TheGlyceroltestkitisa simple,reliable,rapidandaccuratemethodforthemeasurementandanalysisofGlycerolinbeverages,foodstuffsandothermaterials.
Grapeandwineanalysis:Oenologiststoexploitadvancedtestkits.
Charnock,S.C.&McCleary,B.V.(2005).RevuedesEnology,117,1-5.
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Itiswithoutdoubtthattestingplaysapivotalrolethroughoutthewholeofthevinificationprocess.Toproducethebestpossiblequalitywineandtominimiseprocessproblemssuchas“stuck”fermentationortroublesomeinfections,itisnowrecognisedthatifpossibletestingshouldbeginpriortoharvestingofthegrapesandcontinuethroughtobottling.Traditionalmethodsofwineanalysisareoftenexpensive,timeconsuming,requireeitherelaborateequipmentorspecialistexpertiseandfrequentlylackaccuracy.However,enzymaticbio-analysisenablestheaccuratemeasurementofthevastmajorityofanalytesofinteresttothewinemaker,usingjustonepieceofapparatus,thespectrophotometer(seepreviousissueNo.116foradetailedtechnicalreview).Grapejuiceandwineareamenabletoenzymatictestingasbeingliquidstheyarehomogenous,easytomanipulate,andcangenerallybeanalysedwithoutanysamplepreparation.
Megazyme“advanced”winetestkitsgeneralcharacteristicsandvalidation.
Charnock,S.J.,McCleary,B.V.,Daverede,C.&Gallant,P.(2006).ReveuedesOenologues,120,1-5.
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ManyoftheenzymatictestkitsareofficialmethodsofprestigiousorganisationssuchastheAssociationofOfficialAnalyticalChemicals(AOAC)andtheAmericanAssociationofCerealChemists(AACC)inresponsetotheinterestfromoenologists.Megazymedecidedtouseitslonghistoryofenzymaticbio-analysistomakeasignificantcontributiontothewineindustry,bythedevelopmentofarangeofadvancedenzymatictestkits.Thistaskhasnowbeensuccessfullycompletedthroughthestrategicandcomprehensiveprocessofidentifyinglimitationsofexistingenzymaticbio-analysistestkitswheretheyoccurred,andthenusingadvancedtechniques,suchasmolecularbiology(photo1),torapidlyovercomethem.Noveltestkitshavealsobeendevelopedforanalytesofemerginginteresttotheoenologist,suchasyeastavailablenitrogen(YAN;seepages2-3ofissue117article),orwherepreviouslyenzymesweresimplyeithernotavailable,orweretooexpensivetoemploy,suchasforD-mannitolanalysis.
BiosynthesisofethanolandhydrogenbyglycerolfermentationusingEscherichiacoli.
Chaudhary,N.,Ngadi,M.O.,Simpson,B.K.&Kassama,L.S.(2011).AdvancesinChemicalEngineeringandScience,1,83-89.
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Productionofhighvalueproductsfromglycerolviaanaerobicfermentationisofutmostimportanceforthebiodieselindustry.ThemicroorganismEscherichiacoli(E.coli)K12wasusedforfermentationofglycerol.Theeffectsofglycerolconcentrationandheadspaceconditionsonthecellgrowth,ethanolandhydrogenproductionwereinvestigated.Afullfactorialexperimentaldesignwith3replicateswasconductedinordertotestthesefactors.Underthethreeheadspaceconditionstested,theincreaseofglycerolconcentrationacceleratedglycerolfermentation.Theyieldsofhydrogenandethanolwerethelowestwhenglycerolconcentrationof10g/Lwasused.Themaximumproductionofhydrogenwasobservedwithaninitialglycerolconcentrationof25g/Latafinalconcentrationofhydrogenwas32.15mmol/L.Thisstudydemonstratedthathydrogenproductionnegativelyaffectscellgrowth.Maximumethanolyieldwasobtainedwithaglycerolconcentrationof10g/Landwasupto0.40g/gglycerolundermembraneconditionheadspace.Statisticaloptimizationshowedthatoptimalconditionsforhydrogenproductionare20g/Linitialglycerolwithinitialspargingofthereactorheadspace.Theoptimalconditionsforethanolproductionare10g/Linitialglycerolwithmembrane.
TheproteomicresponseofSaccharomycescerevisiaeinveryhighglucoseconditionswithaminoacidsupplementation.
Pham,T.K.&Wright,P.C.(2008).JournalofProteomeResearch,7(11),4766-4774.
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EthanolyieldbySaccharomycescerevisiaeinveryhighglucose(VHG)mediawithanaminoacidsupplementwasinvestigated.Aminoacidsupplementationledtopositivecellresponses,includingreducedlagtimeandincreasedcellviabilityinVHGmedia.AquantitativeshotgunproteomicanalysiswasusedtounderstandhowaminoacidsupplementedS.cerevisiaerespondstohighosmoticconditions.iTRAQdatarevealedthatmostproteinsinvolvedinglycolysisandpentosephosphatepathwayswereup-regulatedunderhighglucoseshock.Reactivationofaminoacidmetabolismwasalsoobservedattheendofthelagphase.Therelativeabundanceofmostidentifiedproteins,includingaminoacyl-tRNAbiosynthesisproteins,andheat-shockproteins,remainedunchangedinthehoursimmediatelyfollowingapplicationofglucoseshock.However,theexpressionoftheseproteinsincreasedsignificantlyattheendofthelagphase.Furthermore,theup-regulationoftrehaloseandglycogenbiosynthesisproteins,firstmaintainingthenlatterlyincreasingglycolysispathwayactivitywasalsoobserved.Thiswasverifiedbyenhancedethanolyieldsat10and12h(0.43and0.45gethanol/gglucose)comparedto2h(0.32gethanol/gglucose).ThesedatacombinedwithrelevantmetabolitemeasurementsdemonstratesthatenhancedethanolfermentationunderVHGconditionscanbeachievedwiththeaidofaminoacidsupplementation.
Comparativetranscriptomicprofileanalysisoffed-batchculturesexpressingdifferentrecombinantproteinsinEscherichiacoli.
Sharma,A.K.,Mahalik,S.,Ghosh,C.,Singh,A.B.&Mukherjee,K.J.(2011).AMBExpress,1(1),33.
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Thereisaneedtoelucidatetheproductspecificfeaturesofthemetabolicstressresponseofthehostcelltotheinductionofrecombinantproteinsynthesis.Forthis,themethodofchoiceistranscriptomicprofilingwhichprovidesabetterinsightintothechangestakingplaceincomplexglobalmetabolicnetworks.Thetranscriptomicprofilesofthreefed-batchculturesexpressingdifferentproteinsviz.recombinanthumaninterferon-beta(rhIFN-β),XylanaseandGreenFluorescenceProtein(GFP)werecomparedpostinduction.Weobservedadepressioninthenutrientuptakeandutilizationpathways,whichwascommonforallthethreeexpressedproteins.ThusglyceroltransportersandgenesinvolvedinATPsynthesisaswellasaerobicrespirationwereseverelydown-regulated.Ontheotherhandtheaminoaciduptakeandbiosynthesisgenesweresignificantlyrepressedonlywhensolubleproteinswereexpressedunderdifferentpromoters,butnotwhentheproductwasexpressedasaninclusionbody(IB).HighlevelexpressionundertheT7promoter(rhIFN-βandxylanase)triggeredthecellulardegradationmachineryliketheosmoprotectants,proteasesandmRNAdegradationgeneswhichwerehighlyup-regulated,whilethistrendwasnottruewithGFPexpressionunderthecomparativelyweakerarapromoter.Thedesignofabetterhostplatformforrecombinantproteinproductionthusneedstotakeintoaccountthespecificnatureofthecellularresponsetoproteinexpression.
ProteomicanalysisofSaccharomycescerevisiaeunderhighgravityfermentationconditions.
Pham,T.K.,Chong,P.K.,Gan,C.S.&Wright,P.C.(2006).JournalofProteomeResearch,5(12),3411-3419.
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SaccharomycescerevisiaeKAY446wasutilizedforethanolproduction,withglucoseconcentrationsrangingfrom120g/L(normal)to300g/L(high).Althoughgrowninahighglucoseenvironment,S.cerevisiaestillretainedtheabilitytoproduceethanolwithahighdegreeofglucoseutilization.iTRAQ-mediatedshotgunproteomicswasappliedtoidentifyrelativeexpressionchangeofproteinsunderthedifferentglucoseconditions.Atotalof413proteinswereidentifiedfromthreereplicate,independentLC-MS/MSruns.Unsurprisingly,manyproteinsintheglycolysis/gluconeogenesispathwayshowedsignificantchangesinexpressionlevel.Twentyfiveproteinsinvolvedinaminoacidmetabolismdecreasedtheirexpression,whiletheexpressionsof12heat-shockrelatedproteinswerealsoidentified.Underhighglucoseconditions,ethanolwasproducedasamajorproduct.However,theassimilationofglucoseaswellasanumberofbyproductswasalsoenhanced.Therefore,tooptimizetheethanolproductionunderveryhighgravityconditions,anumberofpathwayswillneedtobedeactivated,whilestillmaintainingthecorrectcellularredoxorosmoticstate.Proteomicsisdemonstratedhereasatooltoaidinthisforwardmetabolicengineering.
Rapidmonitoringofglycerolinfermentationgrowthmedia:Facilitatingcrudeglycerolbioprocessdevelopment.
Abad,S.,Pérez,X.,Planas,A.&Turon,X.(2014).Talanta,121,210-214.
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Recently,theneedforcrudeglycerolvalorisationfromthebiodieselindustryhasgeneratedmanystudiesforpracticalandeconomicapplications.Amongstthem,fermentationsbasedonglycerolmediafortheproductionofhighvaluemetabolitesareprominentapplications.Thishasgeneratedaneedtodevelopanalyticaltechniqueswhichallowfastandsimpleglycerolmonitoringduringfermentation.Themethodologyshouldbefastandinexpensivetobeadoptedinresearch,aswellasinindustrialapplications.Inthisstudythreedifferentmethodswereanalysedandcompared:twocommonmethodologiesbasedonliquidchromatographyandenzymatickits,andthenewmethodbasedonaDotBlotassaycoupledwithimageanalysis.Thenewmethodologyisfasterandcheaperthantheotherconventionalmethods,withcomparableperformance.Goodlinearity,precisionandaccuracywereachievedinthelowerrange(10or15g/Ltodepletion),themostcommonrangeofglycerolconcentrationstomonitorfermentationsintermsofgrowthkinetics.
RepeatedbatchethanolicfermentationofveryhighgravitymediumbyimmobilizedSaccharomycescerevisiae.
Puligundla,P.,Poludasu,R.M.,Rai,J.K.&Obulam,V.S.R.(2011).AnnalsofMicrobiology,61(4),863-869.
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Themainobjectiveofthisstudywastoevaluatetheeffectofyeastimmobilizationonethanolicfermentationofveryhighgravity(VHG)mediumandtodeterminetheconcentrationsofyeaststoragecarbohydratesliketrehaloseandglycogenduringtheprocess.RepeatedbatchethanolicfermentationofVHGmediumwascarriedoutusingSaccharomycescerevisiaeimmobilizedseparatelywithinCa-alginateandκ-carrageenanpolymers.Immobilizationyields(Y1)werebetween80and90%andethanolyields(YP/S)weremorethan0.41withbothcarriers.Anaveragefermentationefficiencyofnearly70%wasobservedin48-hfermentationbatches.Comparedtofreecells,areductionofmorethan50%intheaccumulatedtrehalose,andatwo-foldincreaseinintracellularglycogenlevelswereobservedinimmobilizedyeastcellsat24and48hoffermentation,respectively,withbothcarriers.Theincreasedviability(uptofour-foldhigher)upon18%ethanoltreatmentfor2h,andthesustainedviabilityoverfoursuccessivebatchesofimmobilizedcellsshowedtheprotectivenatureofthepolymercarriers.Thechemicalnatureofthecarrierswasnotfoundtohaveanyadverseeffectonethanolyields.Applicationofimmobilizedyeastinporousmatricesmayserveasafeasibleandbettertechniqueforethanolproduction,atbothpilotandindustrialscale.
Methaneproductionviaanaerobicdigestionofglycerol:acomparisonofconventional(CSTR)andhigh‐rate(PABR)digesters.
Vlassis,T.,Stamatelatou,K.,Antonopoulou,G.&Lyberatos,G.(2013).JournalofChemicalTechnologyandBiotechnology,88(11),2000-2006.
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BACKGROUND:Biodieselisanalternativetofossilfuelsandcanbeuseddirectlyininternalcombustionengineswhenmixedwithdiesel.Theeconomicfeasibilityofbiodieselproductionnecessitatesthevalorisationofglycerol,whichisproducedinlargequantities(equalto10%ofthebiodieselproduced).Anaerobicdigestionisapplicabletoavarietyoforganicresiduesyieldingbiogasrichinmethane.Inordertoestimatethenetpotentialofglyceroltoyieldmethane,pureglycerolwasselectedtoavoidanyeffectfromtheimpuritiesincrudeglycerol.RESULTS:Theanaerobicdigestionofpureglycerolwasstudiedintwotypesofbioreactors:acontinuousstirredtankreactor(CSTR)andabaffledreactor(periodicanaerobicbaffledreactor,PABR).Bothreactorswereoperatedinmesophilicconditions(35°C)atvariousorganicloadingrates.ThemaximumglycerolloadingachievedinaCSTRwas0.25 gCODL-1d-1,yielding0.074 ± 0.009 LCH4L-1d-1.Ontheotherhand,PABRallowedglyceroldegradationataloadingof3 gCODL-1d-1yielding0.993 ± 0.102 LCH4L-1d-1.CONCLUSION:PABRwasprovedtobemoreefficientsinceitwassubjectedtoa10-foldhigherorganicloadingratethanCSTR.Moreover,itsperformancewasmuchhigherintermsofCODremovalandmethaneproductivity.
Drumdryingperformanceofcondenseddistillerssolublesandcomparisontothatofphysicallymodifiedcondenseddistillerssolubles.
Milczarek,R.R.&Liu,K.(2015).FoodandBioproductsProcessing,94,208-217.
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Condenseddistillerssolubles(CDS)isaviscous,syrupyco-productofethanolproductionfromcornorotherstarchygrains;CDSexhibitsstrongrecalcitrancetodryingduetoitschemicalcomposition,whichincludesasubstantialamountofglycerol.TheobjectivesofthisstudyweretodeterminethedrumdryingperformanceofCDSandtocompareittothatofaphysicallymodifiedCDS(MCDS)havingareducedglycerollevel.Materialtype(CDSvs.MCDS),dwelltime,druminternalsteamtemperature,andgapwidthwereevaluatedfortheireffectsonthefinalmoisturecontent,wateractivity,andcolorofthedriedproduct.WhilebothCDSandMCDScouldbedriedtoarangeofendpointmoisturecontents,driedCDSexhibitedanarrowrangeofwateractivitycomparedtothatofMCDS.Gapwidthwasfoundtobethepredominantfactoraffectingdriedproductcolor.ThisworkdemonstratesthatdrumdryingcaneffectivelyreducethemoisturecontentofCDS,eventhoughthewateractivityofthedriedproductcannotbereducedbeyond∼0.45.Incontrast,MCDScanbereadilydrum-driedintoashelf-stable,flakedproductwithapleasingappearance.
ComparisonofGlucose,GlycerolandCrudeGlycerolFermentationbyEscherichiaColiK12.
Chaudhary,N.,Ngadi,M.O.&Simpson,B.(2012).JournalofBioprocessing&Biotechniques.
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Hydrogenandethanolproductionfromglucose,glycerolandcrudeglycerolfermentationusingEscherichiacoliwasinvestigated.Crudeglycerolusedinthisstudycontained80%glycerol,2.6%ash,12.3%moisture,1.7%freefattyacid,3.4%MONG(matterorganicnon-glycerol),2519mg/kgsulphurand9000ppmsodium.Themaximumyieldofethanolfromcrudeglycerolof0.36g/g,correspondingtoanethanolconcentrationof3.6g/Lwasobtainedat10g/Linitialglycerolconcentration,5g/Ltryptoneconcentrationand100rpmmixingspeed.Comparableyieldswereobtainedatthemixingspeedsof150and200rpm.Oncomparison,thisyieldcorrespondedto105%oftheyield(0.34g/g)obtainedfrompureglycerolatthesameconditionsand85%ofthemaximumyield(0.42g/g)ofethanolobtainedfrompureglycerolat10g/Linitialglycerolconcentration,10g/Ltryptoneconcentrationand200rpmmixingspeed.Additionally,Escherichiacoligrowthforglycerolwascharacterizedandcomparedtothatforglucosewith10%substrateconcentrationat37°Cand200rpmmixingspeed.Thenetgrowthrateforglucoseandglycerolwere0.43and0.26h-1,respectively.Themaximumdryweightattainedforglucoseandglycerolwere0.12and0.04g/L,respectively.
FromcrudeglyceroltocarotenoidsbyusingaRhodotorulaglutinismutant.
Cutzu,R.,Coi,A.,Rosso,F.,Bardi,L.,Ciani,M.,Budroni,M.,Zara,G.,Zara,S.&Mannazzu,I.(2013).WorldJournalofMicrobiologyandBiotechnology,29(6),1009-1017.
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Inthisworkeighteenredyeastswerescreenedforcarotenoidsproductiononglycerolcontainingmedium.StrainC2.5t1ofRhodotorulaglutinis,thatshowedthehighestproductivity,wasUVmutagenized.Mutant400A15,thatexhibiteda280%increaseinβ–caroteneproductioninrespecttotheparentalstrain,wasselected.Acentralcompositedesignwasappliedto400A15tooptimizecarotenoidsandbiomassproductions.Regressionanalysesofthequadraticpolynomialequationsobtained(R2=0.87and0.94,forcarotenoidsandbiomass,respectively)suggestthatthemodelsarereliableandsignificant(P<0.0001)=""in=""the=""prediction=""of=""carotenoids=""and=""biomass=""productions=""on=""the=""basis=""of=""the=""concentrations=""of=""crude=""glycerol,=""yeast=""extract=""and=""peptone.=""accordingly,=""total=""carotenoids=""production=""achieved=""(14.07=""±=""1.45=""mg="">-1)underoptimizedgrowthconditionswasnotstatisticallydifferentfromthemaximalpredicted(14.64±1.57mgl-1)(P<0.05),=""and=""it=""was=""about=""100=""%=""higher=""than=""that=""obtained=""under=""un-optimized=""conditions.=""therefore=""mutant=""400a15=""may=""represent=""a=""biocatalyst=""of=""choice=""for=""the=""bioconversion=""of=""crude=""glycerol=""into=""value-added=""metabolites,=""and=""a=""tool=""for=""the=""valorization=""of=""this=""by-product=""of=""the=""biodiesel=""industry.="">
