Book of abstracts 6-02-20 - EuroMicropH · 19:30 Conference dinner (at Museu da Cerveja, Terreiro...

EuroMicropH1stOpenMeeting 2020

2 COSTActionCA18113:UnderstandingandExploitationtheImpactsofLowpHonMicro-organisms

Dearcolleagues,

Itisourgreatpleasuretohostthe1stOpenMeetingoftheCOSTactionEuroMicropH,devotedto

thestudyofhowlowpHimpactsmicrobialphysiologyandwiththathowitmodulatesmanyissues

inourdailyliferangingfrombiotechnology,tofoodandhealth.Wehopethatthismightbeatime

of fruitful scientific discussions that might foster new interactions, new discoveries and new

projects. You can follow all the aspects related with the action and with the meeting at the

https://euromicroph.euwebsiteandonsocialmediaoftheaction.

WelcometoLisbon,

Onbehalfoftheorganizingcommittee,

NunoPMiraandAnaMendes-Ferreira

OrganizingCommitteeNunoPMira,InstitutoSuperiorTécnico,ChairoftheConferenceAnaAlexandraMendes-Ferreira,UTAD,Vice-ChairoftheconferencePeteLund,UniversityofBirmingham,ChairoftheEuroMicropHActionDanielaDeBiase,SapienzaUniversity,Vice-chairoftheactionMariaJoãoTavares,InstitutoSuperiorTécnicoPatríciaLage,UTADIsabelSeixas,UTADMariaJoanaPinheiro,InstitutoSuperiorTécnicoNunoAlexandrePedro,InstitutoSuperiorTécnico



ConferenceVenueThemeetingwilltakeplaceatInstitutoSuperiorTécnicointheConferenceCenter(locatedinthe-

2 floor of the Civil Engineering Building). Below you can find amapwhere it is highlighted the

differentwaysbywhichyoucanreachtheconferencevenue.



Foodoptions

Inthepicturebelowyoucanfindalistofoptionsforfood.Theseareallinwalkingdistanceoftheconferencevenue.BelowthepictureyoucanfindthedirectlinksfordirectionsviaGoogleMaps.

CaféImpério(directions)Gula´sforKitchenlovers(directions)Vitaminas&Sabores(directions)Alcaparra(directions)Sena(directions)(insideISTAlamedacampus)TascaFit(directions)Sushisan(directions)UDON(directions)KokoroRamenBar(directions)DonCostin(directions)McDonald´s(directions)AtriumShoppingCenter(directions)FrankieHotDogs(directions)ShreeRam(directions)



PROGRAMME:

Wednesday,February12th2020

ForManagementCommitteemembersonly8:00-9:30-ArrivalandregistrationofManagementCommittee(MC)delegates9:30-10:30-MCmeeting(partI):administrationoftheaction10:30-11:00-Coffee-break11:00-12:30-MCmeeting(partII):preparativemeetingsconcerningorganizationofWGactivitiesandpreparationofGAPG2

Offeredlunch

Forallregisteredparticipants14:00-15:10-Welcome notes (Nuno P Mira, conference chair; Professor JoaquimSampaioCabral,FullprofessorofISTandDirectorofInstituteforBioengineeringandBiosciences; Prof João Pedro Conde, Full Professor of IST and Director of theDepartmentofBioengineering)LowpHandtheEuroMicropHaction(PeteLund,ActionChair) Openingconference.AndreasGombert,UniversityofCampinas,BrazilWhatcantheindustrialproductionoffuelethanolfromsugarcaneteachusabouttolerancetolowpHinyeast?15:15-15:45-Coffeebreak15:45-17:30:Session1:IntegrationoffundamentalknowledgeonimpactsoflowpHonmicro-organismsandidentificationofknowledgeandtechnicalgaps.Session chairs: Conor O’Byrne, NUI Galway, Ireland; Oded Liran, Galilee ResearchInstitute,IsraelIntroduction toWorking Group 1 - Connor O’Byrne, NUI Galway, Ireland; (WG1leader)



DanielaDeBiase(SapienzaUniversity,Italy)DieorsurviveunderverylowpHstress:whenanaminoacidcanmakethedifferenceMiguelMachuqueiro(FacultyofSciences,UniversityofLisbon,Portugal)Computational methods to evaluate the impact of low pH on the cellularwater/membraneinterfaceFlashtalksselectedfromtheabstracts–KarenTrchounian,FacultyofBiology,ArmeniaInteraction of membrane-bound enzymes related to proton transport and hydrogenproductioninEscherichiacoliatacidicpH–MatthiasG.Steiger,TechnicalUniversityofWien,AustriaOrganicacidsecretionbyfilamentousfungi– Hana Sychrová, Institute of Physiology of the Czech Academy of Sciences, CzechRepublicPotassium transporters participate in yeast pHhomeostasis and tolerance to lowexternalpH–ArmandsVigants,UniversityofLatvia,LatviaTheaceticacidtoleranceoftheyeastKluveromycesmarxianus–SholemGriffin,UniversityofMalta,MaltaTheEffectofCarbonDioxideasaClimaticParameteronMicrobialFoodContaminantsandSelectiveIsogenicMutants17:30-19:00 - Poster session (at the ground floor of the civil engineeringbuilding)Thursday,February13th20209:30-10:30 – Session 2: Methodologies and expertise in the study of micro-organismsatlowpH.Sessionchairs:GertienSmitts,UniversityofAmsterdam;OttScheler,GalileeResearchInstitute,IsraelIntroduction to Working Group 2 (Gertien Smits, University of Amsterdam, TheNetherlands;WG2leader)



TeutaPilizota,UniversityofEdinburgh,UKTheability tomaintain pH inE. coli is set by themaximumachievable protonmotiveforcePeteLund,UniversityofBirmingham,UKHighthroughputmethodsforgeneticanalysisofacidstressFlashtalksselectedfromtheabstracts–LuísFerraz,UniversityofMilano-Bicocca,ItalyMembrane engineering to improve Saccharomyces cerevisiae robustness towardsorganicacids–MathewMilner,UniversityofBirmingham,UnitedKingdomCanTransposonDirectedInsertion-siteSequencing(TraDIS)beusedtoexploremultipleoutcomesofevolutionunderstressfulenvironments?–OttScheler,GalileeResearchInstitute,IsraelAnalysisofmicrobialgrowthandheterogeneityusingdropletmicrofluidics–EliaTomás-Pejó,IMDEAEnergyInstitute,SpainEvolutionary engineering of Lactobacillus pentosus improves lactic acid productivityfromxylose-richmediaatlowpH–VasilisValdramidis,UniversityofMalta,MaltaQuantitative approaches to address the spore resistance in low pH food processingenvironments:thecaseofAlicyclobacillusacidoterrestris10:30-11:00-Coffee-break11:00-12:30–Session3:LowpHresponsesinmedicine,biotech,andfood:whatcanwelearnandhowcanwehelpeachother(partI)Session chairs: Karolina Rudnicka, University of Lodz, Poland; Rebecca Hall,University of Birmingham, UK; Jana Sedláková, Institute of Biology and Ecology,SlovakiaIntroductiontoWorkinggroups3,4and5(NunoMira,WG3leader;ZeynepGurol,WG4leader;EstefaniaNoriegaFernandez,WG5leader)JaroslavMichalko,SlovakAcademyofSciencesHowsystematicliteraturereviewandmeta-analysismayprovideabetterunderstandingoftheacidicstressresponseinhighlypathogenicfoodbornebacteria12:30-14:00–FreetimeforLunch



14:00-15:30–Session3:LowpHresponsesinmedicine,biotech,andfood:whatcanwelearnandhowcanwehelpeachother(partII)Session chairs: Nuno P Mira, Instituto Superior Técnico, Portugal; Jana Sedláková,InstituteofBiologyandEcology,SlovakiaKarolinaRudnicka,UniversityofLodz,PolandHowHelicobacterpyloriresistsgastricacidandmodulatehostsresponsesBožidarSantek,UniversityofZagreb,CroatiaYeast Trichospron oleaginosus: a producer of microbial lipids from lignocellulosecontainingfeedstocksZeynepGurol,KTHUniversity,SwedenAcidogenicbacteriaasourco-workersformoresustainableproductionprocess15:30-16:00Coffee-break16:00-17:30 Poster session (on the ground floor of the civil engineeringbuilding)19:30Conferencedinner(atMuseudaCerveja,TerreirodoPaço)Friday,February14th20209:30-10:30–Session3:LowpHresponses inmedicine,biotech,andfood:whatcanwelearnandhowcanwehelpeachother(partIII)Sessionchairs:EstefaniaNoriegaFernandez,Nofima,NorwayNicholasBrianJohnson,NestléResearchCenter,SwitzerlandLowpHandHighAcid:achallengeoropportunityfortheFoodIndustryMustafaTurker,Pakmaya,TurkeyPotentialapplicationsoflowpHinFermentationindustryFlashtalksselectedfromtheabstracts–JanaSedlakova-Kadukova,FacultyofScience,SlovakiaAcidophilicmicroorganisms–capableworkersinmetal-bearingwastetreatment– Ljupco Angelovski, University of “Ss. Cyril and Methods”, Republic of NorthMacedoniaEffectoforganicacidsonpathogenmicroorganismsinmeat



–AleksandraDjukić-Vuković,FacultyofTechnologyandMetallurgy,SerbiaLowpHinproductionoflacticacidandprobioticbiomass–LucianC.Staicu,UniversityofWarsaw,PolandRecoveryandcharacterizationofPbSusinganovelstrainofBacillus–AysenurUgurlu,HacettepeUniversity,TurkeyAcclimatizationofanaerobicmicroorganismstolowpHconditionsforenhancementofbiogasproduction10:30-11:00-Coffee-break11:00-12:30–Session4:DisseminationatEuroMicropHSessionchair:MatthiasSteiger,TechnicalUniversityofWien,AustriaMichael Sauer,University ofNaturalResources andLife Sciences,Austria (leader ofWG6)CommunicationandDisseminationinCOSTActionsFinalconferenceSessionchair:DanieladeBiase,UniversityofSapienza,ItalySaraBoverCid,InstituteofAgricultureandFoodResearchandTechnology,SpainPredictive microbiology approaches to quantitatively assess the impact of pH onbehavioroffoodbornebacteria12:30-14:00FreetimeforLunch14:00-16:00 -Openparallel sessions for allWGs (reviewwork performed andestablishgoalsforGP2)16:00-17h30-CoreGroupmeeting



Abstracts



What can the industrial productionof fuel ethanol fromsugarcaneteachusabouttolerancetolowpHinyeast?

AndreasK.Gombert1,2,BiancaE.Della-Bianca2,VijayendranRaghavendran1,FelipeB.Beato1,BrunoL.V.daCosta1,2,ThiagoO.Basso2,MichaelDesai3

1Universidade Estadual de Campinas, Campinas, SP, Brazil ([email protected]); 2Universidade deSãoPaulo,SãoPaulo,SP,Brazil;3HarvardUniversity,Cambridge,MA,USA

Duringindustrialproductionoffuelethanolfromsugarcane,theyeastSaccharomycescerevisiaeconvertssugarsintoethanolinmillionliter-scalefermentors.Thisprocessrunswithout asepsis for ~8months every year and low pH is one of the inherentconditions which ensures that contamination levels remain below an acceptablethreshold.LowpHisnotonlyimportantduringfermentationperse,butalsoduringcell recycling,which includesa sulphuricacidwashingstepatpH~2 for1.5 to2h,leadingtoseverelossofviabilityincontaminatingbacteria,andtosomeextentalsoinyeast.Bycomparingyeaststrains isolated fromthe industrialprocess to laboratory,baker’s and wild strains using different experimental approaches, we gatheredphenotypicdata indicating that tolerance to lowpHbecameoneof thehallmarksofthese industrialstrains. Inamorerecentproject,samples taken fromtwo industrialunits along two whole sugarcane crushing seasons were analyzed by genomesequencing,inordertotrackthemolecularsignaturesinindustrialstrains.ThismightenableustopinpointwhichmutationsoccuringenesinvolvedintolerancetolowpH.



Die or survive under very low pH stress: when an amino acid canmakethedifference

DanielaDeBiase

1Departmentofmedico-surgicalSciencesandBiotechnologies–LaboratoryaffiliatedtoIstitutoPasteurItalia-Fondazione Cenci Bolognetti, Sapienza University of Rome, Corso della Repubblica 79, 04100Latina,Italy([email protected])Bacteria that best grow at pH close to neutrality are referred as neutralophiles.Howeverduringtheirlifecyclethesemicroorganisms,whichcanbeeitherbeneficialor pathogenic, experiencemild-to-harsh acidic pH in natural environments, such asthose encountered in the digestive tract of the animal hosts, in the phagosome ofmacrophages, in fermented foods and in the soil. To contrast the harmful effects ofintracellular acidification, thesebacteriahavedevelopedmolecular strategieswhichpreserve the cellular activities and the membrane potential [1]. The amino acid-dependentacidresistancesystemsveryeffectivelyrelievethecellsexposedtoaverylowpH [1].Theseextremelypowerful systemsrequireenzymesactingprimarilyonamino acids such as glutamine, glutamate, arginine and lysine and the cognateantiporters[1-3].Through structural and functional studies, we and others have shed light on themolecular determinants that in a very elegant way control the activity of the acidresistance systems inneutralophiles. Iwillprovideanoverviewof theeffectivenessand distribution of these systems and show how through a combination ofbiochemical and structural studies and transport assays it has been possible tounderstandthemechanismofactionofthesesystemsandfinally,howthisknowledgecanbeexploitedfordifferentapplications.References:[1]LundP,TramontiA,DeBiaseD.(2014)FEMSMicrobiolRev.38:1091-1125.[2]PennacchiettiE,D'AlonzoC,FreddiL,OcchialiniA,DeBiaseD.(2018)Front.Microbiol.9:2869.[3]TsaiM-F,McCarthyP,MillerC.(2013)PNAS110:5898-5902.



Computational methods to evaluate the impact of low pH on thecellularwater/membraneinterface

MiguelMachuqueiro

BioISI-Biosystems&IntegrativeSciencesInstitute,DepartamentodeQuímicaeBioquímica,FaculdadedeCiênciasdaUniversidadedeLisboa,([email protected])pH isa crucialphysicochemicalproperty thataffectsmostbiomolecules.Changes inprotonationequilibriumofsusceptiblesiteswillmodifytheelectrostaticenvironmentand,consequently,haveaneffectonthemolecularstructure,stabilityandcatalysis.1Adetailed description of a cell membrane has to take in consideration all importantfactors that affect the lipid bilayer behavior and stability. pH is recognizably oneofthosefactorseventhoughitisusuallyignoredduetoitshighcomplexity.ThepKa values of the typical titrable amino acids canbe significantly influencedbychanges in solventmixtureordue to insertion in a lipidbilayer.2-3 In thiswork,wepresent amethodology that allows us to calculate pKa values of peptides, proteins,lipidheadgroups,andevenpharmaceuticaldrugs,at thewater/membrane interface.We take advantage of a CpHMDmethod developed in our group3-4 to estimate pKaprofilesalongthemembranenormal forseveralcompounds,whichshould illustratethe high importance of including pH effects in atomistic simulations at thewater/membraneinterface.References:[1]J.Matthew,Annu.Rev.Biophys.Biophys.Chem.14(1985)387.[2]J.F.Hunt,P.Rath,K.J.Rothschild,D.M.Engelman,Biochemistry36(1997)15177.[3]Teixeira,V.H.,Vila-Viçosa,D.,Reis,P.B.P.S.,Machuqueiro,M.,J.Chem.TheoryComput.,12(2016)930.[4] D.Vila-Viçosa,V.H.Teixeira,A.M.Baptista,M.Machuqueiro, J. Chem.TheoryComput. 11 (2015)2367.



Escherichiacoli’spHhomeostasisissetbythemaximumachievableProtonMotiveForce

GuillaumeTerradot1,EkaterinaKrasnopeeva1,PeterSwain1,TeutaPilizota1

1Synthetic and systems biology Centre, University of Edinburgh, King’s Buildings,Edinburgh,UK([email protected])

Living cells can maintain intracellular composition different from the externalenvironment, andoften, properties of the inside are keptwithin a certain range, socalledhomeostaeses.Forexample,intracellularpHismaintainedclosedtoneutralinneutralophilicbacteria,suchasEscherichiacoli,whichhavebeenreportedtomaintaincytoplasmicpHclose to7.5 ina rangeofexternalpHvalues [1].Morerecently, thisviewhasbeenchallengedwithreportsonpermanentchangesininternalpHupontheenvironmental pH change [2]. Explanations offered for the discrepancy in themeasurementsincludeinaccuraciesinthepHsensorcalibrationandusage,andstraintostraindifferences[2].However,pHhomeostasishasthusfarnotbeenlookedatinthecontextofallthecellularphysiologicalvariablesthatareinfluencedbyit:osmoticpressure, membrane voltage, the electrochemical gradient of protons, and theelectrochemical gradients of all the other ions present in the cell. The intertwinednature of these physiological variables, suggest that we need to look at theirmaintenanceinacoordinatedfashion,inordertounderstandthehomeostasisofevenjust one of them, for instance internal pH. To do so, we build a simplifiedmathematical model of cellular electrophysiology. We find that the cell's ability tomaintain pH is set by the maximum achievable PMF. We employ assays for time-course,single-cell,concurrentmeasurementsofPMFandpH[3,4],tomeasureE.coli'sinternalpHduringexternalpHshiftsattwodifferentPMFvalues,andfindagreementwiththemodelprediction.

References:[1] JoanL.Slonczewski,RobertM.Macnab, JeffryR.Al-ger, andAnnaM.Castle.EffectsofpH

and repellent tactic stimuli on proteinmethylation levels inEscherichia coli; Journal of Bacteriology,152(1):384–399,1982

[2]SmarajitChakraborty,RicksenS.Winardhi,LeslieK.Morgan,JieYan,andLindaJ.Kenney.Non-canonicalactivationofOmpRdrivesacidandosmoticstressresponsesinsinglebacterialcells;NatureCommunications,8(1):1587,2017

[3] Ekaterina Krasnopeeva, Chien-Jung Lo, and Teuta Pilizota. Single-cell bacterialelectrophysiologyrevealsmechanismsofstress-induceddamage.BiophysicalJournal,116(12):2390–2399,2019

[4]Yao-KuanWang,EkaterinaKrasnopeeva,Ssu-YuanLin,FanBai,TeutaPilizota,andChien-JungLo.ComparisonofEscherichiacolisurfaceattachmentmethodsforsingle-cell,invivomicroscopy.bioRxiv,2019.



High-throughputmethodsforgeneticanalysisofacidstress

MathewMilner1,FrancescaBushell1,FatimaAlatar1,HrishirajSen1andPeterALund1,*

1School of Biological Sciences & Institute of Microbiology and Infection, University of Birmingham,BirminghamB152TT,UK*[email protected]

Highthroughputmethodsareusuallybasedontheuseofdifferent‘omicstechniquestocollectdatafromorganismscultivatedunderspecificconditionsofinterest,suchasinthepresenceofadefinedstress.Theycanbeinvaluableasasourceofcandidates(in the shape of gene lists) for more detailed study, but do not necessarily inthemselves provide a direct measure of the importance of different genes underparticular stresses. For example, a gene which is needed for survival or optimalgrowthinastressfulenvironmentwillnotalwaysshowincreasedexpressioninsuchanenvironment,andsowouldnotbedetectedbyRNA-basedmethods.Recently,thesequencing of very dense transposon libraries before and after stress treatments(knownastraDISorTnseq,[1,2])hascometotheforeasawayofdirectlymeasuringgene fitness, while laboratory-based evolution coupled with whole genomesequencing is also becoming popular as a way of detecting mutations that enableadaptationstodifferentstresses[3].WehaveusedallthreemethodstostudyE.coligrownunderlowpHstress[4,5],andinthistalkIwillpresentsomeofthedatafromthisanalysis,includingacomparisonbetweenthemethods.

References:[1] vanOpijnenTandCamilliA.2013.Transposoninsertionsequencing:anewtoolforsystems-levelanalysisofmicroorganisms.NatureRevMicrobiol11:435-442[2] Chao MC et al. 2016. The design and analysis of transposon insertion sequencingexperiments.NatureRevMicrobiol14:119-128[3] DragositsM and MattanovichD. 2013. Adaptive laboratory evolution: principles andapplicationsforbiotechnology.MicrobCellFact.12:64.[4]StinconeAetal.2011.AsystemsbiologyapproachshedsnewlightonEscherichiacoliacidresistance.NucleicAcidsRes.39:7512-7528[5] Johnson MD et al. 2014. Characterization of mutations in the PAS domain oftheEvgSsensorkinaseselectedbylaboratoryevolutionforacidresistanceinEscherichiacoli.MolMicrobiol.93:911-927



Howsystematic literaturereviewandmeta-analysismayprovideabetter understanding of the acidic stress response in highlypathogenicfoodbornebacteria

JaroslavMichalko1*,OdedLiran2,DirkHofreuter3,DavidAttuyVeydaSilva3,SaschaAlDahouk31Mlynany Arboretum, Institute of Forest Ecology, Slovak Academy of Sciences, Slepcany, Slovakia;2MIGAL–GalileeResearch Institute,KiryatShmona, Israel; 3DepartmentofBiological Safety,GermanFederalInstituteforRiskAssessment,Berlin,Germany*[email protected]

Meta-analysis represents amethod for quantitative synthesis of research results. Itusually comprises a systematic literature review and statistical analysis of dataextractedfromtheliterature.Followingasetofestablishedguidelines,summarizedinPRISMA[1],meta-analysisenables: (i) tomergeknowledgeonaparticularresearchtopic/questioninatransparentandreproduciblewaybycombiningoutcomesacrossdifferentdatasets,(ii)toexaminepatternsofresponseacrossavailabledata,and(iii)to explore sources of heterogeneity in outcomes. Systematic reviews incorporatingmeta-analysis are therefore a valuable tool for in silico hypothesis testing based onalready available literature data and, if done properly, can be a source of novelknowledge which is more informative than the lessons that can be learned fromclassicalnarrativereviewswhicharesubjecttoahighdegreeofsubjectivity[2].Inourpresentation,wewill demonstrate the suitability and general applicability ofmeta-analysis for synthesizingknowledgeon the impactof lowpHonmicrobes.WeusedBrucella spp. as a model for highly pathogenic foodborne bacteria, which have toevade acidic stress on their way from stable to table (environment, food hygienemeasures,gastricacid,phagosome).Theaimofourstudywastoanalyzethegrowthresponseofbrucellaeinsidemacrophageswhereacidicstressplaysadecisiveroleintheearlyphaseofinfection[3].Basedonthisexample,wewillexplaintheindividualstepsintheprocessofameta-analysisfollowingstandardizedandgenerallyacceptedguidelines[1]andhowwecanaddressquestionsrelatedtotheaimsandgoalsoftheCOSTActionEuroMicropHinamorefocusedmanner.References:[1]Liberati,A.,Altman,D.G.,Tetzlaff, J.,Mulrow,C.,Gøtzsche,P.C., Ioannidis, J.P. et al. (2009).ThePRISMAstatementforreportingsystematicreviewsandmeta-analysesofstudiesthatevaluatehealthcareinterventions:explanationandelaboration.Annalsofinternalmedicine,151(4),W-65.[2] Gurevitch, J., Koricheva, J., Nakagawa, S., & Stewart, G. (2018). Meta-analysis and the science ofresearchsynthesis.Nature,555(7695),175.[3]Damiano,M.A.,Bastianelli,D.,AlDahouk,S.,Köhler,S.,Cloeckaert,A.,DeBiase,D.,&Occhialini,A.(2015). Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution andcontributiontofitnessunderextremelyacidicconditions.Appl.Environ.Microbiol.,81(2),578-586.



HowHelicobacter pyloriresists gastric acid and modulates hostsresponses

KarolinaRudnicka1,AgnieszkaMatusiak1,MałgorzataSiwińska2,WeronikaGonciarz1,Adrian Gajewski1, Eliza Mnich1, Marcin Włodarczyk1,3, Maria Walencka1, AgnieszkaKrupa1,MagdalenaChmiela1

1LaboratoryofGastroimmunology,DepartmentofImmunologyandInfectiousBiology,FacultyofBiologyandEnvironmentalProtection,UniversityofLodz,Lodz,Poland;2LaboratoryofGeneralMicrobiology,FacultyofBiologyandEnvironmentalProtection,UniversityofLodz,Lodz,Poland;3Divisionof Cellular Immunology,Department of Immunology and InfectiousBiology, Faculty ofBiologyandEnvironmentalProtection,UniversityofLodz,Lodz,PolandHelicobacterpylori(H.pylori) isabacteriumthatsuccessfullycolonizesgastricmucuslayerofmore thanhalf ofhumanpopulation.Thisbacterium inhabits themucus-linedsurfaceoftheantrumofthehumanstomachwhereit inducesamildinflammation,butitspresenceisotherwiseasymptomatic.However,mostlyin10-15%ofcases,itleadstoa development of gastric inflammation, gastric and duodenal ulcers or even gastriccancer. AlthoughH. pylori is not an acidophile it possess mechanisms to survive andadopt to stomach acid that allow this bacterium to induce not only local but systemicconsequences(1-4).Thepresenceorabsenceofsymptomsandtheirseveritydependonmultiple bacterial components, host susceptibility and environmental factors, whichallow H. pylori to switch between pathogenicity and commensalism. A variety ofvirulence factors appear to play a role in pathogenesis. These include the vacuolatingcytotoxin VacA, cytotoxin-associated proteins CagA, urease, flagellum andlipopolysaccharide.H.pyloriaccompanyhumanshundredsthousandsofyears-thus,itisno surprising that the bacterium “learnt” how to use its structures tomanipulate theimmunecellstoremainunrecognizedandavoideradication.ThemechanismsoftheseH.pylori-driven immunemanipulationsand theirconsequenceson the localandsystemiclevels,inthelightofourrecentfindingswillbediscussed(1-4).

TheresultspresentedduringthelecturewerefinanciallysupportedbyNationalScienceCentre(Poland)-SONATA12 no. 2016/23/D/NZ6/02553, PRELUDIUM 2013/09/N/NZ6/00805 and 2015/17/N/NZ6/03490as well as NN401015136andNN303451738. References:[1]RudnickaK., Backert S., ChmielaM.: Genetic polymorphisms in inflammatory andother regulators ingastriccancer:Risksandclinicalconsequences.CurrentTopicsinMicrobiologyandImmunology2019,421,53-76. Book chapter in „Molecular mechanisms of inflammation: induction, resolution and escape byHelicobacterpylori”.Springer,2019.[2] ChmielaM, Kupcinskas J. Pathogenesis ofHelicobacter pylori infection.Helicobacter. 2019, 24 Suppl1:e12638.[3]ChmielaM.,MiszczykE.,RudnickaK.StructuralmodificationsofHelicobacterpylorilipopolysaccharide:Anideaforhowtoliveinpeace.WorldJournalofGastroenterology,2014,7;20(29):9882-9897.[4]ChmielaM,GonciarzW.MolecularmimicryinHelicobacterpyloriinfections.WorldJGastroenterol.2017Jun14;23(22):3964-3977.doi:10.3748/wjg.v23.i22.3964.



Yeast Trichosporon oleagninosus - a producer of microbial lipidsfromlignocellulosecontainingfeedstocks

MirelaIvančićŠantek1,MarinaGrubišić1,BožidarŠantek1*

1DepartmentofBiochemicalEngineering,FacultyofFoodTechnologyandBiotechnology,UniversityofZagreb,Pierottijeva6,Zagreb,Croatia*email:bsantek@pbf.hrMicrobiallipidsarealternativefeedstockforbiodieselorpolyunsaturatedfattyacids(PUFA) production. Yeast Trichosporon oleaginosus is one of the oleaginousmicroorganisms that has the capacity to grow and accumulate lipids on differentlignocellulose containing feedstocks. In this study, corn cobs were used aslignocellulose containing feedstockwhichwas pretreated by NaOH at loading from0.08 to1.6g/gdryweightof rawcorn cobs (gNaOH/gDW)at121 °C for30min. Infollowed step, alkaline pretreated corn cobs were hydrolyzed by using commerciallignocellulolyticmulti-enzymescocktails.ThehighestglucoseandxyloseyieldswereobservedatNaOHloadingrateof0.16g/gDW.Theseenzymaticcorncobshydrolysateswere used for lipids production by T. oleaginosus in the following bioprocessconfigurations: separate hydrolysis and lipid production (SHLP) and simultaneoussaccharificationand lipidsproduction(SSLP).Higher lipidyield(88.88mg/gDW)andproductivity[2.4g/(Lday)]aswellasconsiderablereductionofbioprocesstimewereobservedintheSSLPcomparetotheSHLPconfiguration.Afterlipidsextractionfromyeast biomass, the fatty acids composition of these lipids was defined in order toevaluatetheirpotentialforbiodieselorPUFAproduction.ObtainedresultsshowthatT.oleaginosushasgreatpotentialtobeusedasa lipidsproducerfromlignocellulosecontainingfeedstocks.



Acidogenic bacteria as our co-workers for more sustainableproductionprocess

ZeynepCetecioglu1

1KTHRoyalInstituteofTechnology,Dept.ofChemicalEngineering,Sweden([email protected])Environmentalsustainabilityisaglobalgoaltodayandtoaccomplishit,transitionintoacirculareconomywhichincludesresourcerecovery,reuseandrecyclingisessential.Therefore,renewability,degradability,andsustainabilityarethekeywordsofthebio-basedproductionandtheyarekeyparameterstolimitglobaltemperaturerisetowellbelow2°Candgiventhegraverisks, tostrive for1.5°Cwhichwasaddressed inTheParis Agreement (2016)[1]. Feedstock and energy recovery are also encouragedunder the “Resource Efficiency Roadmap”[2]. This puts high demands on thedevelopment of next generation production (NGP) technologies to turn carbon-richsubstrate/wastestreamsintochemicalsandmaterials insteadofpetrochemicalbasedproduction.Oneofthemostpromisingbio-basedproductsisvolatilefattyacid(VFA)which can be produced by acidogenic bacteria as intermediate fromwaste-streamsand has high market demand [3]. Nevertheless, to increase the bio-based VFAproduction yield is compulsory to make it competitive with the petrol-basedproduction.ThestudiesshowedthatpHandwaste/substratetypeintermsofcontentandmatrix such as liquid, slurry, solid, etc. aremain parameterswhich affect bothbioproduction efficiency of VFAs and VFA composition in mixed fermentation andshiftingofbacterialcommunityinprocess[4–10].

References:[1] UNFCCC, Paris Agreement, Conf. Parties Its Twenty-First Sess. (2015) 32. doi:FCCC/CP/2015/L.9/Rev.1; [2]Roadmap; [3]M.Atasoy, I.Owusu-Agyeman,E. Plaza, Z. Cetecioglu,Bio-based volatile fatty acidproduction andrecoveryfromwastestreams:currentstatusandfuturechallenges,Bioresour.Technol.(2018)0–1;[4]M.Atasoy,O.Eyice,A.Schnürer,Z.Cetecioglu,Volatile fattyacidsproductionviamixedculture fermentation:Revealing thelinkbetweenpH,inoculumtypeandbacterialcomposition,Bioresour.Technol.292(2019)121889;[5]M.Zhou,B.Yan, J.W.C. Wong, Y. Zhang, Bioresource Technology Enhanced volatile fatty acids production from anaerobicfermentationof foodwaste :Amini-review focusingonacidogenicmetabolicpathways,Bioresour.Technol.248(2017) 68–78; [6] J. Yin, X. Yu, Y. Zhang, D. Shen, M. Wang, Y. Long, T. Chen, Enhancement of acidogenicfermentationforvolatilefattyacidproductionfromfoodwaste:Effectofredoxpotentialandinoculum,Bioresour.Technol.216(2016)996–1003;[7]G.Strazzera,F.Battista,N.H.Garcia,N.Frison,D.Bolzonella,Volatilefattyacidsproductionfromfoodwastesforbiorefineryplatforms:Areview,J.Environ.Manage.226(2018)278–288;[8]S.Bengtsson, J. Hallquist, A. Werker, T. Welander, Acidogenic fermentation of industrial wastewaters: Effects ofchemostat retention timeandpHonvolatile fatty acidsproduction,Biochem.Eng. J. 40 (2008)492–499; [9]M.Atasoy,Ö.Eyice,Z.Cetecioglu,VolatileFattyAcidsProduction:effectsofbacterialcommunityshiftundervariousoperationalconditions,in:3rdIWAResour.Recover.Conf.8-12Sept.2019.Venice,Italy,Venice,n.d;[10]I.Owusu-Agyeman,E.Plaza,Z.Cetecioglu,Anaerobicco-digestionofsewagesludgeandexternalorganicwaste:strategytoshift production frombiogas to volatile fatty acids, in: 3rd IWAResour. Recover. Conf. 8-12 Sept. 2019. Venice,Italy,2019.



Low pH and High Acid: a challenge or opportunity for the FoodIndustry?

NickJohnson

Food Safety Microbiology, Quality Management, Société de Produits Nestlé SA., Nestlé ProductTechnology Centre Nutrition, Nestlé Research and Development Konolfingen, Nestlé Strasse 3, 3510Konolfingen,Switzerland([email protected])Nestlé, and thewider food industry, employ the acidity ofmany food andbeverageproducts toassist inprovidingstabilityandsafety.Theability touse loweredpH infoodandbeverageproductshasanenormousbenefit ineitherreducing theneedtodeliverhighamountsofheator inneedinganasepticfillingenvironmenttopreventspoilage or both. Consequently, the costs of manufacture and wastage are greatlyreduced. However, lowered pH as a sole hurdle would make most productsunpalatable.Therefore,arangeofproduct typeswithvaryingdegreesofcomplexityhavebeendevelopedwithinthefoodindustry,toemployproductrecipeandprocessdesigns that combine antimicrobial hurdles. The combined use of hurdles enable arelativelyhigherpHwhilstdeliveringreducedperceivedsourness,butstillprovidinga robust, safe and stable product. These approaches use the term multi-hurdletechnology(MHT).References to recent reported spoilage incidences in low pH food products will beused to demonstrate the impact these have on the industry, and how they haveassistedinidentifyingandmanagingspoilagerisk.TwocasestudiesoflowpHproductdesigns(i.e.acidifiedsaucesandready-to-drinkbeverages)willbeusedasexamplesof the application, challenges and opportunities offered by MHT. Finally, we willbriefly cover some of the current applications using ‘novel’ technologies in low pHfoodandbeverageproduction,withattentiononwhy thesealternativenon-thermaltechnologiesarestilloftenseenasnichebythefoodindustry.



Potential Applications and Opportunities in Low pH in Food andIndustrialBiotechnology

MustafaTurker*,BurcuTuranli-Yildiz

Pakmaya,Kocaeli,Turkey*Presentingauthor:[email protected] pH, interfering with pH gradient and proton motive force across cellularmembranes, influences a vast number of physiological functions and deleteriouslyaffects bioprocesses involved in a variety of industrial applications. Thus, from theindustrialpointofview,itisacrucialnecessitytolearnhowtocopewithacidstressincellular level. Although, there are a number of studies elucidating the acquiredhomeostatic mechanisms for relieving the disruptive effects of steep pH gradientacross cell membrane (Baker-Austin and Dopson 2007; Lund, Tramonti, and Biase2014),translationtoindustryisofteninterrupted.In thispresentation, potential opportunities of ameliorations in lowpH response intermsof industrialneedsandchallengeswillbediscussed.Industrialdemandsuponeconomical and technical perspectives will be highlighted in the basis of directexamples from food and industrial biotechnology including dairy, food, beveragefermentations, control of pathogens/contaminants, biomining, bioremediation,generation of bioenergy and robust cell factories for production of various bio-products.IndustrialexploitationofimprovementsinlowpHresponsewilleventuallyleadtoadvancedgrowthofworld-widebio-basedeconomy.References:[1]Baker-Austin,Craig,andMarkDopson.2007.“LifeinAcid :PHHomeostasisinAcidophiles”15(4).https://doi.org/10.1016/j.tim.2007.02.005.[2] Lund, Peter, Angela Tramonti, and Daniela De Biase. 2014. “Coping with Low PH: MolecularStrategiesinNeutralophilicBacteria”38:1091–1125.https://doi.org/10.1111/1574-6976.12076.



Predictive microbiology approaches to quantitatively assess theimpactofpHonbehavioroffoodbornebacteria

SaraBover-Cid1,CristinaSerra-Castelló1,AnnaJofré1

1IRTA.FoodSafetyProgram.FincaCampsiArmets/n;17127Monells.Spain([email protected])

Predictive microbiology, also known as quantitative microbial ecology, aims tocharacterize the behavior of microorganisms as a function of the environmentalfactors, through the development of mathematical algorithms. In food, theenvironmentalconditionsincludeintrinsic(pH,wateractivity,typeandconcentrationof antimicrobials, etc.), extrinsic properties (composition of atmosphere packaging,temperature, pressure, etc.) and intrinsic (interaction with other microorganisms).The mathematical algorithms contribute to understand the response ofmicroorganisms to stresses associated with food production and preservationtechnologies, for instance in terms of growth, inactivation or metabolite (toxin)production.Thequantitativeapproachallows the identificationof themostrelevantfactors as well as their interactions. When properly validated, predictive modelsconstitute key tools for developing quantitativemicrobial risk assessments (QMRA)anddecisionsupport systems formicrobial riskmanagementaiming to reduce foodspoilage and improve food safety all along the food chain. The presentation willintroduce the main features of the predictive microbiology discipline. DifferentapproachesthatcanbeusefultoassesstheimpactofpHonthebehavioroffoodbornemicroorganisms will be addressed. Special attention will be paid on specific case-studiesdealingwith foodbornepathogens: (i) theapplicationofavailablepredictivemodelstounderstandtheroleofthepHonthegrowthinhibitionincomparisonwithother factors; and (ii) modeling the impact of pH as an enhancing factor of highpressureprocessingwithinafoodsafetyassurancestrategy.



CommunicationandDisseminationinCOSTActions

MichaelSauer

1BOKU-University of Natural Resources and Life Sciences, Department of Biotechnology, Institute ofMicrobiology and Microbial Biotechnology, Muthgasse 18, 1190 Vienna, Austria; CD Laboratory forBiotechnologyofGlycerol;ACIBGmbH([email protected]) The declared aim of COST actions is to “help connect research initiatives acrossEuropeandbeyondandenableresearchersandinnovatorstogrowtheirideas[…]bysharingthemwiththeirpeers.”ThecreatedCOSTnetworks“offeranopenspaceforcollaboration among scientists across Europe […] and thereby give impetus toresearch advancements and innovation.” COST funding “intends to complementnational research funds, as they are exclusively dedicated to cover collaborationactivities,suchasworkshops,conferences,workinggroupmeetings,trainingschools,short-term scientific missions, and dissemination and communication activities.”Hence,connectionsandcommunicationarethecentralaspectsofallCOSTactions.Dissemination is a task which can be addressed in a quite forward manner. Thedisseminationofher/hisresultsisacentraltaskforeveryresearcher.Manyroutesfordisseminationexistandsuccessofdisseminationisessentiallyaquestionofdiligenceandperseveranceofthepersonsinvolved.Communication to the contrary is amuchmore difficult andmuch less understoodtask. Building reliable and sustainable connections between researchers or – evenmoredifficult-insideandbetweenresearchfieldsbearsmanyobstaclesandvariousunsolvedproblems.Whilebeingintheleadoftherespectiveworkpackage,myownpictureofhowwecanknitasoundcommunicationnetwork, isstillkindofblurred.Here, I set out to summarize some doubts and questions to which I cannot givesatisfyinganswersat the timebeing. Iwould like tokindly invite allparticipants tojoinmyeffortandclarifytheneedsofourcommunitiesandsuggestusefulmeasureshowtomeetthem.



POSTERS



P1. Influence of acidic stress on biohydrogen production in darkfermentation

AdamCenian*,GawełSołowski

InstituteofFluid-FlowMachinery,PolishAcademyofSciences(*[email protected])Biohydrogen production technologies are nowadays widely investigated (see e.g.[Chaganti et al., 2012; Solowski et al. 2018] and references therein). This results fromhydrogenwideapplicationinchemistry,energetics(energystorageandfuel-cellCHP)ortransport industry (hydrogen cars). The dark fermentation (DF) is related to methanefermentation process, with methanogenesis being inhibited e.g. by low pH value. It isknown thatmethanogenicbacteriapreferpHvalues7 –8.Acidic conditions (pH<6,5)althoughsupport substratesdecomposition (withhydrogengeneration) strongly inhibitmethanegeneration.TheoptimalpHvalue forhydrogengeneration(inDF)dependsonsubstrates and technology but seems to be in the range 5.0 to 6.0 [Muñoz-Páez et al.,2019].However,highhydrogenyieldsduringDFofwheatstrawarereportedforpH2.4[Nasirian et al., 2011] but also 9.0 [Li et al., 2018]. Figure 1 presents results of cotton-waste fermentation under condition of controlled pH 6 value and without pH control(valuerisesabove7,favoriteformethanogenesis).Itisclearlyseenthatcontrolofacidicconditionsmaysignificantlyincreasehydrogenproduction.

Fig.1CumulativehydrogenproductionfromcottonforVSS40g/Lwith(pH6orwithoutpHcontrol)References:[1] Chaganti, S.R. et al. 2012. Dark fermentative hydrogen production by mixed anaerobic cultures: Effect ofinoculumtreatmentmethodsonhydrogenyield.Renew.Energy48,117–121;[2] Li, Y. et al. 2018. Biohydrogen production from fermentation of cotton stalk hydrolysate by Klebsiella sp.WL1316newlyisolatedfromwildcarp(CyprinuscarpioL.)oftheTarimRiverbasin.Appl.Microbiol.Biotechnol.102,4231–4242;[3] Muñoz-Páez, K.M., et al. 2019. Distinct effects of furfural, hydroxymethylfurfural and its mixtures on darkfermentationhydrogenproductionandmicrobialstructureofamixedculture.Int. J.HydrogenEnergy,44,2289-2297;[4] Nasirian, N. et al., 2011. Development of amethod for biohydrogen production fromwheat straw by darkfermentation.Int.J.HydrogenEnergy36,411–420;[5]G.Sołowski,etal.2018.Productionofhydrogenfrombiomassanditsseparationusingmembranetechnology,RenewableandSustainableEnergyReviews,82,3152-3167.

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P2.BioactivecompositecoatingsonTi-6Al-4V

Agnieszka Sobczak-Kupiec1,Wioletta Florkiewicz1, Anna Drabczyk1, Sonia Kudłacik-Kramarczyk1,BożenaTyliszczak1

1CracowUniversityofTechnology,FacultyofMaterialsEngineeringandPhysics,InstituteofMaterialsScience([email protected])

Metalsandtheiralloyshaveplayedapredominantroleasstructuralbiomaterials inreconstructive surgery. Nowadays, titanium alloys are biometals of particularinterests,duetotheirgoodmechanicalproperties[1].However,thecontactofTiwithpatient tissue may induce unspecific protein adsorption and bacterial adhesioncausing poor implant osseointegration. Among the known materials used as anantifoulingagent,apromisingoneispolyethyleneglycol(PEG)[2].Design and fabrication ofmaterialswith surface adjusted for a particular biologicalapplication is of essential importance to the synthesis of biocompatible materials.Taking intoconsideration thedesiredcharacteristicsof thevariousbiomaterials thegreatinterestoftheresearchersfocusedoncompositematerialsiswell-justified[3].The combination of antifouling polymer with bioactive substances can enhancecellular interaction ofmaterial with the host bone and reduce the risk of bacterialbiofilm formation. Among all inorganic materials, hydroxyapatite (HA) has beenconsideredasapropercandidateforenhancingregeneration-supportivepropertiesofscaffoldmaterials[4].

References:[1]Q.Chen,G.A.Thouas,Reports.Mater.Sci.87,2015,1-57.[2]G.Zeng,R.Ogaki,R.L.Meyer,ActaBiomater.24,2015,64–73.[3] D.A. Wahl, J.T. Czernuszka, Eur. Cells Mater. 11, 2006, 43–56.[4]N.Ramesh, S.C.Moratti,G.J.Dias, J.Biomed.Mater.Res. -PartBAppl.Biomater.106,2018,2046–2057.

This study was supported by grant from the National Science Centre in the frame ofUMO-2016/21/D/ST8/01697.



P3.Wetmethodofsynthesiscalciumphosphate

AgnieszkaSobczak-Kupiec1,DagmaraSłota1,MagdalenaGłąb1,WiolettaFlorkiewicz1,BożenaTyliszczak1

1Cracow University of Technology, Faculty of Materials Science and Physics, Institute of MaterialsScience([email protected])Osteoporosis is a disease of the 21st century that affects an enormous amount ofpeople,bothsexesbutmainlywomenandtheelderly.Itisestimatedthatthisproblemcurrentlyaffectsasmanyas200millionpeopleworldwide,causingpainandloweringthe comfort of life [1]. It is a skeletal system disease characterized by loss of bonedensity and deterioration of the microarchitecture of bone tissue, which leads toincreasedbonefragilityandleadstofractures[2].One of the basic materials of the mineral phase of bone is hydroxyapatiteC10(PO4)6(OH)2 [3]. Its greatest advantages are bioactivity, biocompatibility andosteoconductionproperties.Therefore,itiswidelyusedintheregenerativemedicineof the skeletal system, including bioactive coatings, bone filler, bone tissueengineeringscaffoldsaswellasacomponentofcomposites[4].Presentedresearchisfocusedonsynthesisandcharacterizationofhydroxyapatite.Inthiswork,HApwasobtainedby twowetmethods.The first onewas carriedout atroom temperature, using ammonium dihydrogen phosphate (NH4H2PO4) andhydrated calciumnitrate (Ca(NO3)2 as reagents. Another one, at boiling point usingcalciumacetate((CH3COO)2Ca)anddisodiumhydrogenphosphate(Na2HPO4).Inbothcases, the syntheses were performed under different conditions, changing pH andreagentconcentrations.Acknowledgment:References:[1] T. Sozen, L. Ozisik, andN. Calik Basaran, “An overview andmanagement of osteoporosis,” Eur. J.Rheumatol.,vol.4,no.1,pp.46–56,2017.[2]R.Eastelletal.,“Postmenopausalosteoporosis,”Nat.Rev.Dis.Prim.,vol.2,pp.1–17,2016.[3]A.L.Boskey, “Bonecomposition: relationship tobone fragilityandantiosteoporoticdrugeffects,”BonekeyRep.,vol.2,p.Articlenumber:447,2013.[4] K. Lin and J. Chang, Structure and properties of hydroxyapatite for biomedical applications, vol.4214,no.8.ElsevierLtd.,2015.ThisstudywassupportedbygrantfromtheFoundationforPolishScienceintheframeofTEAMNETPOIR.04.04.00-00-16D7/18



P4.LowpHinproductionoflacticacidandprobioticbiomass**

AleksandraDjukić-Vuković1,DraganaMladenović1,LjiljanaMojović1

1Department of Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy,UniversityofBelgrade,Serbia([email protected])

Lactic acid (LA) is an important chemical with wide application range in food,cosmetic and pharmaceutical industries as acidifying agent and preservative. LA isalso used for production of poly-lactides - thermostable, biocompatible polymerssuitable forbiomedical applications.Physicochemicalpropertiesofpoly-lactides aredeterminedbyratioofL-andD-LA.SomeLAbacteriaareproducingL-orD-LAwithgreat stereospecificitywhichmakesbiotechnological LAproductionpreferred routetoday1.Decrease in pH during LA fermentation is a result of produced LA and can befavorableforpreventionofcontaminationandenableopenlacticacidfermentation2,which is applied in silaging. But also, it heavily affects productivity of the processwhenhighLAproductionisthemaingoalinprocess.CaCO3andNaOHwerethemosteffective neutralizing agents in LA fermentation on stillage and similar agri-foodindustrysubstrates3,4.ControlofpHandhighLAproductionisevenmorechallengingwhen LA producing strain has probiotic characteristics and remaining probioticbiomassshouldbevalorizedinfeed.References:[1]Djukić-VukovićA,MladenovićD,IvanovićJ,PejinJ,MojovićL.Towardssustainabilityoflacticacidand poly-lactic acid polymers production. Renew Sustain Energy Rev. 2019;108:238-252.doi:10.1016/J.RSER.2019.03.050[2] Djukic-Vukovic A, Lazovic S,Mladenovic D, Knezevic-Jugovic Z, Pejin J, Mojovic L. Non-thermalplasma andultrasound-assisted open lactic acid fermentation of distillery stillage. Environ Sci PollutResInt.2019;26(35):35543-35554.doi:10.1007/s11356-019-04894-9[3] Djukić-Vuković AP, Mojović LV, Vukašinović-Sekulić MS, et al. Effect of different fermentationparameters on l-lactic acid production from liquid distillery stillage. Food Chem. 2012;134(2):1038-1043.doi:10.1016/j.foodchem.2012.03.011[4]ZhangY,CongW,ShiS.ApplicationofapHfeedback-controlledsubstratefeedingmethodinlacticacidproduction.ApplBiochemBiotechnol.2010;162(8):2149-2156.doi:10.1007/s12010-010-8989-x

**Posterselectedforpresentationinaflashtalk



P5.TheaceticacidtoleranceofyeastsKluveromycesmarxianus**

JekaterinaMartinova1,KristianaKovtuna1,ArmandsVigants1

1University of Latvia, Institute of Microbiology and Biotechnology. Jelgavas str.1, Riga, Latvia.armands,[email protected]. marxianus is non-conventional food grade yeast with great potential inbiotechnology industry (1).Acetic acid is aby-product in fermentationprocessesofthese yeasts. There is growing interest in studies of acetic acid tolerance of theseyeasts (2,3). The inhibition of biomass growth by acetate of several K. marxianusstrainswasstudied.Itwasfoundthatincreasedconcentrationofacetateslowsdownyeast growth and this depends frommediumpHand carbon source. InhibitionwasmorepronouncedwhenK.marxianus growthon lactose as comparewith glucoseorglucose-galactose mix. The growth inhibition degree correlate with undissociatedacetic acid concentration inmedium. Increase of concentration of acetate results inlag-phase prolongation and decrease in biomass yield. The acetic acid tolerancesignificantlydiffersbetweenstrains.ThecorrelationbetweenaceticacidtoleranceofK.marxianus grown on lactose and localization of b-galactosidase (cytoplasm orperiplasm)wasobserved.ThestrainK.marxianusCBS712withb-galactosidaselocatedonly incytoplasmhadmostpronouncedinhibitingeffectsofacetatewhengrownonlactose.IncreasedtoleranceofK.marxianuscellstoelevatedconcentrationsofacetatewas observed through adaptation during an extended lag phase. The tested K.marxianusstrainshaddifferentadaptedcellsamountintheculturepopulation.References:[1]FonsecaGG,HeinzleE,WittmannCandGombertAK(2008)“TheyeastKluyveromycesmarxianusanditsbiotechnologicalpotential”ApplMicrobiolBiotechnol79:339–354[2]MartynovaJ,KokinaA,Kibilds J,Liepins J,ScerbakaRandVigantsA(2016)“EffectsofacetateonKluyveromyces marxianus DSM 5422 growth and metabolism” Applied Microbiology andBiotechnology100:4585–4594[3]JayaprakashP,MartaniF,MorrisseyJ,BranduardiP(2019)“Enhancingorganicacidtoleranceinthenon-Saccharomyces yeast Kluyveromyces marxianus” 7th Conference on Physiology of Yeasts andFilamentousfungi(24-27June2019,Milan,Italy)AbstractBook127p **Posterselectedforpresentationinaflashtalk



P6. Acclimatization of anaerobic microorganisms to low pHconditionsforenhancementofbiogasproduction**

AysenurUgurlu,EceKendir

1Hacettepe University, Department of Environmental Engineering, Beytepe, Ankara([email protected])Anaerobic digestion is amature technology that produces energy through biomassincludingwood,energycrops,wastes,andalgae.Itisanoxygenfreebiologicalprocessin which microorganisms work in consortia. Acidogenesis and methanogenesis aretwo importantstagesofanaerobicdigestionthatareproneto inhibition.Acidogenicandmethanogenicmicroorganismshavedifferentgrowthkinetics,physiologyandpHtolerance[Ref1].MethanogensareknownasmostsensitivemicroorganismsworkingwithanoptimumpHclose toneutral. Imbalancebetweenvolatile fattyacids(VFAs)production and reduction bymethanogens results in accumulation of VFAs. As thealkalinity is not sufficient, pH drop was observed and methanogenic activity isinhibited [Ref2]. NaOH or alkalinity addition may provide pH stability, however;operationalcostsandtoxicityduetoNaOHarethemajorconcerns[Ref3].So,recentinvestigationshavefocusedonuseofacclimatedculturestolowpHasacheaperandoutstandingalternative.Therearesomestudiesproveduseofacclimatedculturesforbiogasproductionfromdifferenttypesofwastesincludingfoodwaste,cheesewhey,vegetables fruit waste, dairy waste at low pH [Ref4]. So, detailed investigationsshouldbe carriedout in the future to better comprehend thenature of theprocessapplicabilityinlargescaleapplications.References:[1]YiJ.,DongB.,JinJ.,DaiX.,2014.EffectofIncreasingTotalSolidsContentsonAnaerobicDigestionofFoodWaste underMesophilic Conditions: Performance andMicrobial Characteristics Analysis. PLOSONE9(7),e102548.[2] Xiao K.K., Guo C.H., Zhou Y., Maspolim Y., Wang J.Y., Ng W.J., 2013, Acetic acid inhibition onmethanogensinatwo-phaseanaerobicprocess.BiochemicalEngineeringJournal75,1-7.[3] Charalambous P., Shin J., Sin S.G., Vyrides I., 2020. Anaerobic digestion of industrial dairywastewaterandcheesewhey:PerformanceoninternalcirculationbioreactorandlaboratorytestatpH5-6.RenewableEnergy147,1-10.[4]KuradeM.B., SahaS.,Kim J.R.,TohH-S., JeonB-Y., 2020.Microbial communityacclimatization forenhancementinthemethaneproductivityofanaerobicco-digestionoffats,oil,andgrease.BioresourceTechnology296,122294.**Posterselectedforpresentationinaflashtalk



P7. Evolutionary engineering of Lactobacillus pentosus improveslacticacidproductivityfromxylose-richmediaatlowpH**

EnriqueCubas-Cano1,CristinaGonzález-Fernández1,EliaTomás-Pejó1

1IMDEA Energy Institute, Biotechnological Processes Unit, 28935, Móstoles, Spain.([email protected])Havinginmindthatxylose,thesecondmostabundantsugarinlignocellulose,cannotbemetabolized bymost ofwild typemicroorganisms, the use of this sugar for theproduction of chemicals is a challenging approach. In this context, lactic acidproduction fromC5hasbeenreported inLactobacilluspentosus(1-3).Thisacid isarising platform chemical that can be used for the production of pharmaceuticals,chemicals and biopolymers (4). The pH drop produced during fermentation due tolactic and acetic acid accumulation can produce a detrimental effect on bacterialgrowth.Inthisstudy,L.pentosusCECT4023Twasevolutionaryengineeredtoimproveits acid pH tolerance in xylose fermentation. After a long-term non-pH controlledsequential batch cultivationwith increasing xylose concentration, a new strainwasobtained(namedMAX2).Thisstrainshowedbetween1.5and2-foldincreaseinlacticacidproductioninxylosedefinedmediaindependentlyoftheinitialpH.WhenthepHwas controlled in bioreactor, 1.4-fold increase in productivitywas attained both inxylose defined and in wheat straw hydrolysate. These results demonstrated thepotentialofMAX2straintoproducelacticacidfromhemicellulosicsubstratesatlowpH,reducingtheneedofusingneutralizingagentsintheprocess.References:[1] Cubas-Cano E, González-Fernández C, Ballesteros M, Tomás-Pejó E. Biotechnol Prog. (2019)35(1),e2739[2]Cubas-CanoE,González-FernándezC,BallesterosM,Tomás-PejóE.BioresourTechnol,(2019)288,121540[3]HuJ,LinY,ZhangZ,XiangT,MeiY,ZhaoS,etal.LiangY,PengN.BioresourTechnol.(2016)214:74-80[4] Cubas-Cano E, González-Fernández C, Ballesteros M, Tomás-Pejó E. Biofuels Bioprod Biorefin(2018)12:290-303.**Posterselectedforpresentationinaflashtalk



P8. Efficient production of lactic acid from gardening residues:importanceofpHcontrolfortheresistanceofinhibitorycompounds

EnriqueCubas-Cano1,CristinaGonzález-Fernández1,IgnacioBallesteros2,EliaTomás-Pejó1

1IMDEA Energy Institute, Biotechnological Processes Unit, 28935, Móstoles, Spain.([email protected])2CIEMAT,BiofuelsUnit,28040,Madrid,SpainTheexpansionofurbangreenareashasboostedanewsourceofgardeningresidues.Althoughhighvolumesofthesematerialsaregeneratedannually,theirvalorizationisstillhinderedduetoitsseasonalityandheterogeneity(1).Inthissense,bothC6andC5 sugars from gardening residues can be used to produce platform chemicals likelactic acid, as reported with other lignocellulosic materials (2-3). This acid is aversatilebuildingblockwithplentyof industrial applications like food,pharmaandbiopolymers(4).Inthiscontext,anefficientlacticacidfermentationofhemicellulosicsugars from gardening hydrolysates with Lactobacillus pentosus CECT4023T isaddressed.Togetherwithsugars,someinhibitorydegradationcompoundsareusuallyfound in the hydrolysates due to the sugar and lignin degradation duringpretreatment of lignocellulose. In this work, xylose consumption was found to behampered due to a pH drop because of the presence of acids, both formed duringpretreatmentandfermentation.AutomaticNaOHadditioninbioreactortocontrolthepHresultedin44%increaseinlacticacidproduction,reaching95%ofthemaximumtheoretical yield. These results provide new insights for process optimization tovalorise emerging lignocellulosicmaterials like gardening residues intohigh added-valueproducts.References:[1]BoldrinA,ChristensenTH,2010.Seasonalgenerationandcompositionofgardenwaste inAarhus(Denmark).WasteManag,30(4),551-557(2010).[2] Cubas-Cano E, González-Fernández C, Ballesteros M, Tomás-Pejó E. Lactobacillus pentosus CECT4023Tco-utilizesglucoseandxylosetoproducelacticacidfromwheatstrawhydrolysate:anaerobiosisasakeyfactor.BiotechnolProg,e2739(2019).[3] Cubas-Cano E, González-Fernández C, Ballesteros M, Tomás-Pejó E. Evolutionary engineering ofLactobacilluspentosus improves lacticacidproductivity fromxylose-richmediaat lowpH. BioresourTechnol,288,121540(2019).[4] Cubas-Cano E, González-Fernández C, BallesterosM, Tomás-Pejó E. Biotechnological advances inlactic acid production by lactic acid bacteria: lignocellulose as novel substrate. Biofuels BioprodBiorefin,12,290-303(2018).



P9.PotassiumtransportersparticipateinyeastpHhomeostasisandtolerancetolowexternalpH**

MasarykJakub,PapouškováKlára,ZimmermannováOlgaandSychrováHana

DepartmentofMembraneTransport,InstituteofPhysiologyoftheCzechAcademyofSciences,Prague,CzechRepublic([email protected])Potassium is a keymonovalent cationnecessary formultiple aspects of cell growthand survival, for example compensation of negative charges of macromolecules tomaintain electroneutrality, cell turgor and volume, enzyme activity, and proteinsynthesis. Tightly regulated potassium fluxes across the plasma and organellarmembranesarealsoindispensableforthemaintenanceofpropermembranepotentialandintracellularpH.Inyeastcells,potassiumisaccumulatedagainstitsconcentrationgradienttorelativelyhighamounts(usually,theaccumulationratioismorethan105;yeastsgrowinginthepresenceofmicromolarKClaccumulateintracellularly200-300mM K+). Yeast cells use three types of transporters to accumulate potassium ingrowing anddividing cells [1,2].Analysis of transporters and correspondingknock-outmutants revealed that themain important isTrk1,a systempresent inall yeastspecies. The mal-function of Trk1 affects crucial physiological parameters andinfluencesmanyprocesses includingintracellularpHregulationandcellresponsetoosmotic and pH stresses [1,2]. This mal-function also diminishes virulence ofpathogenicyeastspecies[3,4].

References:[1] Arino J, Ramos J& SychrovaH (2010) AlkaliMetal Cation Transport andHomeostasis in Yeasts.MicrobiolMolBiolRev74,95-120;[2] Ramos J, Arino J & Sychrova H (2011) Alkali-metal-cation influx and efflux systems innonconventionalyeastspecies.FEMSMicrobiolLett317,1-8;[3] Llopis-Torregrosa V, Husekova B & Sychrova H (2016) Potassium uptake mediated by Trk1 iscrucialforCandidaglabratagrowthandfitness.PLoSOne11,DOI:10.1371/journal.pone.0153374;[4]Llopis-TorregrosaV.,VazC.,MonteolivaL.,RymanK.,EngströmY.,GacserA.,GilG.,O.LjungdahlP.O.,Sychrová H. (2019) Trk1-mediated potassium uptake contributes to cell-surface properties andvirulenceofCandidaglabrata.Sci.Rep.9:7529.**Posterselectedforpresentationinaflashtalk



P10. Acidophilic microorganisms – capable workers in metal-bearingwastetreatment**

JanaSedlakova-Kadukova,PeterPristaš

Department ofMicrobiology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef SafarikUniversityinKosice,Srobarova2,04154Kosice,Slovakia([email protected])Microorganisms are capable of transforming many metals by catalysing redoxreactions.Manyofthemconsequentlypromotethemobilisationorimmobilisationofmetals in ecosystems due to their dissolution or precipitation. Their capability tomobilisemetalsissuccessfullyusedinlow-gradeorebioleaching,however,itcanbeapplied in metal-bearing waste treatment. Bioleaching represents eco-friendly andcosteffectivealternativetoconventionalmetallurgicalprocess.Inourwork,wehaveisolatedmultiplesulphuroxidizingacidophilicbacteria,amongthem,Acidithiobacillusalbertensis,forthefirsttimereportedfromEuropeterritory.Theisolatewasappliedfor indium bioleaching from LCD monitors. Several other acidithiobacilli e.g.Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans were tested forbioleaching abilities of various metals from metal bearing waste (spent alkalinebatteries, Ni-Cd batteries, Li-accumulators, mother boards from computers etc.),bioleachingofLifromlowgradeorelepidolitewehavecarriedeitherinpurecultureor in combination of acidophilic bacteria (A. ferrooxidans, A. thiooxidans) withacidophilicfungusAspergillusniger,oryeastRhodotorularubra.Accordingtoobtainedresults,microorganismshavegreatpotentialindevelopmentofinnovativeprocessesofmetalrecoveryfromvariouswastematerials.TheworkwasfinanciallysupportedbygrantsVEGA1/0229/17,SK-PL-18-0012andCOSTCA18113




P11. The European Network for Optimization of VeterinaryAntimicrobialTreatment(ENOVAT)

JonathanGomez-Raja1,TheofilosPapadopoulos2,ElsBroens3,AlainBousquet-Melou4,ChantalBritt5,PetraCagnardi6, JeroenDewulf7,AnnetHeuvelink8,LisbethR. Jessen9,Ana Mateus10, Aneliya Milanova11, Gudrun Overesch12, Constanca Pomba13, NikolaPuvaca14,DorinaTimofte15,PeterPanduroDamborg9

1Funde-Salud,Merida, Spain ([email protected] ), 2AristotleUniversity ofThessaloniki, Thessaloniki,Greece, 3UtrechtUniversity,Utrecht,Netherlands,4VeterinarySchoolofToulouse,Toulouse,France,5Swiss3RCC,Bern,Switzerland,6UniversityofMilan,Milan,Italy,7GhentUniversity,Ghent,Belgium,8GDAnimalHealth,Deventer,Netherlands,9UniversityofCopenhagen,Frederiksberg,Denmark,10RoyalVeterinaryCollege,London,UnitedKingdom,11TrakiaUniversity,StaraZagora,Bulgaria,12UniversityofBern,Bern,Switzerland,13UniversityofLisbon,Lisbon,Portugal,14UniversityBusinessAcademyinNoviSad,NoviSad,Serbia,15UniversityofLiverpool,Liverpool,UnitedKingdom

The global antimicrobial resistancecrisis has been the driver of severalinternational strategies onantimicrobial stewardship, thattypically are only slowly beingimplemented into “real life”. This isparticularly unfortunate for veterinarymedicine, which is challenged by (i) ashortage of experts in key disciplinesrelated to antimicrobial stewardship,(ii) few antimicrobial treatmentguidelines, and (iii) inferior diagnostictestscomparedtohumanmicrobiology.The European Network forOptimization of VeterinaryAntimicrobial Treatment (ENOVAT)COST Action network comprisesparticipants from universities, knowledge institutes and companies across thirty-oneEuropeancountriesandrepresentsthemostcomprehensivegroupinginthisparticularfield.The Action will 1) survey the state-of-the-art in microbiological diagnostic practices andveterinary treatment guidelines across Europe, 2) develop an extensive European straindatabase and a standard for making antimicrobial treatment guidelines, 3) refinemicrobiological diagnostic methods and European treatment guidelines. All will bedisseminated to national and international stakeholders (Figure). Furthermore, the Actionwillrecommendpriorityresearchareasforfutureoptimizationofantimicrobialtreatmentinanimals, and develop a roadmap outlining howEuropean countries can advance towards acommonhighlevelofveterinaryantimicrobialstewardship.

ENOVATActionchart



P12. Response mechanisms of model organism Saccharomycescerevisiaetoyeastviruses

Juliana Lukša1, Ravoitytė Bazilė1, Konovalovas Aleksandras2, Aitmanaitė Lina2,YurchenkoVyacheslav3,ServaSaulius2,4,ServienėElena1,4

1Nature Research Centre, Lithuania; 2Vilnius University, Lithuania; 3University of Ostrava, CzechRepublic;4VilniusGediminasTechnicalUniversity([email protected])SimpleeukaryoticorganismSaccharomycescerevisiae,hostingdsRNAvirus-originatedkiller systems, is used as an attractive model for understanding the molecularmechanisms of viral infection and pathways viruses exploit to overtake the hostcellularmachinery. Inthisstudy,weprovideatranscriptomeprofilingofTotiviridaeviruses (M-2 and L-A-lus) possessing strain M437 by applying RNA-seq. WedemonstratethateliminationofbothdsRNAsaffectedtranscriptionalchangesof715genes, while M-2 free cells differentially expressed 486 genes. Most of thetranscriptional responses inducedbyviraldsRNAsaremoderateanddonot exceedthe limit of four-fold change. Enriched GO terms of positively regulated genes arerelated to stress response, namely cellular response to oxidative stress, oxidation-reduction process carbohydrate metabolic process, and ribosomal biogenesis.Negativelyregulatedgenesinvirusdeficientbakingyeastcellsarerelatedtocellularaminoacidandlipidbiosyntheticprocesses,RNAmetabolism,andcellularrespiration.InsightsonthealterationofhostgeneexpressionwillhelptounderstandthebiologyofdsRNAmycoviruses,andtheirimpactonthehostcells.



P13. Interaction of membrane-bound enzymes related to protontransportandhydrogenproductioninEscherichiacoliatacidicpH**

HeghineGevorgyan1,2,3,ArmenTrchounian1,2andKarenTrchounian1,2,3*

1Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan StateUniversity, 0025 Yerevan, Armenia; 2Scientific-Research Institute of Biology, Faculty of Biology,Yerevan StateUniversity, 0025Yerevan,Armenia; 3MicrobialBiotechnologies andBiofuel InnovationCenter,YerevanStateUniversity,0025Yerevan,Armenia*Email:k.trchounian@ysu.amBacteriahavestrategiestosurviveandgrowunderacidicconditionsbychangingthecell structure, metabolism and transport systems [1,2]. Escherichia coli has fourmembranereversiblehydrogenases(Hyd),whichcatalyzethereversiblereductionofprotonstomolecularhydrogen[3].InthisstudyinteractionbetweenprotonATPase,Hyd, formate dehydrogenase-H (FDH-H) and potassium transport system is firstinvestigatedinE.coliduringfermentationofmixtureofglucose,glycerolandformateatpH5.5.ThelackofFDH-Hand/orHydsreducedFOF1-ATPaseactivity.However,K+ionshada stimulatingeffectonprotonATPaseactivity.Thiseffecthasnot foundatslightlyacidicpH6.5[3].Accessible–SHgroupsweredecreasedinthemembraneofmutant strain with defect of FDH-H, compared with wild type. Besides, significantchangesof–SHgroupswereshowninthepresenceofDCCD,formate,ATPandK+ions.Taking together, the data obtained show that proton ATPase and FDH-H mightinteract together through dithiol-disulfide (-SH-HS to –S-S) interchange. Moreover,interaction betweenmentionedmembrane-bound enzymes suggest a new survivalmechanism for E. coli bacteria for maintaining intracellular pH and proton motiveforcegenerationatacidicpH.References[1]KrulwichT.A.,SachsG.,Padan,E.MolecularaspectsofbacterialpHsensingandhomeostasis.NatureRevMicrob,2011;9:330–343[2] Trchounian A., Trchounian K., Fermentation revisited: how do microorganisms survive underenergy-limitedconditions?,TrendsBiochemSci2019;44:391-400.[3]GevorgyanH.,TrchounianA.,TrchounianK.UnderstandingtheroleofEscherichiacolihydrogenasesandformatedehydrogenasesintheFOF1-ATPaseactivityduringthemixedacidfermentationofmixtureofcarbonsources,IUBMBLife2018;70:1040-1047**Posterselectedforpresentationinaflashtalk



P14. Natural Killler T-cells respond to Helicobacter pylori LPSLewisXYpresentedbyCD1d-loadeddextramer

RudnickaK.1,KilcoyneM.2,MatusiakA.1,ChmielaM.1

1Laboratory of Gastroimmunology, Department of Immunology and Infectious Biology, The Faculty ofBiologyandEnvironmentalProtection,UniversityofLodz,Lodz,,Poland2CarbohydrateSignallingGroup,MicrobiologySchoolofNaturalSciences,NationalUniversityofIreland,GalwayIrelande-mail:[email protected](NKT)cellsconstituteasubpopulationoflymphocytesthatsharecharacteristics of NK cells and lymphocytes able to respond to lipid antigens. TheinvolvementofunconventionalNKTcellsinresponsetoH.pylori(Hp)glycolipidshasnotbeenwelldescribed.TheaimofthestudywastoevaluatepossiblebindingofHplipopolysaccharide(LPS)withCD1dreceptorbyflowcytometry.Theperipheralbloodmononuclear leukocytes were isolated from asymptomatic volunteers with definedHpstatus,adjustedtothedensityof2.5x107/mlandstimulatedovernightwithHpLPS(or standard LPS from E.coli). The percentage of NKT cell subpopulations andsecretionofcytokineswereevaluatedbyflowcytometryandELISA,respectively.NKTcells isolated by a MidiMACS™ Separator (Milteny Biotec) were then co-incubatedwithFITCconjugatedCD1ddextramer(Immudex)previouslyloadedwithHpLPS(orLPS of E.coli). The binding was visualized by flow cytometry. Stimulation withstandardE.coliandHpLPSledtoaproliferationofclassicCD3+CD56+NKTcells,withnosuchimpactonCD3+CD16+subset.ExclusivelyHpLPSinducedtheproliferationofCD3+Nkp46+ NKT subset only in cell cultures derived from Hp(+) donors, incomparison tononstimulated cells.This effectwas accompaniedby the elevationofCD25+ among CD3+Nkp46+ NKT cells able to produce IL-10. NKT cells responses toLPSofHpdependonbindingoflipidAwithCD1dmoleculeandinteractionofsugarmoietieswithNKTreceptors.Thisstudywas financiallysupportedbyNationalScienceCentre(Poland) -SONATA12no.2016/23/D/NZ6/02553.



P15.Effectoforganicacidsonpathogenmicroorganismsinmeat**

LjupcoAngelovski,SandraMojsova,DeanJankuloski

1Foodinstitute,Facultyofveterinarymedicine,Universityof“Ss.CyrilandMethodius”,Skopje,[email protected]

InterventionsappliedatslaughterintheU.S.andCanadacomprisephysical,chemicalor biological treatments (1). In Europe interventions for carcass decontaminationwith substances other than potablewater are not categorically banned (Regulation(EC)No.853/2004)butEFSAapprovalisneeded(2).Organicacidshaveconsiderablepotential for industry use because they are quite inexpensive and generallyrecognizedassafe(3).Inthisstudylactic,citricandaceticacid,eachat3%(v/v)concentrationwereusedastwodecontaminationagents,andeachacidwassprayed(30secondsduration,25°C)on ground beef meat (portions of 300 g) inoculated before with Salmonellatyphimurium, Escherichia coli, Listeria monocytogenes, Enterobacteriaceae andcoliformbacteria(inconcentrationfrom8.3to8.5log/cfu).Duringthe9daystorageat4°Casamplewastakenon1,3,5,7and9dayandanalyzedforallofthementionedmicrobiologicalparameters.The results indicated a range of reduction in the bacterial populations from 0.6log/cfu to the highest of 3.0 log/cfu. In the samples inoculated with Salmonellatyphimurium the highest reduction of 2.3 log/cfuwas noted in the samples treatedwithaceticacid.ForListeriamonocytogenes,highestreduction(1.5log/cfu)wasnotedinthesamplestreatedwithlacticacid.References[1]Gill,C.O.(2009).Effectsonthemicrobiologicalconditionofproductofdecontaminatingtreatmentsroutinelyappliedtocarcassesatbeefpackingplants.JournalofFoodProtection,72,1790-1801.[2] Hugas, M. and Tsigarida, E. (2008). Pros and cons of carcass decontamination: The role of theEuropeanFoodSafetyAuthority.MeatScience,78,43-52.[3] Calicioglu,M., Kaspar, C.W., Buege, D.R., & Luchansky, J.B. (2002). Effectiveness of sprayingwithtween 20 and lactic acid in decontaminating inoculated Escherichia coli O157:H7 and indigenousEscherichiacolibiotypeIonbeef.JournalofFoodProtection,65,26-32. **Posterselectedforpresentationinaflashtalk



P16. Recovery and characterization of PbS using a novel strain ofBacillus**

LucianC.Staicu1,PaulinaJ.Wojtowicz1,AdrianGorecki1,LarryL.Barton2

1FacultyofBiology,UniversityofWarsaw,Warsaw,Poland;2DepartmentofBiology,UniversityofNewMexico,Albuquerque,USA([email protected])Lead(Pb)ranksasamajoranthropogenicpollutantbecauseitisusedextensivelybyindustrysuchasoremining, lead-acidbatterymanufacturingandfossil fuelburning(CRC,2018).Spentlead-acidbatterywastewaterisparticularlychallengingtotreatasitcontainshighlevelsofsulfate(g’sL-1),Pb(<5mgL-1)andischaracterizedbylowpH (1-3) (Vuet al., 2019).Most of the studies carriedout todateon this industrialwastewater type focusedon its treatmentandmarginallyonresourcerecovery.OurworkinvestigatesnovelmetabolicpathwaysbacteriausetodetoxifyPbandrecoveritin the form of valuable minerals. We study a novel Bacillus strain (Abq.) able todegrade cysteine to sulfide, accompanied by the biomineralization of Pb(II) to PbS(galena) (Fig).Within48hof incubation the strain reduced1mMPb(II) completelyusingastoichiometricconcentrationofcysteine.ThemineralogicalidentifyofPbSwasconfirmed by XRD and further characterization is in progress. Its negative surfacecharge indicates the presence of a biopolymer layer, indicative of high colloidalstability. The biomineralization process occurs extracellularly, which opens thepossibilitytoefficientlyrecoverPbS.

Fig.ControlexperimentandbiogenicPbSproducedbyBacillussp.Abq.

References[1]CRCHandbookofChemistryandPhysics(2018)(ed)Rumble,J.R.99thEd.CRCPress,BocaRaton,USA.[2]VuHHetal.(2019)Sustainabletreatmentforsulfateandleadremovalfrombatterywastewater.Sustainability11,3497.Acknowledgments: This work is supported by the National Science Center (Poland) research grant2017/26/D/NZ1/00408.**Posterselectedforpresentationinaflashtalk



P17. Membrane engineering to improve Saccharomyces cerevisiaerobustnesstowardsorganicacids**

LuísFerraz1,NadiaMariaBerterame1,MariaJoãoSousa2,PaolaBranduardi1

1UniversityofMilano-Bicocca,DepartmentofBiotechnologyandBiosciences,Milan,Italy2UniversityofMinho,CentreofMolecularandEnvironmentalBiology,Braga,Portugal([email protected])During bio based industrial processes yeasts are challenged to perform in adverseconditions, such as low pH, high temperatures and in the presence of inhibitorsderivedfrombiomass.Organicacids(OAs)playauniqueroleintheseprocessessincetheyareoftenreleasedfrombiomasstreatmentsand,wheninthefermentativebroththey cancompromise the cellmetabolismandcausegrowtharrest.OAs canalsobeproducts of interest obtained frommicrobial factories, with applications in severalindustrialsectors.Therefore,controllingthecellularinward/outwardfluxofOAscanimprovefermentationperformances.Plasmamembraneplaysapivotal role in thisprocess,actingasaselectivegateandchangingitscompositioninresponsetothepresenceofOAs.Thus,itsengineeringhasbeen envisaged as a strategy to increase yeast performance. We are evoking amembrane rewiring focusing on themodulation of the transcription factor ECM22,involvedintheregulationofergosterolbiosynthesis.HereweshowhowchangesinthecellularcontentofergosterolcanhaveapositiveornegativeeffectonyeastrobustnessdependingonthemolecularstructureoftheOAs.Furthermore, these effects can vary according to other parameters relevant forindustrialprocesses,suchastemperature.**Posterselectedforpresentationinaflashtalk



P18.CanTransposonDirectedInsertion-siteSequencing(TraDIS)beused to explore multiple outcomes of evolution under stressfulenvironments?**

MathewMilner,HrishirajSen,PeterLund

1InstituteofMicrobiologyandInfection,UniversityofBirmingham([email protected])Laboratory-based evolution has become a widely used method to explorefundamental questions about evolution as a process, and is also a powerful tool tostudythelinkbetweengenotypeandphenotype.Weareinterestedinunderstandingshortandlongtermsadaptationsofbacteriatostressfulenvironments,andtostudythis we have evolved six populations of E. coli MG1655 for in a dynamic pHenvironment,byiterativegrowthanddailydilutionover5monthsinunbufferedLB,starting at pH 4.5. Whole genome sequencing of the evolved populations and ofindividual clones from each population revealed many striking similarities in theevolutionary trajectories in the evolved strains.We are interested in exploring theimpactofmultipledifferentparametersonevolutionarytrajectories,butasevolutionexperiments take a long time, we are currently investigating whether traDISexperiments can partially replicate evolution experiments in a relatively short timeframe. Since TraDIS provides ameasure of relative contributions to fitness of eachgene (by comparison of read counts after growth in two conditions), in principle itshouldbepossibletouseTraDIStoidentifygeneswhoselossof functionprovidesafitnessbenefit.Herewepresentourlatestresults.**Posterselectedforpresentationinaflashtalk



P19.Organicacidsecretionbyfilamentousfungi**

MatthiasG.Steiger1,2,AliceRassinger2,MichaelSauer2,3

1InstituteofChemical,EnvironmentalandBioscienceEngineering,TUWien,Vienna,Austria;2AustrianCentreofIndustrialBiotechnology(ACIB),Vienna,Austria,DepartmentofBiotechnology,3UniversityofNaturalResourcesandLifeSciences(BOKU),Vienna,Austria([email protected])

Filamentous fungi are excellentproducersof organic acidsunder extremely lowpHconditions.Aspergillusniger is capableofproducing citric acidand isused for largescale fermentations formore than a century.We identified themain gene for citricacid secretion in Aspergillus niger, cexA, which belongs to belongs to the majorfacilitator superfamily subclass DHA1. Members of this family work as drug-H+antiporter. The disruption of this gene completely abolishes citric acid secretion,OverexpressionofcexAleadstoasignificantincreaseinsecretedcitricacidinA.niger.NewevidencehowthegeneexpressionisregulatedinA.nigerispresented.References

[1] Steiger, M.G., Rassinger, A., Mattanovich, D., Sauer, M., 2019. Engineering of the citrate exporterprotein enables high citric acid production in Aspergillus niger. Metab. Eng. 52, 224–231.https://doi.org/10.1016/j.ymben.2018.12.004**Posterselectedforpresentationinaflashtalk



P20. Effects of lactic acid bacteria inoculation in pre-harvestingperiodonalfalfa(MedicagosativaL.)silage

MustafaKIZILSIMSEK1,IbrahimERTEKİN2

1University of Kahramanmaras Sutcu Imam, Fac. of Agric., K.Maras, Turkey;([email protected])2UniversityofMustafaKemal,Fac.ofAgric.,Hatay,Turkey;Theaimofthecurrentstudywastodeterminetheeffectoflacticacidbacteria(LAB)asmicrobialadditiveonfermentationfeaturesandfeedqualityparametersofalfalfasilage.Alfalfa foragewas inoculatedwith different LABbacteria on thedaybefore cutting.Bacterialinoculationsignificantlyaffectedthefermentationpropertiesandsomefeedqualityparametersofalfalfasilage.Theuseofthebacterialinoculantinalfalfasilageincreased drymatter recovery, decreased silage pHmore effectively and preventedproliferationofsomeunwantedmicroorganisms insilagescomparedtocontrol.TheLAB isolatesbelonging toLactobacillusbifermentansspeciesgave thebestresultallsilageandqualitycharacterswastakenintoconsider.

References[1] Agarussi, M.C.N., Pereira, O. G., da Silva, V. P., Leonardo, E.S., Ribeiro,K.G. and Santos, A.A. 2019.“Fermentative profile and lactic acid bacterial dynamics in non-wilted and wilted alfalfa silage intropicalconditions”,MolecularBiologyReports,46,451-460.[2] Dordevic, S., Mandic, V. and Stanojevic, D. 2016. “The effect of bacterial inoculant on chemicalcompositionandfermentationofalfalfasilage”,BiotechnologyinAnimalHusbandry,32(4),431-423.[3]Kızılşimşek,M.,R.J.Schmidtt,R.J.,andKung,L.,Jr.2007.“Effectsofamixtureoflacticacidbacteriaappliedasafreeze-driedorfreshcultureonthefermentationofalfalfasilage”,J.DairyScience,90(12),5698-5705.



P21. Influenceof lowpH food ingredients and sport on thehumanmicrobiomeandhealth,monitoredbymobileapplicationandtaste

NorbertBabcsán1,3,ÁdámBoday2,PéterFritz3,JuditBabcsánKiss1,3

1InnobayHungaryLtd([email protected]).,2HerbafermLtd.,3UniversityofMiskolcWe propose a new nutrition concept using available food databases and ownexperienceoflowpHfoodqualityingredientsonthemicrobiomeonhumansurfaces.Every day average 700 kcal sport using aerobic exercising like swimming and bikeridingcanessentiallyimprovehumanhealththroughactivatingthelymphaticsystemon the gut skin axis. Proper selection of acidic food ingredients, elimination and/orconsumption of certainmicrobiome containing living foods theweight balance andthepersonalmoodcanbecontrolledusingsimplemobileapplications,andcouplingtheavailablefoodingredientcomponentsintheavailabledatabasesanditstastes.Theacid containing foodswere selected frompickles, yoghurt, citrus fruits and berries,wines and coffees; and certain yeast containing food like beer and bread wereeliminated.Bythisnewmethodasignificant,adjustableandsustainablebodyweight,moodandhealthcontrolwereachieved.Duringthestudytheskinwascleanedonlyby food quality acids, water and various infra-red radiations which drasticallydecreasing theprevious skinproblems likeacneanddandruff.Our futureplan is towiden our study on a clinical group trial with available serum test protocols forvitamins and certain acids anddeveloping spectral tasting as a biochemical sensingmethod for the human body which can be the key triggers of the digestion andimmuneprocesses.



P22. Analysis ofmicrobial growth andheterogeneity using dropletmicrofluidics**

SimonaBartkova1, Immanuel Sanka1,PillePata1,ToomasTeekivi1,MarkoVendelin1,OttScheler1*

1Department of chemistry and biotechnology, Tallinn University of Technology, Akadeemia tee 15,Tallinn12618,Estonia([email protected])Dropletmicrofluidicsdealswithgeneration,manipulationandanalysisofsmall (pL-nLsized)dropletsthatactastinytesttubes,wherebiologicalandchemicalreactionstake place. Typical droplet microfluidic experiment can involve generating andanalyzinghundredsofthousands(orevenmillions)ofdroplets.1

The detection and analysis of (bio)chemical reactions or cell growth in droplets isusually achieved via fluorescence. Droplet fluorescence can be analyzed with user-friendly freeware, for example CellProfiler that is often used in fluorescentmicroscopy.2Dropletsareverysuitableplatformfortheanalysisofsinglecellgrowthpatterns.Forexample,theyenablepreciseanalysisofantibioticsusceptibilityatsinglecell(CFU)level,thatcanrevealthephenotypicheterogeneityingrowth.3

References[1]Dropletmicrofluidicsformicrobiology:techniques,applicationsandchallenges.Kaminski*,Scheler*andGarstecki,LabonaChip,2016,16,2168-2187[2]CellProfiler:Afittoolforimageanalysisindropletmicrofluidics.Bartkova,Vendelin,Sanka,PataandScheler.biorXiv2019,doi:https://doi.org/10.1101/811869[3]Antibiotic inhibition of bacteria growth in droplets reveals heteroresistancepattern at the singlecelllevel.Scheler,Makuch,Debski,Horka,Ruszczak,Pacocha,Sozanski,Smolander,PostekandGarstecki.biorXiv2018,doi:https://doi.org/10.1101/328393




P23.Effectofbacteriocinsonpathogenmicroorganismsincheese

SandraMojsova,LjupcoAngelovski,DeanJankuloski

1Food institute, Faculty of veterinary medicine, University of “Ss. Cyril and Methodius”, Skopje,RepublicofNorthMacedonia([email protected])

Due to the decreasing of pH value and the formation of organic acids and theirpreservativeeffect,lacticacidbacteria(LAB)forcenturieshavebeenusedinfoodandfeed conservation (1, 2). By preventing the increase of non-pathogen and pathogenmicroflorabacteriocins can inhibit fooddeterioration.Because theyareharmless toeukaryoticcellsthebacteriocinsorbacteriocinproducingLABexceptcytolysincanbeusedassafealternativestochemicalpreservativesinfoods(3).The aims of this study were to identify bacteriocin producing enteroccoci isolatedfrom North Macedonian artisanal cheeses (200 samples) and to determine theexistence of bacteriocin structural genes (4,5). Furthermore, the following culturesweretestedforsensitivitytoenterocins:L.monocytogenes(NCTC11994),L.innocua(cheese isolate), S. aureus (cheese isolate), P. aeruginosa (NCTC 10662), B. cereus(NCTC 7464), S. enteritidis (meat isolate),E. coli (NCTC 9001) andY. enterocolitica(ATCC11303).Bacteriocinproducing isolateswere identifiedasE. faecalis (n=8),E.faecium (n=2) and E. hirae (n=2). The 13 isolates were screened for antibacterialactivity,sincetheymayproduceenterocinsthatcancontrolthegrowthofthetestedindicator bacteria. All of the isolates expressed their antibacterial activitypredominantly against L. monocytogenes in comparison to L. innocua. None of theisolateswerefoundtobeinhibitoryagainstS.enteritidis,Y.enterocoliticaandE.coli.

References[1]O’Sullivan, L., Ross,R.P.,Hill, C. (2002). Potential of bacteriocinproducing lactic acidbacteria forimprovementsinfoodsafetyandquality.Biochimie84,593–604.[2]Klaenhammer,T.R.,Barrangou,R.,Buck,B.L.,Azcarate-Peril,M.A.,Altermann,E.(2005).Genomicfeatures of lactic acid bacteria effectingbioprocessing andhealth. FEMSMicrobiolRev. 29, 393–409.http://dx.doi.org/10.1016/j.fmrre.2005.04.007[3] Casaus, P., Nilsen, T., Cintas, LM., Nes, IF., Hernández, PE., Holo, H. (1997). Enterocin B, a newbacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A.Microbiology143(Pt7):2287-2294.[4] Dutka-Malen S, Evers S, Courvalin P. (1995). Detection of glycopeptide resistance genotypes andidentificationtothespecieslevelofclinicallyrelevantenterococcibyPCR.JClinMicrobiol.,33(1):24-27.[5]Jackson,C.R.,Fedorka-Cray,P.J.,Barrett, J.B.(2004).Useofagenus-andspecies-specificmultiplexPCRforidentificationofenterococci.JClinMicrobiol.42,3558–3565.



P.24 Regulatory networks involved in Jen1 lactate transporterendocytosistriggeredbyalkalistress

Barata-Antunes C1, Talaia G1, Gournas C2, Saliba E2, Casal M1, André B2, GeorgeDiallinas3andPaivaS1*

1CentreofMolecularandEnvironmentalBiology,DepartmentofBiology,UniversityofMinho,CampusofGualtar,Braga,4710-057,Portugal;2MolecularPhysiologyoftheCell,UniversitéLibredeBruxelles(ULB), IBMM, Gosselies, Belgium 3Department of Biology, National and Kapodistrian University ofAthens,Panepistimiopolis15784,Athens,Greece.*[email protected] Saccharomyces cerevisiae cells modulate extracellular pH, being able toalkalinizeitssurroundingswhengrowninthepresenceofalternativecarbonsources.We have recently shown that, in S. cerevisiae, prolonged growth in lactate triggersJen1 lactate transporter endocytosis. This phenomenon is associated with a pH-dependent effect and requires a functional Jen1 transporter. Proteins of the alpha-arrestinfamilyarekeyplayers in linkingsignallingeventsandnutrienttransportersendocytosis.Wefoundthatamemberof theconservedalpha-arrestin family,Bul1p,mediates Jen1 endocytosis in response to alkali stress[1]. This downregulationinvolvestheNpr1pkinaseandtheSit4pphosphataseanditisonlyoperatinginarichnitrogen source, as replacing ammonium with L-proline leads to reduced Jen1pturnoverinprolongedgrowthinlactate.HowanincreaseinpHleadstotheturnoverofJen1pandpossiblyothertransporters,especiallyH+symporters,isstillunknown.

References[1]TalaiaG,GournasC;SalibaE;Barata-AntunesC;CasalM;AndréB;DiallinasG;PaivaS.(2017).Theα-arrestin Bul1p mediates the endocytic turnover of Jen1p lactate transporter in response toalkalinizationanddistinctphysiologicalsignals.JournalofMolecularBiology.429(23):3678-3695.



P25. The diffusion of the viral-derived membrane-active peptidepepR along Staphylococcus aureus biofilms causes a gradedspatiotemporalprofileofbacterialdisruption

SandraN.Pinto ([email protected]) (1), SusanaA.Dias (2),AnaF. Cruz (2),DalilaMil-Homens(1),FabioFernandes(1),JavierValle(3),DavidAndreu(3),ManuelPrieto(1),MiguelA.R.BCastanho(2),AnaCoutinho(1,4),AnaSaloméVeiga(2)

(1)iBB-InstituteforBioengineeringandBiosciences,DepartmentofBioengineering,InstitutoSuperiorTécnico,UniversidadedeLisboa,Av.RoviscoPais1049-001Lisboa,Portugal;(2)InstitutodeMedicinaMolecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa,Portugal;(3) DepartmentofExperimentalandHealthSciences,UniversitatPompeuFabra,BarcelonaBiomedical Research Park, 08003 Barcelona, Spain; (4) Departamento de Química e Bioquímica,FaculdadedeCiências,UniversidadedeLisboa,CampoGrande1749-016Lisboa,Portugal.Infections caused by bacterial biofilms are a major healthcare problem given theirreduced susceptibility to conventional antibiotics. Development of effectiveantibiofilm agents is urgent. In this work, we show that pepR, a multifunctionalpeptide derived from the Dengue virus capsid protein, is able to both prevent theformation and act on preformed Staphylococcus aureus biofilms. Thorough anddetailed mechanistic studies using flow cytometry and confocal microscopy revealthat pepR targets the bacterialmembrane.More importantly, the peptide is able todiffuse through a preformed biofilm and rapidly kill biofilm-embedded bacteriathroughthesamemechanism.However,thediffusionofthepeptidealongthematrixofS.aureusbiofilmsshapedaspatiotemporalgradientofpepRwhichwasresponsiblefor its reduced activity at the inner layers of thebiofilm.As shown recently [1] theinnerlayerofthebiofilmcorrespondstoareasoflowpH(~5.1)andtheareaswherethe peptide is more active corresponds to an increase in the pH value towards aneutral pH. Overall, our work demonstrates the potential of pepR towards thedevelopment of novelmembrane-active antibiofilm peptides and the importance ofdiffusion/biofilmstructureforthebiologicalactivityofantimicrobialmolecules.Reference[1]FulazS,HiebnerD,BarrosCHN,DevlinH,VitaleS,QuinnL,CaseyE(2019)RatiometricImagingoftheinSitupHDistributionofBiofilmsbyUseofFluorescentMesoporousSilicaNanosensors.ACSApplMaterInterfaces11:32679-32688.



P26. The Effect of Carbon Dioxide as a Climatic Parameter onMicrobialFoodContaminantsandSelectiveIsogenicMutants**

SholeemGriffin1,GuillaumeDubios1,VasilisP.Valdramidis1.

1Department of Food Sciences and Nutrition, Faculty of Health Sciences, University of Malta, Msida,MaltaCarbondioxide dissolves in aqueous solutions existing in a chemical equilibrium ascarbonic acid and bicarbonate, lowering its pH. An increase in accumulation ofatmosphericCO2levelscanleadtotheloweringofpH,whichmayprovideaselectivepressure causing adaptational changes to someorganisms. Inparticular, pathogenicmicroorganisms which have a high replicative index; such as Escherichia coli, maystimulatetheirgrowth.Theglutamatedecarboxylase(GAD)systemmaintainsthepHinE.coliunderacidicconditions.MutationsintheGADsystemcanleadtochangesinthe growth of E. coli leading to its enhanced growth under acidic conditions. ToevaluatetheeffectofincreasingCO2concentrationonbacterialgrowthandassessitsmechanismof action,wild-typeE. coli and itsmetabolic or stress responsemutantsΔdnaK,ΔgadB,ΔgadC,ΔgadDandΔrpoS,weregrowninaplatereaderintrypticsoyabroth until stationary phase at atmospheric gas conditions, 2.5, 5 or 10%CO2. Theoptical densitywas recorded at 600 nm for 24 h and used to calculate the growthresponse and the timeof detection (TTD) to assess growth. Increase in CO2 to 5%significantlyincreasedtheTTDofE.coliΔdnaK,ΔgadC,ΔgadDandΔrpoSby1h,andsignificantly decreased the growth ofE. coliΔdnaK,ΔgadB andΔgadC compared towildtype E. coli (p < 0.05). This highlights the importance of the GAD system andmetabolicstressresponsegenesintheenvironmentaladaptationtoincreasingCO2.**Posterselectedforpresentationinaflashtalk



P27. Genome-wide mutational profile of acidophilic bacteriumAcidobacteriumcapsulatum

SibelKucukyildirim1,SamuelF.Miller2,andMichaelLynch2

1DepartmentofBiology,HacettepeUniversity,Ankara,TURKEY;2BiodesignCenterforMechanismsofEvolution,ArizonaStateUniversity,Tempe,USA([email protected])Analysesofspontaneousmutationhaveshownthattotalgenome-widemutationratesarequalitativelysimilarformostprokaryoticorganisms(Lynchetal.2016;Longetal.2018). However, this view is mainly based on organisms that grow best aroundneutral pH values (6.0-8.0). However, some results for archaea adapted to life inextreme habitats have suggested relatively a lower genomic mutation rate andpredominanceofinsertion-deletionevents(Groganetal.2001;Mackwanetal.2007;Drake 2009). Therefore, in order to understand how environmental factors(temperature, pH, etc.) and intrinsic mechanisms (DNA replication and repair)cooperate and determine the genome-widemutation rate and spectrum across thetreeoflife,itisnecessarytoexpandexperimentalassaystospecieslivinginextremeenvironments. Here, we have determined the genome-wide rate of spontaneousmutations in theacidophilicAcidobacteriumcapsulatumusingadirectandunbiasedmethod:mutation-accumulationexperimentsandwhole-genomesequencing.Whole-genome sequencing of 70 mutation accumulation lines of A. capsulatum after anaverageof~2900celldivisionsyieldedabase-substitutionmutationrateof1.22×10-10persitepergenerationor5x10-4pergenomepergeneration,whichissignificantlylowerthantheconsensusvalueofmesophilic(approx.15-40C)andneutrophilic(pH6-8)prokaryoticorganismswithDNAgenomes.However,theinsertion-deletionrateis high relative to the base-substitution mutation rate. Organisms with a similareffective population size and a similar expected effect of genetic drift should havesimilarmutationrates.Sinceselectionoperatesonthetotalmutationrate, itmaybesuggested that relatively high insertion-deletion rate may compensate low base-substitutionrateinA.capsulatum.References[1] Drake, J.W., 2009Avoidingdangerousmissense:Thermophilesdisplayespecially lowmutation rates.PLOSGenet5:e1000520.[2] Grogan, D. W., G. T. Carver and J. W. Drake, 2001 Genetic fidelity under harsh conditions: analysis ofspontaneousmutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Proc Nat Acad Sci USA 98:7928-7933.[3]Mackwan,R.R.,G.T.Carver,J.W.DrakeandD.W.Grogan,2007AnunusualpatternofspontaneousmutationsrecoveredinthehalophilicarchaeonHaloferaxvolcanii.Genetics176:697-702.[4]Long,H.,W.Sung,S.Kucukyildirim,E.Williams,S.F.Milleretal.,2018Evolutionarydeterminantsofgenome-widenucleotidecomposition.NatEcolEvol2:237-240.[5]Lynch,M.,M.S.Ackerman,J.-F.Gout,H.Long,W.Sungetal.,2016Geneticdrift,selectionandtheevolutionofthemutationrate.Nat.Rev.Genet.17:704-714.



P28.QuantitativeapproachestoaddressthesporeresistanceinlowpH food processing environments: the case of Alicyclobacillusacidoterrestris**

IvanLeguerinel1,NoémieDesriac1,VasilisP.Valdramidis2

1UniversitédeBrest,EA3882,LaboratoireUniversitairedeBiodiversitéetEcologieMicrobienne,UMTACTIA19.03ALTER’iX, 6 ruede l'Université, F-29334Quimper, France; 2 Faculty ofHealth Sciences,Departmentof FoodSciences andNutrition,UniversityofMalta,MsidaMSD2080,Malta (presentingauthor:[email protected])

The elimination and control of sporulatingmicroorganisms, such asAlicyclobacillusacidoterrestris, from the processing environment is of great importance, as itsendosporesaredifficulttodetectandareabletosurvivethepasteurizationprocesses.Theirgrowthisassociatedwithoff-flavoursaswellasthereductionoftheshelf-lifeofacidic fruit juices and acidic beverages (pH<4) during distribution and storage. Tocontrolitsdevelopmentandoptimizetheapplicationofheattreatments,insuchfoodproducts, it is necessary to characterize its heat resistance and growth dynamics,particularly its recovery following heat treatments in relation to environmental pH.The current study presents quantitative approaches to assess the impact of theheating temperature and the pH of the products. Concurrently, the impact of therecoverymediumon theapparentheat resistance isassessed.For thatpurpose, thethermosensitivity parameter ofA. acidoterrestris is estimatedby theuse ofBigelowtypemodel, while the impact of the heatingmedium on the heat resistance is alsoevaluated.Quantitativeapproachesonassessingtheeffectoftheacidicenvironmentinrelationtotherecoverymediaarealsopresented.Forthatpurpose,aRossotypeofmodel that incorporates the estimation of cardinal value parameters is used topresentquantitativelytheimpactoftherecoverymedia.




P29.ImpactofgastricpathogenH.pylorionMUC5ACproductionanddevelopmentofchronicinfection

WeronikaGonciarz1,AgnieszkaKrupa1,MagdaleniaChmiela1

1UniversityofLodz,FacultyofBiologyandEnvironmentalProtection,DepartmentofImmunologyandInfectiousBiology,LabolatoryofGastroimmunology;email:[email protected]

Helicobacterpylorisurviveinstomachinacidicandcolonizegastricepithelialcellsofmore than half of the human population, causing chronic inflammatory response,peptic ulcer or gastric cancer. We asked whether H.pylori and their solublecomponents may promote or maintain gastric colonization due to enhancement ofmucin5(MUC5AC)production.WeusedCaviaporcellusexperimentallyinfectedwithH.pylori7/28daysafter inoculation.Furthermore primarygastricepithelial cellsofCaviaeporcelluswerepropagatedinvitrofor24hintheculturemediumaloneorwithH.pyloricomponents:glycineacidextract-(GE),10μg/ml;cytotoxin-associatedgeneA-(CagA) protein, 1μl/ml; UreA urease subunits, 5μg/ml; H.pylori or Escherichia colilipopolysaccharide-(LPS) 25ng/ml or with the liveH.pylori CCUG17874 (2h, 2×107CFU/ml). Primary gastric cells or gastric tissue were stained with anti-MUC5ACantibodiesandsecondaryFITCantibodiestoassessMUC5ACproductionorwithanti-H.pylori FITC antibodies to show adhesion. H.pylori components or live bacteriaenhanced the production ofMUC5AC by gastric epithelial cells, in vivo and in vitro,which was related to increased adhesion ofH.pylori.MUC5ACwas producedmoreintensively during acute (7 days) than chronic (28 days) phase of infection.Upregulation of MUC5AC production in response to H.pylori is an importantmechanismthemaintenanceofinfection.



P30. Physico-chemical and microbiological characterization oftypicalBulgariansourdoughs

GotchevaV.1,KlimentovaE.1,PetkovaM.2,StefanovaP.1,AngelovA.1

1University of Food Technologies, Plovdiv, Bulgaria; 2Agricultural University, Plovdiv, [email protected]

Inthepast15years,consumer’sinterestinBulgariatosourdough(SD)bakedgoodsisgrowingfastbecauseoftheirdistinctflavor,textureandhealthyattributes.Asaresult,thereisahighindustrialdemandforadequatesourdoughstartercultureswithgoodtechnological performance. Yeasts and lactic acid bacteria form stable mixedpopulations in artisanal sourdoughs, with composition mainly defined by the rawmaterials used, geographical factors, preparation methods, and the productionenvironment. The aim of this studywas to study typical artisanal sourdoughs fromvarious locations in Bulgariawith focus on the lactic acid bacteria (LAB) and yeastspeciesrepresented intheirmicrobiota.Somephysico-chemicalcharacteristicswerealso assessed. Physico-chemical analyses of the 16 SD samples showed dry mattercontentfrom25.17to53.6%,pHvalueswithin3.1and5.3,andtotaltitratableacidityfrom6.8 to32.6.The total LAB counts varied from5x105 to4x109 cfu/g, andyeastcountswerebetween6x104and4x108cfu/g.Amongthe11LABspeciesidentified,themost commonly found isolates were Lactobacillus brevis (31%), Lactobacillusplantarum(24%),andPediococcuspentosaceus (14%).Saccharomycescerevisiaewasthemostabundantyeastspeciesfoundinall16sourdoughs(86%).Pichiafermentans,Kluyveromycesmarxianus,Yarrowia lipolytica, Kazachstania humilis,K. barnettii andCandidaglabratawerealsoidentified.



P31. Influence of soy milk fermentation with Lactobacillus andBifidobacterium strains on the protein immunoreactivity andmineralavailability

LidiaH.Markiewicz,AgataSzymkiewicz,BarbaraWróblewska

Institute of Animal Reproduction and Food Research PAS, Department of Immunology and FoodMicrobiology;Tuwima10,10-748Olsztyn,Poland,([email protected])

The aim of the study was to investigate the ability of 26 Lactobacillus and 26Bifidobacterium strains to grow in the soymilk and acidify the medium. Moreover,their ability to decrease the immunoreactivity of β-conglycinin and glycinin – twomainsoyallergensandtoinfluencethemineralavailabilitywereinvestigated.Among52 investigated strains tested, 8 Lactobacillus and 6 Bifidobacterium strains werestandingoutintermsofdecreasingsoyproteinimmunoreactivity.Amongtheselectedstrains, lactobacilli lowered thepH to the level of 4.3-4.9,whereasbifidobacteria tothelevelof4.6to6.4.Bifidobacteriumstrainscharacterizedwiththelowestacidifyingabilitiesreleasedthelowestamountsofmagnesium,calcium,manganeseandzincandthehighest amounts of copper.All testedbifidobacteria releasedmanganese,whichwas at the lowest amounts in most of Lactobacillus-fermented soymilk. Theconcentrationofironwaslowerthaninacontrolinallsampleswhatindicatesontheutilization of this element by growing bacteria. To sum up, the influence ofLactobacillus and Bifidobacterium strains on the immunoreactive properties of soyproteins was pH-independent, whereas their ability to release micro- andmacroelements from thematrixwaspH-dependent.The investigated strainswill befurther characterized for their technological properties useful in the production offermentedsoymilk.



P32.Highcontentanalysisoffruitandberryfungalmicrobiota

Elena Servienė1, Juliana Lukša1, Ramunė Stanevičienė1, Bazilė Ravoitytė1, ŽivilėStrazdaitė-Žielienė11NatureResearchCentre,Vilnius,Lithuania([email protected]) Carposphereisauniqueanddynamichabitat,withmicrobialcommunitiessubjectedto relatively large environmental changes. Microorganisms flourishing on fruit andberry surface are exposed to ripeningconditioned acidity variations affecting theirsurvivalandshifting thepatternofmicrobiota. In thepresentstudy, theabundance,structure, anddiversityof the fungalmicrobiotaassociatedwithcarposphereof seabuckthorn berries were investigated. High-throughput amplicon-based sequencingapproachwasusedtoreveal the fungalcommunityalterations.Analysisof themostabundant representative OTUs showed a clear separation among microorganismsinhabiting the unripe and mature berries. In parallel, culture-dependent methodswereused to isolate and characterize yeasts and yeast-like fungi distributedon thesurface of sea buckthorn and cherry at different maturation stages. Potentiallybeneficial, inducing resistance in the hosting plant, or pathogenic, responsible fordisease development, microorganisms were detected. The information on theprevalence of fungal microorganisms on the carposphere of tested plants is highlyrelevant for the development of strategies for plant cultivation and diseasemanagementaswellasthequalityofberrieswithpotentialinfoodproduction.



P33.MolecularapproachestounderstandtheeffectofaceticacidinuropathogenicE.coli

Fatemah ALatar1, Francesca Bushell1, Thippesh Sannasiddappa1, John Herbert2,FrancescoFalciani2,PeterLund1

1University of Birmingham/Insitute of Microbiology and Infection, Birmingham, UK 2Institute ofIntegrativeBiology,UniversityofLiverpool,UK(Email:[email protected])

Aceticacidhaslongbeenknownforitsantibacterialactivity.WeareusingtraDIStoinvestigate themolecularmechanisms bywhich acetic acid acts as an antibacterialagent.Todothis,wegrewahigh-densitytransposonlibraryinuropathogenicE.coliEO499serotype131inM9mediaatpH7andpH5.5withaceticacidconcentrationsof40mMand 4mM, respectively, or without added acetic acid. Sequencing libraries weregenerated from total bacterial populations after growth, and sequenced using atransposon-specificprimertogeneratepositionsandfrequenciesforeachtransposon.Bycomparingnumbersof readsbeforeandafter thestress,we identifiedcandidategeneswhere transposon inserts led toadecreaseof fitnessunderaceticacidstress.Eightofthesewerechosenforfurtherstudy:nuoM,nuoG,sucA,sthA,pitA,apaH,rssBandytfP.Becauseofthedifficultiesofconstructinggenedeletionsintheuropathogenicstrainsfor validating TraDIS results, we tested the relative fitnesses of the correspondinggene deletionmutants from the Keio library (in strain BW25113),with the growthconditionsusedforEO499. Interestingly,onlya fewknockoutsshowedareducedinrelative fitness in time course competition at pH with acetic acid, while TraDISidentifiedrelativelyhighernumberofgenes.ThismayduetothedifferencesbetweenstrainsusedinTraDISandcompetition.Toovercomethisissue,wehavealsoisolatedtransposonmutants fromE. coli EO499 transposon library for thedeterminationofrelativefitness.Theresultswillbepresented.



P34.Physicochemicalandbiologicalcharacterizationofpyomelaninisolated from P. aeruginosa in regard of antimicrobial and pro-regenerativeactivity

Mateusz M. Urbaniak1,2, Weronika Gonciarz1, Karolina Rudnicka1, Przemysław Płociński1,MagdalenaChmiela1

1UnivesityofLodz,FacultyofBiologyandEnvironmentalProtection,Departmentof ImmunologyandInfectiousBiology,LaboratoryofGastroimmunology;2UniversityofLodz,TheBio-Med-ChemDoctoralSchool of the University of Lodz and Lodz Institutes of the Polish Academy of Sciences([email protected])

Numerous studies report on the biomedical potential of melanines, however little isknown on the chemical structure and bioactivity of pyomelanin from Pseudomonasaeruginosa. The aimof the studywas to determine the physicochemical andbiologicalproperties of pyomelanin isolated from P.aeruginosaMel+ strain by acid precipitation.Chemicalpropertieswereassessedwithstandardoxidizersandreducers.Solubilitywastested in polar, polar aprotic and non-polar solvents and thermostability in thetemperaturerangeof4-500°C.ThecytotoxicitywasevaluatedintheMTTandresazurinreduction assays using mouse L929 fibroblasts. Cell migration and pro-regenerativeproperties were assessed in the in vitro wound healing assay. Antibacterial activitytowardsGram-positivecocciandGram-negativerodswasassessedinbrothmicrodilutionmethodandresazurinreductionassay.The invitrostudiesrevealedthatpyomelanin(innon-toxic concentrations) exhibit the highest antimicrobial potential towards H.pylori(MIC=64 μg/ml), and that it stimulates (1-32 μg/ml) the regeneration of previouslydamaged fibroblasts. Pyomelanin manifests thermostabile, reducing and oxidizingproperties. The polarity of the solvent do not affect the solubility of the pigment. Lowcytotoxicity and the ability to support cell migration and anti-H.pylori activity allowsconsideringpyomelaninasapotentialnaturalagentsupportinggastriculcershealingandlimitingmicrobialexpansion.

These studies were performed within the project “Multifunctional composites biologically active for

applications in regenerative medicine of bone system (POIR.04.04.00-00-16D7/18)” carried out within the TEAM NET programme of the Foundation for Polish Science co-financed by the European Union under the

European Regional Development Fund.



P35. General Stress Response of Salmonella enterica TyphimuriumtoEnvironmentalStress

MilaArapcheska*1,JovankaTuteska2,AnneJolivet-Gougeon3

1FacultyofBiotechnicalSciences,“St.KlimentOhridski”University-Bitola,R.NorthMacedonia;2HighMedical School, “St. KlimentOhridski” University - Bitola, R. NorthMacedonia; 3Faculté des SciencesPharmaceutiques et Biologiques, Université de Rennes 1, Rennes, France (*e-mail:[email protected])Salmonella enterica Typhimurium is an enteric pathogen and etiological agent inbacterialfood-bornediseases.TheabilityofSalmonellatosurviveinthefoodchainisdue,inpart,toitsabilitytorespondeffectivelytoenvironmentalchanges.S.entericaTyphimuriumresponsetowidevarietyofenvironmentalstressesisaresultofhighlycomplexandtightlyregulatedprocessofgeneexpression.Thestress-inducedchangesin S. enterica Typhimurium bacterial cells were analyzed at the level of geneexpression.Forthatpurpose,thesimultaneousanalysisofgeneexpressionofasetof369genesdirectlyor indirectly involved inregulatorymechanismsand invirulencewas performed by PCR-basedmicroarray technique. Expression profile of bacterialcells submitted to oligotrophic stress (24 hours incubation in distilled water) wascompared with expression profile of bacterial cells in exponential growth phase,considered as control condition. A number of induced genes under studied stressconditions,whichratioofgeneexpression(stresscondition/controlcondition)hadavalueabove1.05weregenesthatmediateinageneralstressresponse.Infact,generalstress response renders bacterial cells broadly stress resistant in a way in whichdamageisrathertobeavoidedthantoberepaired.

References[1] Álvarez-Ordóñez, A., Begley, M., Prieto, M., Messens W.,López M., Bernardo, A., Hill, C. (2011):Salmonella spp. survival strategieswithin thehostgastrointestinal tract.Microbiology.Vol.157 (12):3268–3281.[2] Gruzdev N., Pinto R., Sela S. (2011): Effect of desiccation on tolerance of Salmonella enterica tomultiplestresses.AppliedandEnvironmentalMicrobiology.Vol.77(5)pp:1667–1673.[3]Ibanez-RuizM.Robbe-SauleV.,HermantD.,LabrudeS.,NorelF.(2000):IdentificationofRpoS(σS)-regulatedgenesinSalmonellaentericaserovarTyphimurium.J.Bacteriol.182,5749–5756.[4]KvintK,NachinL.,DiezA.,NystromT.(2003):Thebacterialuniversalstressprotein:functionandregulation.CurrOpinMicrobiol.6:140-5.[5] Monack D.M (2012): Salmonella persistence and transmission strategies. Current Opinion inMicrobiology.Vol.15(1):pp:100–107.[6] Runkel S.,Wells H.C., Rowley G. (2013): Livingwith Stress. A Lesson from the Enteric PathogenSalmonellaenterica.AdvancesinAppliedMicrobiology.pp:87-144.[7] Spector M.P., Kenyon W.J. (2012): Resistance and survival strategies of Salmonella enterica toenvironmentalstresses.FoodResearchInternational.Vol.45(2)pp:455-481.



P36. Regulation of endothelial cells activation / apoptosis byHelicobacter pylori antigen components – link to cardiovascularheartdisease

PaulinaRusek1,2,AgnieszkaKrupa1,MagdalenaChmiela1

1UniversityofLodz,FacultyofBiologyandEnvironmentalProtection,InstituteofMicrobiology,BiotechnologyandImmunology,DepartmentofImmunologyandInfectiousBiology,LaboratoryofGastroimmunology;2UniversityofLodz,TheBio-Med.-ChemDoctoralSchoolof theUniversityofLodzandLodzInstitutesofthePolishAcademyofSciences([email protected])

Coronaryheartdisease(CHD)usuallyassociateswithhyperlipidemiacoexistingwithobesity,hypertension anddiabetes. Involvement ofmicrobiological components in thepathogenesisofatherosclerosis is also considered. Helicobacter pylori (HP) colonizes gastric epitheliumcausing acute local inflammation and gastric epithelial damage. HP infectionmay also stayasymptomaticanddevelopchronicinflammatoryreactionininfectedorganism.Interestingly,HP antigenic components can influence endothelial cells causing disorder of endothelialhomeostasis anddysregulation of its activity and apoptosis. The aim of our research wasfocused on investigating mechanisms initiated in vascular endothelial cells during HPinfection. Experiments were performed using primary endothelial cells developed fromCaviae porcellus aortic tissue. Cells were stimulated with HP bacterial components:lipopolysaccharide and acid glycine extract – GE, containing surface components. To date,analysisconsistofexpressionofpErkandpro-oranti-apoptoticproteins:Bax,Bcl-2,Bcl-xL,caspase3inprimaryvascularendothelialcells.Additionally,cellDNAwastestedforanysignsofdamage(DAPIstaining)andendothelialcellswereexaminedformetabolicactivitychange(MTT assay) upon stimulation with HP components. Obtained results suggest that HPcomponentsactivateendothelialcellsviaErk-dependentpathwayandregulatetheirsurvival.EndothelialdysfunctioncausedbyHPmaysignificantlyimpactthedevelopmentofCHD.These studieswere performedwithin the project “Multifunctional composites biologically active for applications inregenerativemedicineofbonesystem(POIR.04.04.00-00-16D7/18)”carriedoutwithintheTEAMNETprogrammeofthe Foundation for Polish Science co-financed by the European Union under the European Regional DevelopmentFund.

References[1] Alzahrani S. et al. Effect of Helicobacter pylori on gastric epithelial cells. World J Gastroenterol20(36):12767-12780(2014);[2]MatusiakA.etal.PutativeconsequencesofexposuretoHelicobacterpylori infection in patients with coronary heart disease in terms of humoral immune response andinflammation.ArchMedSci12(1):45-54 (2016); [3]OlaM.S. et al.RoleofBcl-2 familyproteinsandcaspases in the regulation ofapoptosis. Mol Cell Biochem 351: 41-58 (2011); [4] Vijayvergiya R.,Vadivelu R. Role of Helicobacter pylori infection inpathogenesis ofatherosclerosis, World J Cardiol,7:134-143(2015);[5]WangM.etal.Isolationandprimarycultureofmouseaorticendothelialcells.JVisExp118,e52965(2016



P37. Adaptation of Crohn’s disease related bacteria AIEC LF82 toprotectintracellularmicrocoloniesfromphagolysosomesattacks

SylvieRimsky,OlivierEspéli,VictoriaPrudent,GaelleDemarre

Center for Interdisciplinary Research in Biology, Collège de France, CNRS, Paris France([email protected])

Patients with Crohn's disease present an abnormal colonization of the intestine byproteobacteria.AmongthemthefamilyofAdherentInvasiveE.coli(AIEC)arefoundassociatedwith intestinal lesions inpatientswith inflammatoryboweldisease.AIECarepredominant in themucus, adhere to epithelial cells, colonize themand surviveinsidemacrophages.WeareinterestedintheadaptationoftheAIECallowingsurvivalandgrowthinmacrophages.TheAIECresidesinsidematurephagolysosomeswheretheenvironmentishighlystressful(acidpH,oxidativestress,nutrientstarvation,etc.).This environment dramatically limits the growth of other E. coli strains. We haverecentlydemonstratedthatstringentresponseandSOSinductionarecriticalforAIECsurvival and multiplication within macrophages (1). The aim of this project is todecipheracidstressinterplaywithvirulenceresponseofAIECinphagolysosomes.WeobservedthatintracellularLF82produceanextra-bacterialmatrixpresentingtraitsofabiofilmthatgovernstheformationofLF82intracellularbacterialcommunities(IBC)inside phagolysosomes. The expression of the High Pathogenicity Island (HPI),allowingironcapturebytheYersiniabactinsiderophore,isessentialfortheformationof the IBCandLF82survivalwithinmacrophages.WeexploreHPI role inacidicpHgrowth.References[1]Demarre G, Prudent V, SchenkH, Rousseau E, BringerM-A, BarnichN, et al. (2019) The Crohn’sdisease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive,multiply and tolerate antibiotics within macrophages. PLoS Pathog 15(11): e1008123. https://doi-org.insb.bib.cnrs.fr/10.1371/journal.ppat.1008123



P38. Postharvest antimicrobial treatments with organic acids toimprovetheshelflifeoffreshblueberries

PlesoianuAlinaMadalina1,TutulescuFelicia2,NourVioleta2

1DunareadeJos”UniversityofGalati,FacultyofFoodScienceandEngineering,“DomneascaStreet111,800201 Galati, Romania ; 2 University of Craiova, Department of Horticulture & Food Science, 13A.I.CuzaStreet,200585,Craiova,Romania([email protected])Fresh highbush blueberries(VacciniumcorymbosumL.)areone of the most popularsoft fruits characterised by attractive sensorial attributes and by high antioxidantpotentialdue to their richness inphenolics, especiallyanthocyanins [1,2].Theyarehighlyperishableastheyaresusceptibletovariousmicrobialinfections,bothpre-andpostharvest,theirshelflifebeingestimatedbetween35and40days[3].Thepresentresearchwasconductedtoinvestigatetheeffectsofpostharvesttreatmentswithcitric(2%), benzoic (0.2%) and sorbic (0.2%) acids on physicochemical, biochemical andmicrobiologicalevolutioninfreshblueberriesundercoldstorageconditions.Sampleswere evaluated initially and at 7-day interval for dry matter, total soluble solids,titratable acidity, total phenolic content, total flavonoid content, antioxidant activityand microbial load for six weeks storage time. Chemical treatments significantlyreduced thedevelopmentofmicroorganismson thesurfaceof the fruits throughoutthestorageperiodascomparedtothecontrolsamples,buttheycausedasignificantincrease inmoisture loss(sorbicacid>benzoicacid>citricacid>water),probablydue to the partial damage of the natural cuticular wax layer covering the fruit.Antimicrobial effects of chemical treatments were more noticeable than theirbiochemical effects. The results showed that chemical treatments did not affect thetotal phenolics, total flavonoids and antioxidant activity since no significantdifferenceswereobservedamongtreatmentsandcontrol.References[1]GiovanelliG.,BrambillaA.,RizzoloA.,SinelliN. (2012).Effectsofblanchingpre-treatment andsugar composition of the osmotic solution on physicochemical, morphological and antioxidantcharacteristicsofosmodehydratedblueberries(VacciniumcorymbosumL.).FoodRes.Int.49,263-271.[2]MozeS.,PolakT.,GasperlinL.,KoronD.,VanzoA.,PoklarUlrihN.,AbramV.(2011).Phenolicsin Slovenian bilberries (VacciniummyrtillusL.) and blueberries (Vaccinium corymbosum L.). J. Agric.FoodChem.59,6998-7004.[3]PolsS.,WilliamsE.,BotesA.,Vries,F.(2019).Developmentofnovelpostharvesttechniquesforthepreservationofblueberryfruit.ActaHortic.1256,575-582.



P39.Roleofmitochondrialphospholipidsinaceticacid-inducedcelldeath

VítorMartins1*,TâniaFernandes1*,DianaLopes2,RosárioDomingues2,ManuelaCôrte-Real1andMariaJoãoSousa1

1CentreofMolecularandEnvironmentalBiology(CBMA)/DepartmentofBiology,UniversityofMinho,CampusdeGualtar,4710-057Braga,Portugal;2MassSpectrometryCentre,UIQOPNA/DepartmentofChemistry,UniversityofAveiro,CampusUniversitáriodeSantiago,3810-193Aveiro,Portugal*authorscontributedequallyinthisworkYeastcelldeath inducedbyaceticacidstresshasbeencorrelatedwithaplethoraofcell death effectorswhosemammalian orthologues have been themselves linked tothe apoptotic process [1]. Most recently, we identified the endoplasmic reticulum-mitochondria encounter structure (ERMES) components as players in acetic acid-induced apoptosis, with deletion mutants displaying a prominent delay in theappearanceof severalapoptoticmarkers [2].Additionally,we found thatabsenceofERMES induces changes inmitochondrial phospholipid profile, and that acetic acidtreatment in bothwild-type andmutant strainswas accompaniedbymitochondrialphospholipid remodeling. Indeed, mitochondrial phospholipids such as cardiolipinandphosphatidylserinehavealreadybeen linkedtomammalianapoptosis [3,4],butsofartherearenostudiesregardingtheroleoftheselipidsinaceticacid-inducedcelldeath.Hence,weusedyeaststrainsdeficientineitherthecardiolipinsynthaseCrd1p,the lyso-phosphatidylcholine acyltransferase Taz1p, the phosphatidic acid transferproteinUps1por thephosphatidylserinesynthaseCho1p.Resultsshowasignificantdelay in loss of cell survival and plasma membrane integrity, as well as inmitochondrialdepolarizationanddegradation,andinsuperoxideanionaccumulation,supportinga role forphospholipidmetabolism inyeast celldeath inducedbyaceticacid.[1]Sousa,M.J., Ludovico,P.,Rodrigues,F., Leão,C. andCôrte-Real,M. "StressandCellDeath inYeastinducedbyaceticacid"inCellHomeostasisandStressResponse,PaulaBubulya(Ed.),2012,ISBN:978-953-307-978-3,InTech,DOI:10.5772/27726.[2]Martins,V.M.,Fernandes,T.R.,Lopes,D.,Afonso,C.B.,Domingues,M.R.,Côrte-Real,M.,&Sousa,M.J.(2019).ContactsinDeath:TheRoleoftheER–MitochondriaAxisinAceticAcid-InducedApoptosisinYeast.JournalofMolecularBiology,431(2),273-288.[3]X.X.Li,B.Tsoi,Y.F.Li,H.Kurihara,R.R.He,CardiolipinandItsDifferentPropertiesinMitophagyandApoptosis,J.Histochem.Cytochem.63(2015)301–311[4]K.Segawa,S.Nagata,AnApoptotic“EatMe”Signal:PhosphatidylserineExposure,TrendsCellBiol.25(2015)639–650.



P40. Adaptive response and tolerance of the yeast SaccharomycescerevisiaetostressinducedbysulfurdioxideunderlowpH:theroleofthetranscriptionfactorCom2

Patrícia Lage1,2, Belém Sampaio-Marques3,4, Paula Ludovico3,4, Nuno P Mira5*, AnaMendes-Ferreira1,2*

1Universidade de Trás-os-Montes e Alto Douro, Escola de Ciências da Vida e Ambiente; Vila Real,Portugal. 2BioISI - Biosystems & Integrative Sciences Institute, Campo Grande, Lisboa, Portugal.3Lifeand Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057Braga, Portugal. 4ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal.5Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Department ofBioengineering,UniversityofLisbon,Portugal.

During vinification Saccharomyces cerevisiae cells are frequently exposed tohigh concentrations of sulfur dioxide (SO2) that is used to avoid overgrowth ofunwantedbacteriaorfungipresentinthemust.UptonowthecharacterizationofthemolecularmechanismsbywhichS.cerevisiaerespondandtolerateSO2wasfocusedontheroleofthesulfiteeffluxpumpSsu1andinvestigationontheinvolvementofotherplayershasbeenscarce,especiallyatagenome-widelevel.Inthiswork,weuncoveredtheessential roleof thepoorlycharacterized transcription factorCom2 in toleranceandresponseofS.cerevisiaetostressinducedbySO2attheenologicallyrelevantpHof3.5. Transcriptomic analysis revealed that Com2 controls, directly or indirectly, theexpressionofmorethan80%ofthegenesactivatedbySO2,apercentagemuchhigherthan the one that could be attributed to any other stress-responsive transcriptionfactor. Large-scale phenotyping of the yeast haploid mutant collection led to theidentificationof50Com2-targetscontributingforprotectionagainstSO2includingallthegenesthatcomposethesulfatereductionpathway(MET3,MET14,MET16,MET5,MET10)andthemajorityofthegenesrequiredforbiosynthesisoflysine(LYS2,LYS21,LYS20, LYS14, LYS4, LYS5, LYS1 and LYS9) or arginine (ARG5,6, ARG4, ARG2, ARG3,ARG7,ARG8,ORT1andCPA1).OtheruncovereddeterminantsofresistancetoSO2(notunderthecontrolofCom2)includedgenesrequiredforfunctionandassemblyofthevacuolar proton pump and enzymes of the antioxidant defense, consistentwith theobservedcytosolicandmitochondrialaccumulationofreactionoxygenspeciesinSO2-stressedyeastcells.



P41. Effect of low pH in the denitrification pathway and nitrousoxidereductaseofMarinobacterhydrocarbonoclasticus

MartaS.P.Carepo1,CíntiaCarreira1,2,IsabelMoura1,SofiaR.Pauleta2

1Biological Chemistry Lab, LAQV, REQUIMTE, Departamento de Química, Faculdade de Ciências eTecnologia,UniversidadeNovadeLisboa,CampusdaCaparica,2829-516Caparica,Portugal.2MicrobialStress Lab, UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,Universidade Nova de Lisboa, Campus da Caparica, 2829-516 Caparica, Portugal.([email protected],[email protected])

Increasing atmospheric concentration of N2O has been a concern, as it is apotentgreenhousegasandpromotesozonelayerdestruction[1].ReleaseofN2Ofromsoils increases at low pH. Here,Marinobacter hydrocarbonoclasticus was grown inbatchmode in the presence of nitrate, to study the pH effect in the denitrificationpathway by gene expression profiling, quantification of nitrate and nitrite, andevaluate the ability of whole-cells to reduce NO and N2O [2]. At pH 6.5, nitriteaccumulatesinthemediaandthecellswereunabletoreduceN2O.TheamountofN2Oreductase at acidic pH was lower than that at pH 7.5 and 8.5, pinpointing a post-transcriptionalregulation,thoughpHdidnotaffectgeneexpressionofN2Oreductaseaccessory genes. The enzyme isolated fromacidic growthhas its catalytic center asCuZ*(4Cu1S),while theenzymefrompH7.5or8.5has itasCuZ(4Cu2S).ThisstudyevidencesaninvivosecondarylevelofregulationrequiredtomaintainN2Oreductaseinanactivestate.

Acknowledgments:WethankFundaçãoparaaCiênciaeTecnologiaforthefinancialsupport through the project PTDC/BIA-PRO/098882/2008 (SRP) and PTDC/BBB-BQB/0129/2014(IM),andthescholarshipSFRH/BD/87898/2012(CC).

References:[1]PauletaSR,CarepoMSP,MouraICoordChemRev.2019;387:436[2]CarreiraC,MestreO,NunesRF,MouraI,PauletaSR.PeerJ.2018;6:e5603.



P42. Pathway prospection for the implementation of microbe-based production of levulinic acid: a computational approach

AnaVila-Santa1,AhsanIslam2,FredericoCFerreira1,KristalaPrather3,NunoPMira1

1Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Department ofBioengineering, University of Lisbon, Portugal. 2Department of Chemical Engineering, LoughboroughUniversity, Loughborough, UK. 3Department of Chemical Engineering and Center for IntegrativeSyntheticBiology(CISB),MassachusettsInstituteofTechnology,Cambridge,USA.

LevulinicAcid(LA)isaC5carboxylicacidwithalargerangeofindustrialapplications,from cosmetic products to fuel additives[1]. This commodity chemical may beproducedthroughapetrochemicalrouteorviachemicalcatalysisusingpuresugarsor lignocellulosic residues as substrates, while the biological production of LA in abiorefinerycontexthasyettobeexplored[2].Inthisworkweusepathwaypredictionand retrobiosynthetic methodologies to pinpoint new-to-nature biochemical routesthat canbe imported into themodelorganismsEscherichia coli andSaccharomycescerevisiaeleadingtoLAproduction.Thecandidatepathwaysareselectedandfurtherevaluated in silicoon thebasisofparameters suchas theavailabilityofenzymes tocatalyze the required synthetic reaction steps, thermodynamics, pathway yield andpossibilitytocouplechemicalproductiontogrowth.Withthedescribedmethodologyfourcandidatenew-to-natureLAproducingpathwaysarereportedhere.

References

[1] D.EspositoandM.Antonietti,“Redefiningbiorefinery:thesearchforunconventionalbuildingblocksformaterials,”Chem.Soc.Rev.,vol.44,no.16,pp.5821–5835,2015.

[2] D.J.Hayes,J.Ross,M.H.B.Hayes,andS.Fitzpatrick,“TheBiofineprocess:productionoflevulinicacid,furfuralandformicacidfromlignocellulosicfeedstocks,”Biorefineries-IndustrialProcess.Prod.,vol.1,2005.

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