologicalprocesses.Theworkflowisshowntobesuitedforcomplexmicrobialcommunityanalysisoneverydesiredtimeandinvestigationlevelwithhigherautomatizationpotentialasdescribedelsewhere.Applicationsareshownforwastewatertreatmentplantsandbiogasreactors.

References:Kochetal.2013.NatureProtocols8/1,190-202.Kochetal.2013.EnvironmSciTechnol47,1753-1760.Güntheretal.2012.EnvironmSciTechnol46(1),84-92

Programme

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 – alison.brognaux@doct.ulg.ac.be

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, anika.bockisch@tu-berlin.de

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 – jvenus@atb-potsdam.de2teleBITcom 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 – je@brain-biotech.de

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 – Henk.noorman@dsm.com

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 – reuss@ibvt.uni-stuttgart.de

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 – lyberatos@chemeng.ntua.gr

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. Neubauer2

1 CELLution Biotech BV, Assen, The Netherlands – nico.oosterhuis@cellutionbiotech.com2Technische 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 – thomas.dreher@sartorius-stedim.com

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 – benjamin.minow@rentschler.de

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, juergen.hubbuch@kit.edu

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 – ngorret@insa-toulouse.fr

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, Ingrid.Schmid@infoteam.de

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. kvg@kt.dtu.dk

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 – peter.neubauer@tu-berlin.de

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. Neubauer1

1 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, buchholz@ibvt.uni-stuttgart.de

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, buchholz@ibvt.uni-stuttgart.de

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<2min,asaconsequenceoftheappliedCO2/HCO3-variations.Moreover,itwasobservedthattheabsolutenumberofdifferentlyexpressedgenesincreasedinanalogytotheextentoftheCO2/HCO3-gradientwithCR2vs.MR>>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, valentina.man-giafridda@novartis.com. 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.3

1 University for Technology Berlin, Germany, eric.lorenz@campus.tu-berlin.de 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, michael.loeffler@ibvt.uni-stuttgart.de

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, sebastian.hans@campus.tu-berlin.de

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 Greiner2

1 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, f.hillig@tu-berlin.de

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 Rios1

1Bioprossess Research Group, University of Antioquia, Colombia2Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, howardrm13@gmail.com

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.

Mobile multi-sensor systems for the 3-D characte-rization of industrial scale processes and the inve-stigation of gradients (P12)Anika Bockisch, Erich Kielhorn, Stefan Junne, Peter Neubauer Technische Universität Berlin, Chair of Bioprocess Engineering, Berlin, Germany, anika.bockisch@tu-berlin.de

Anaerobicprocessesareoftencharacterizedbylaminarflow.Hence,gradientsintheliquidphaseoccur.However,theknowledgeoftheirimpactonprocessperformanceispoor.Thetraditionalsamplingspotsareusuallymountedatthewallofthebioreactors.Samplesorvalues,whichhavebeentakenwithouttheconsiderationofgradients,mightthereforenotreflecttheconditionsofthecoreoftheliquidphase.Therefore,mobilesamplingsystemsandminiaturizedmultipositionsensorshavebeendevelopedandappliedinbrewingtanksandbiogasreactors.IthasbeenshownthattheonlinemonitoringofgradientsisachievablewithmicrosensorsforthepH-value,theredoxpotential,O2-andCO2-concentration,andtemperature,respectively.Thecouplingofsensorswithaprocess-statedependentsam-plingfrequencyatthespotwhereprocessdisturbancescanbedetectedveryearlyoffersthepossibilitytosavetimeandcostsforofflineanalytics.Thecombinationofthisdataandstudiesonthecell’smetabolismandphysiologyallowsanimprovedunderstandingofgradientsandtheirimpactonaprocess,andafurtherimproveddesignofscale-downexperimentsclosetotheconditionsintheindustrialscale.

Multiparameter sensor system for process monito-ring in biogas plants (P13)Sandra Päßler, Ute Enseleit, Winfried Vonau Kurt-Schwabe-Institut für Mess- und Sensortechnik e.V. Meinsberg, Wald-heim, Germany, paessler@ksi-meinsberg.de

Theposterpresentselectrochemicalsensorswhichcanbeusedtoinvestigatebiotechnologicalprocessese.g.inbiogasplants.Themonitoringisfocusedoninsufficientlymixedareasandinhomogeneitiesinthebiogasmedia.Thesensorshavetobeadaptedtothemediacompositionandthemeasure-mentrequirements.Consequently,twodifferentsystemswereconstructed.Forthemeasurementinahydrolysisbasinalancewiththreesensorheadsindifferentdepthswasdeveloped.EachsensorheadcontainselectrodestomeasurepH,redoxpotentialandtemperature.Thesecondsystemisamulti-parameterprobewithminiaturisedsensorsforthesameparameterswhichisusedinthefermenterofabiogasplant.

Monitoring of Bacillus sporulation via electo-opti-cal measuring method (EloTrace®) (P14)Dounia, Sandra1, Schütze, O.2, Mucha, O.2, Angersbach, A.3, Bunin. V.3, Stahl, U.1, Bader, J.4

1 Research and Teaching Institute for Brewing in Berlin (VLB), Research Insti-tute for Microbiology, Berlin, Germany, s.dounia@vlb-berlin.org 2 ABiTEP GmbH, Berlin, Germany3 EloSystems GbR, Berlin, Germany4 Beuth University of Applied Sciences, Life Sciences and Technology, Depart-ment of Biotechnology, Berlin, Germany

Newtoolsformonitoringoffermentationprocessesarerequiredtoachieveadditionalprocesscontrolandtomeettheincreasingdemandonreproducibilityandqualitycontrolinbiotechnologi-calproduction.Inthisworktheelectro-opticalmoduleEloTracewasusedforonlinemeasurementandcharacterizationofchangesincellphysiologyandmorphologyduringaerobicgrowthandsporu-lationofBacillusamyloliquefaciens.

SomerepresentativesofthespeciesBacillusamyloliquefaciensbelongtothegroupofplant-growth-promotingrhizobacteria(PGPR)andweresuccessfullyusedasbiofertilizersinagricultureotherswereusedfortheproductionofenzymesandantibiotics.DuringfermentationofBacillihighaerationratesarerequiredtoachieveoptimalgrowthandhighproductformation.Thesede-mandsonthefermentationconditionsresultinfinelydispersedgasbubblesandfoamformationwhichcausesevereproblemsduringautomaticsamplingandonlinemeasurement.Keypointofthepresentedworkwastodevelopanautomaticsamplingtoolabletohandlesamplesincludingfoamandgasbubbles.ThesuccessfuldevelopmentofthesamplingunitbythecollaborationofEloSystemsGbR(Berlin),ABiTEPGmbH(Ber-lin),TechnicalUniversityofBerlinandHSMannheimenabledon-linemeasurementofcelllength,opticaldensityandanisotropyofpolarizability(AP),aparameterforthephysiologicalstateofcells,withEloTraceeveninstronglyaeratedfermentationproc-esses.SpecificcurvesoftheAP-levelofthecellsreflectaltera-tionsofintracellularsubstancesandmodificationsofmembranesurface.GainedAP-signalscouldbecorrelatedwithonsetofsporulation,whichisbasedonconversionofcellularstructures.ThisworkwassupportedbyAiFe.V.(Arbeitsgemeinschaftindus-triellerForschungsvereinigungen“OttovonGuericke”e.V.).

Sensor equipment for quantification of spatial heterogeneity in large bioreactors (P15)Anders Nørregaard1 (andno@kt.dtu.dk), Luca R. Formenti1, Stuart M. Stocks2, Brian Madsen3, John M. Woodley1 and Krist V. Gernaey1

1 Department of Chemical and Biochemical Engineering, Technical Universi-ty of Denmark, Building 229, DK-2800 Kgs. Lyngby, Denmark 2Novozymes A/S, Krogshoejvej 36, DK-2880 Bagsværd, Denmark; 3Novo Nordisk A/S, Hallas Allé, DK-4400 Kalundborg, Denmark.

Suspensioncultivationinlargestirredtankreactorssuffersfromimperfectmixingandpressuregradientsduetothelargesizeoftheliquidcolumninthebioreactors.Thisleadstogradientsofsubstrateconcentrationsandinturncellpopulationheteroge-neity.Theprocessesinlargescalecannotbedirectlycomparedtolaboratoryscaleexperimentsduetothesereasons,andthus,inordertounderstandthelargescaleprocessesexperimentaldatahastobecollectedatlargescale.Thecostofacquiringdataatlargescaleishigh.Thebioreactorsareusuallyrunwithalimitedarrayofsensorsandinordertoapplymoresensorequipmentthebioreactorhastobemodifiedwhichisbothcostlyandresultsinproductiondowntime.Thepresenceofthreephases(gas,liquid,andsolid),andtheopaquenatureofthefermentationbrothtogetherwiththenecessityofheatsterilizationfurtherincreasestherequirementstothesensorequipment.Inordertooptimizetheexperimentalsetup,differenttypesofsensorequipmentarecurrentlyinvestigatedwithregardtodescribingtheflowpatternsandshearstress,masstransferofoxygenandsubstrategradientsinthefermentationbroth.Ulti-mately,datadescribingthespatialheterogeneitycanbeusedtoenhanceexistingprocessmodelsandtocreatebetterscale-downstrategiesforlab-scaleexperiments.Accurateprocessmodelsandlab-scaleexperimentscouldinturnleadtoamorescientificapproachtoscalingofbiotechnologicalprocesses.

3rd BioProScale Symposium 2014

Poster abstracts

Continuous production of biobutanol: Design and evaluation of bioreactor configurations (P16)Peter Götz1, Katja Karstens1, Horst Niebelschütz2, Richard Görlitz2, Giuseppe Olivieri3, Francesca Raganati3, Antonio Marzocchella3

1 Beuth University of Applied Sciences, Berlin, Germany, goetz@beuth-hochschule.de 2 ARGUS Umweltbiotechnologie GmbH, Berlin, Germany 3 Università degli Studi di Napoli - Federico II, Napoli, Italy.

FermentativesolventproductionbytheAcetone-Butanol-Ethanol(ABE)fermentationhaditspeakinthefirsthalfofthelastcentury.Initialprocessdevelopmentstartedfrombatchoperation,butduetotheadvantagesofcontinuousproduction,therehavebeenseveralattemptstoimplementthisoperatingmode.ThebiphasicnatureoftheABEfermentation,causedbyametabolicswitchfromacidtosolventproduction,degenera-tionofproductionstrainsbyplasmidlossandtheoccurrenceofcontaminations(bacteriophages)aresomeofthemainchal-lengeswhichareencounteredindevelopmentofacontinuousABEfermentation.Areviewofcontinuousreactorconfigurations,includingsinglestageCSTR,seriesofCSTRsand(fixed-bed)flowreactorswillbegiven.BasedonresearchofmetabolismofC.acetobutylicum,alaboratorysetupforinvestigatingnewreactorconfigurationswillbepresented.Problemsaddressedbyusingthisconfigura-tionforresearchareregenerationofdegeneratedcells,induc-tionofsolventproductionandsuppressionofcontaminations.Specialattentionwillbegiventoscalabilityoftheprocess.Thepresentedworkisrelatedtotheauthors´researchintheEra-NetEuroTransBio7ProjectOPTISOLV:GermanpartnersaresupportedbytheBMBF,theItalianpartnerissupportedbyMSE.

InfluenceofbiotransformationmediaandC-Sourceonbiotransformationwithfed-batch-cultivatedyeasts(P17)Thomas Grimm, Technische Universität Berlin, Chair of Bioprocess Enginee-ring, Berlin, Germany, thomas.grimm@berlin.de

In24squaredeepwellplatesyeastwerecultivatedwithfed-batchmediatogetahighcelldensityforafollowingwhole-cellscreening.Withthebestvolumeforoptimalaerobicgrowthin24squaredeepwellplatessixpromisingyeaststrainsweregrownonEnPressomedium(Biosilta).Aftercultivationthewhole-cellbiotransformationfortheenantioselectivereductionof2-butanonetoR/S-2-butanolwasinvestigated.Theinfluenceofbiotransformationconditionsontheconversionandenantioselectivityofthesixculturedyeastswereexaminedunderanaerobicconditionsafter24h.Ourresultsshows,thatwithfed-batchlikeEnPressocultivationahigherbiomasspervolumeandahigherenzymeactivityperbiomasswithhigherselectivitycouldbereached.TheresultsoftheinfluenceofthebiotransformationmediaandtheC-sourceduringthebiotransformationwhereshown.

Production and partial characterization of exopoly-saccharide from Rhodococcus erythropolis grow-ing brewer´s spend grain from pilot plant (P18)Enriqueta Martinez-Rojas Versuchs- und Lehranstalt für Brauerei in Berlin (VLB) e.V., Berlin, Germany, e.martinez@vlb-berlin.org

BiopolymersliketheExopolyssacharide(EPS)havebeenextensivelystudiedbutonlyafewofthemhavebeenindustri-allydevelopedoverthelastdecades.Exopolyssacharidearehighlyheterogeneouspolymerscontaininganumberofdistinctmonosaccharidesandnoncarbohydratesubstituentsthatarespeciesspecific.Duetothebroadmetabolicdiversityandarray

ofuniqueenzymaticcapabilities,RhodococcuserytbropolishasbeenusedtoproduceEPSinsubmergedfermentationwithBrewer’sspentgrainfrompilotplantasalternativecarbonsource.Theresultsobtainedsuggestanopportunityforexplor-ingthisalternativestrategytoproduceEPSandotherhighvalu-ablechemicalsusingeconomicallyfeasiblesubstrates.

Two compartment reactor concepts for bioprocess scale-down studies (P19)Michael Limberg, Viola Pooth, Wolfgang Wiechert, Marco Oldiges Forschungszentrum Jülich; Institute of Bio- and Geosciences IBG-1: Biotech-nologie, Jülich, Germany, m.limberg@fz-juelich.de

Therelevanceofbiotechnologicalproductionincreaseswiththedemandforsustainablebulkproducts.Butperformancelossesduringthescale-upfromlaboratoryintoproductionscaleandtheassociatedincreaseofproductioncosts,imperilsthecompetitivenessofbiotechnologicalproduction.Onereasonforthelossesisbasedonthedecreasingmixingqualityandtheresultinggradientformationattheindustrialscale.Toconsidertheinfluenceofinhomogeneouscultivationconditionsduringbioprocessdevelopmenttheyhavetobetransferreddowntolaboratoryscale.Thisisthesocalledscale-downreactorcon-cept.Astherearenocommercialscale-downsystemsavailable,themostwidelyusedsetupsarecustom-madedeviceswiththeirindividualscopeofapplication.Predominantlythesetupsarebasedonthetwocompartmentreactorconcept[1].Thecellsuspensionoscillatesbetweenanidealandadisturbedcom-partmentwithdifferentresidencetimes.Inthisstudyatwocompartmentscale-downdevicecomposedoftwoconnectedstirredtankreactors(STR)issetupandchar-acterized,forflowandresidencetimedistribution.Thereactorcompoundsconsistofstandardlaboratoryscalepartsinacom-mercialcultivationplatform.AsreferencesetupanestablishedandwellcharacterizedtwocompartmentreactorwhichconsistsofanaerobicSTRconnectedtoananaerobicplugflowreactor(PFR)isused.ThePFRcontains18%oftotalworkingvolumeof5Landisequippedwiththreesamplingportsindefinedintervals[2].AcrossthePFRaspace/timeresolutionofthelimitingconditionscouldbeachieved,buttheoccurrenceofsecondaryeffects,likepHoscillations,makesitverydifficulttoallowaseparatediscussionofthecriticalparameters.Bothscale-downsetupswereusedforcomparativecultivationswithC.glutamicumproductionstrainsunderoscillatingoxygensupplyconditions.TheSTR-STRtwocompartmentscale-downreactor,whichwasbuildupinthiswork,isbasedonaDASGIPparallelfermentationplatformandenablestherebyahighlevelofflexibility.Bycom-biningdifferentvesselsizes,fluidlevelsandsensorequipment,thevolumeratiobetweenthetwocompartmentsaswellastheonlineanalyticscouldbeadaptedtoabroadscopeofapplica-tions.AdditionallythepossibilityofmonitoringandcontrollingtheconditionsineachcompartmentenablestheobservationofseparatedorcombinedeffectsofoscillatingpH,temperature,pO2andsubstrateconcentration.Thisstudyaimsatcompara-tiveanalysisoftheconceptsofadefinedspace/timeresolution(STR-PFR)withtheconceptofanaverageresidencetimeresolu-tion(STR-STR)foranindustriallyrelevantmicrobialsystem.

References: [1]Neubauer&Junne.CurrentOpinioninBiotechnology2010.21:114-

121[2]Käßetal.BioprocessBiosystEng.,2013

Microfluidic chip development for sampling of metabolites secreted by a single cell (P20)Katrin Rosenthal, C. Dusny, V. Oehling, F. Fritzsch, O. Frick, A. Schmid TU Dortmund University, Laboratory of Chemical Biotechnology, Germany, katrin.rosenthal@bci.tu-dortmund.de

Classicbiotechnologyoffersseveralanalyticalmethods,basedonpopulationlevelwithaplentyofavailableapplicationranges.However,exactinvestigationofcell-to-celldifferencesandindividualeffectsoncellphysiology,aswellasmetabolicandproductionperformancemayonlybeobtainedbysinglecellanalysis(SCA).1,2Specificsinglecellresearchquestionsnecessitatespecificandnewlab-on-a-chipsystems.Inordertoestablishasystemsuitablefortheprecisedefinitionofphysicalandchemicalparameters,themicrofluidicchipEnvirostathasbeendevelopedpreviously.IntheEnvirostat,celltrappingisappliedbynegativedielectrophoresis(nDEP)allowingthecontactlesscultivationofmicroorganisms.3Thismethodprovidestheadvantageofavoidingunspecificeffectsoncellphenotyperesultingfromcell-surface-interactions.Additionally,acontinuousmicroflu-idicmediumflowenablestheapplicationofspecificchemicalperturbationstothetargetcell.ProofoftheEnvirostatconceptwasdemonstratedbycultivatingvariousmicroorganismsandbygrowthstudiesofsinglebacteriaandyeastcells.3,4Themanu-facturingprocessoftheEnvirostatchiphasbeenoptimizedpar-ticularlywithregardtotheestablishmentofanalyticalmethodsfortheevaluationofcellularproductivityatsinglecelllevel.Thepreviouslyusedindirectgluebondingprocesswasreplacedbyadirectfusionbondingprocess,whichenhancesthemicrofluidicstabilityofthechipandtherebythereproducibilityofsinglecellexperiments.Twoglasssubstratesarecovalentlybound,leadingtoahomogeneousconnectionexhibitinghighbondstrengthandchemicalresistance.Besidestheirbiocompatibility,glassmadechipsarecompatibletofurtheranalyticsofexcretedcellmetabolites.Theelectricandmicrofluidicdesignwaschangedtoimprovethemicrofluidicstabilityandthereproducibilityofsinglecellisolation.Unreliableelectricalisolationsectionswerereplacedbyvalidatedelectrodegeometries,whichsimplifycellseparation,aswellastheremovalofinterferingcellsormediumfromtheinoculum.WepresentthemostrecentdevelopmentofthemicrofluidicchipEnvirostat,whichwasoptimizedtoenableanalysisofex-cretedmetabolitesofasinglecell.

References: 1.Fritzschetal.AnnuRevChemBiomolEng3,129-155(2012)2.Delvigne&Goffin.BiotechnolJ9,61-72(2014).3.Fritzschetal.Labonachip13,397-408(2013)4.Dusnyetal.ApplEnvironMicrobiol(2012).

From shake flasks to pilot-scale production and application of the plant-growth-promoting bacte-rium Azospirillum brasilense for a liquid inoculant formulation (P21)Norma A. Valdez-Cruz, Cesar Gonzalez-Monterrubio, Carlos Martinez-Salinas, Ramses I. García-Cabrera, Ramses A. Gamboa-Suasnavart, Luz D. Marín-Palacio, Jesus Villegas, Abel Blancas-Cabrera and Mauricio A. Trujillo-Roldán Unidad de Bioprocesos, Departamento de Biología Molecular y Biotec-nología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. Biofabrica Siglo XXI S.A. de C.V. México, adrivaldez1@gmail.com

Azospirillumbrasilenseisthebestwellknownplantgrowthpromotingrhizobacteriaforitscapacitytofixnitrogen,andalsofortheabilitytoproducekeysignalsandcomponentsofplantgrowthpromotion.Thisbacteriaarewidelyusedinseveral

plantations,mainlywheat,sorghum,barley,rice,andmaize.However,thereisnoreportintheliteraturedisclosingaliquidproductproducedinpilotscalebioreactors,andabletobestoredatroomtemperatureformorethantwoyears.Theaimofthisworkwasscaled-upaprocessfortheproductionofaliquidinoculantformulationbasedonA.brasilense,fromshakeflaskto1,000Lpilot-scalebioreactorusingtheoxygenmasstransfercoefficient(KLa)ascriteria.Furthermore,weaimedtodeterminetheshelflifeoftheliquidformulationstoredatroomtempera-ture,andtheincreaseinmaizecropsyieldsingreenhouses.Aconcentrationrangingfrom3.5to7.5x108CFU/mLwasob-tainedinshakeflasksandbioreactors,andaftertwoyearsstoredatroomtemperature,theliquidformulationshowedaoneorderofmagnitudedecrease.Applicationsoftheculturedbacteriainmaizeyieldsresultedinincreasesofupto95%incorncobs,and70%inabovegroundbiomass.

Microbial analysis of a biofiltration system – Do heterotrophic bacteria contribute to metal removal? (P22)Lorika S. Beukes, Stefan Schmidt, Discipline of Microbiology, School of Life Sciences, University of KwaZulu-Natal, Private Bag X01, Pietermaritzburg 3209, South Africa, lorikabeukes@live.co.za

Theobjectiveofthisstudywastoverifythepresenceofironandmanganeseoxidizingbacteriainabiofiltrationsystememployedtopurifyboreholewaterandtoassesstheirpotentialcontributiontometalremoval.Inadditiontothequantifica-tionofthesetargetmicroorganismsinbiofiltersamples,thecontributionofindividualisolatestotheremovalofFe(II)andMn(II)fromboreholewaterandthepresenceofmetabolicallyactivebiofilmsonthefiltermatrixwereverified.PresumptiveFe(II)andMn(II)oxidizingbacteriawerequantifiedusingMSVPmediumpluseitheraddedironormanganese.Selectedisolateswerecharacterizedbylightandelectronmicroscopy,physiologi-caltests,MALDI-TOFMSand16SrRNAgenesequenceanalysis.ColorimetricassaysusingferrozineandleucoberbelinblueaswellasXRDanalysiswereemployedtoconfirmFe(II)andMn(II)oxidationusingselectedbacterialisolates.ThepresenceofmetabolicallyactivebiofilmswasverifiedbyCLSManalysisofbiofiltermatrixsamples.Anon-motile,Gramandoxidase-negativeplumprod-shapedisolateabletoformbiofilmswasidentifiedasamemberofthegenusAcinetobacterwhichwasconfirmedbyMALDI-TOFMSand16SrRNAgenesequenceanalysis.TheabilityofthisisolatetooxidizebothFe(II)andMn(II)wasconfirmedbyXRDanalysisofcrystalsformedinaerobicbatchculturescontainingMSVPplusaddedironormanganese.InthepresenceofactivecellsofAcinetobactersp.strainLB1,therateofMn(II)oxidationwassignificantlyhigheratneutralpHthanthatdeterminedforabioticcontrols.However,atneutralpHtherateofFe(II)oxidationinthepresenceofactivecellsofAcinetobactersp.LB1wasinthesamerangeasthatdeterminedusingabioticcontrols.CLSManalysisofbiofiltermatrixsamplesconfirmedthepresenceofmetabolicallyactivebiofilmsviaCTCstaining.Ourdataindicatethatheterotrophicbacteriasuchasmem-bersofthegenusAcinetobacterpresentinthesystemwaterorwithinmetabolicallyactivebiofilmsonthebiofiltermatrixcancontributetotheoxidativeremovalofMn(II)atneutralpH.However,theremovalofironbymicrobialactivityappearstobeindistinguishablefromabioticironoxidationatneutralpH.

3rd BioProScale Symposium 2014

Pharmaceutical protein synthesis and screening in vitro with a cell free platform (P23)Marco G. Casteleijn, Centre for Drug Research, University of Helsinki, Helsin-ki, Finland, marco.casteleijn@helsinki.fi

Therefore,thecell-freesystemsbypasscellwalls,organellestructuresandavoidtheissuesofcellulargeneticregulation.Theabsenceofthecellularbarriersallowseasyincorporationofsubstrates,efficientproductremoval,rapidsystemmonitoringandconvenientsampling.Thisshouldpromoteproductionofsyntheticproteinswithnovelpropertiesaswellastheproduc-tionofsustainablefuels,chemicalsandnewclassesofdesigner(nano)materialswithtuneableproperties.Herewedescribetheexperimentaloutlinetodevelopaproteinexpressionandscreeningplatformgearedtotranslatebiologi-callyactiveproteinstowardspharmaceuticaldrugcandidates.Ouraimistoproductproteinsasnativeaspossibleandtoaddfunctionalitytotheproteinspost-transitionallybymeansofconjugationtechniques.Thefocusisondevelopmentofinnova-tivetoolsforbiobaseddrugdiscoveryanddrugdelivery.

Wealth from waste? A case study using some se-lected citrus peels (orange, lime and lemon) (P24)Esonu Marvellous, Department of Animal Science and Technology, Federal University of Technology, Owerri, P.M.B.1526, Imo State, Nigeria, ugwum1@yahoo.com

AstudyontheextractionofessentialoilsfromLemonpeels,LimeandOrangeusingN-Hexanewascarriedout.TheyieldofoilfromLemon,LimeandOrangewas3.6%,6.87%and6.13%respectively.TheeffectsofTemperature,Timeandparticlesizeswerealsostudied.Theresultobtainedshowthattheyieldofoilincreasedinsmallparticlesizeandlongercontacttime.Theyieldatatemperatureof25°Cin24hrswith0.5mmparticlesizefororangeandlemonwas0.846and0.69%respectively,at40°Cfor1.0mmparticlesizeand30minuteswas0.615and0.31%respectivelyandthatof60°Cfor1.5mmparticlesizefor1hourwas0.39and0.015%respectively.Furtherexperi-mentsweredoneconsideringtwodifferentsolvent.Theyieldwas0.2and0.7%respectivelyforlemonandorangeusingMethanoland0.9and1.5%respectivelyforN-Hexane.TheoilwascharacterizedandSpecificgravitywasfoundtobe0.9016,0.9020,0.9039respectivelyforlemon,limeandorange,Freefattyacidvaluewas2.62,2.06,1.78respectively,Peroxidevalueinmillequivalentperkilogramwas9.26,9.46,9.54respectively,Saponificationvaluewas173.48,179.10,174.33respectively,Unsaponificationwas18.3%,16.5%,17%respectivelyandAcidvaluewas5.213,4.11,3.55respectively.

Effect of overexpression of inhibin α fragment on subsequent maturation of preantral follicles (P25)Ahmed Allam, KSU, South Africa, aallam@ksu.edu.sa

Thepresentstudyaimedtodetecttheeffectofinhibinαonthegrowthandapoptosisofthebovinepreantralfolliclesandontheexpressionofgenesoffolliclestimulatinghormonereceptor(FSHR),luteinizinghormonereceptor(LHR),insulin-likegrowthfactorreceptor(IGF-1R)andActivingenesaswell.Toenhanceexpressionoftheinhibinαfragment,differenttypesofpreantralfolliclesweretransfectedwithpEGISI(Fig.1)theninvitrocul-turedfor25days(D).TheinhibinDNAvaccinewasconstructedwithinhibinαfragmentinsertedintotheCterminusofHBsAg-S.TransfectionincreasedthegrowthanddevelopmentofalltypesofpreantralfolliclestillD10butoccurredtofollowedbysharpdecreasingatD15.Theincidenceofapoptosiswashigher(P<0.01)intransfectedpreantralfolliclesthanincontrolsatD15andincreasedfurther(P,0.01)byD25.Thelevelsofgeneexpressionintransfectedfollicleswereincreasedbythefirst10

daysofculturethendramaticallydecreasedintheremainingtimesofcultureperiodtoreachtotheminimumlevelofexpres-sion.Inconclusion,overexpressionofinhibinαfragmentinducepreantralfolliclegrowth,justtillD10ofinvitroculturebutoccurredsignificantdecreasinginthegrowthduetoapoptosisanddecreasedgeneexpression.

Expression efficacy of dual gene mediated by the internal ribosome entry site element of Encephalo-myocarditis virus (P26)Chao-Lin Liu, Ming Chi University of Technology- New Taipei City, f2402002@ms16.hinet.net

Internalribosomeentrysites(IRESs)allowthetranslationofatranscriptindependentofitscapstructure.However,itisunclearwhethertheefficiencyofIRES-mediatedtranslationiscom-parabletothatofcap-dependenttranslation.Here,wegener-atedvariousbicistronicconstructswithquantifiablereportergenes,luciferasesfromfirefly(Fluc)aswellasjellyfish(Rluc)andenhancedgreenfluorescenceprotein(EGFP),asupstreamanddownstreamcistronsoftheencephalomyocarditisvirus(EMCV)IREStopreciselyevaluatetheefficacyofIRES-mediatedtransla-tioninmammaliancells.Therewasnosignificantdifferenceinproteinproductionwhenthereportergenewasclonedasanupstreamcistron.However,lowerlevelsofproteinproductionwereobtainedwhenthereportergenewaslocateddownstreamoftheIRES.Moreover,inthepresenceofanupstreamcistron,amarkedlyreducedlevelofproteinproductionwasobserved.Inconclusion,ourfindingsdemonstratewhatEMCVIRES-mediatedtranslationisrelativelylessefficientthancap-dependenttransla-tionandprovidevaluableinformationregardingtheutilizationofIREStofacilitatetheexpressionofmorethanoneproteinfromatranscript.

Novel environmental friendly deacetylation me-thod for chitin (P27)Min-Lang Tsai, Department of Food Science, National Taiwan Ocean Univer-sity- Keelung, Taiwan, tml@mail.ntou.edu.tw

Commercialpreparationofchitosanapplieswithhot-alkalinemethod.Itisnotonlytoproducelotswastewater,butalsotoinefficientlyconsumeenergy.Thisstudyutilizesexplosivepuff-ingtotreatchitinperforminglargescaleofdeacetylation.ThisGreenProcessingwithadvantagesofenergy-save,environment-friendlyandconvenience-simpleisexpectedtomakechitin-chitosanproductsgreatreactionactivityduetotheirporousstructures.Thecolorofpuffedchitinwillbebrownerduetoincreasingmoisturecontents,puffedpressure,puffedtimes,andpH.Thedegreeofdeacetylation(DD)ofexplosivepuffedchitinwasfrom32.2%increasedto41.4%.TheDDofpuffedchitinwasincreasedwithpuffedpressureincreasing.X-raydiffrac-tionpatternsofchitinandpuffedchitinsexhibitedtwochar-acteristiccrystallinepeaks,closeto10°and20°,andexhibitedthesmallcrystallinepeaksat27°.Thecharacteristiccrystallinepeaksofpuffedchitinsweredecreasedwithmoisturecontents,puffedpressureandpuffedtimesincreasing.However,thesmallcrystallinepeaksat27°wereincreasedaspuffedpressureincreasing.Thestructuresofbreakandwrinklesofpuffedchitinswereincreasedduetomoisturecontentsandpuffedpressureincreased.Thepuffingtreatmentcouldreduceparticlesizesofpuffedchitins.

Poster abstracts

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