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RESEARCHOpenAccessMetabolicfluxandtranscriptionalanalysiselucidatehigherbutanol/acetoneratiofeatureinABEextractivefermentationbyClostridiumacetobutylicumusingcassavasubstrateXinLi*,Zhi-GangLiandZhong-PingShiAbstractBackground:Inacetone-butanol-ethanol(ABE)fermentationbyClostridiumacetobutylicumATCC824usingcorn-basedsubstrate,thesolventsaregenerallyproducedataratioof3:6:1(A:B:E,w/w).
Results:Ahigherbutanol/acetoneratioof2.
9:1wasfoundwhencassavawasusedasthesubstrateofanin-situextractivefermentationbyC.
acetobutylicum.
Thisratiohada64%incrementcomparedtothatoncorn-basedsubstrate.
Themetabolicfluxand(keyenzymes)genestranscriptionalanalysisindicatedthatweakenedmetabolicfluxesinorganicacids(especiallybutyrate)formationandre-assimilationpathways,whichassociatedwithlowerbukandctfABtranscriptionallevels,contributedtohigherbutanolandloweracetoneproductionrateinfermentationsoncassava.
Moreover,NADHgenerationwasenhancedundertheenrichedreductiveenvironmentofusingcassava-basedsubstrate,whichconvertedmorecarbonfluxtobutanolsynthesispathway,alsoleadingtoahigherratioofbutanol/acetone.
Tofurtherincreasebutanol/acetoneratio,tinyamountofelectroncarrier,neutralredwassupplementedintocassava-basedsubstrateat60hwhenbutonalproductionratereachedmaximallevel.
However,neutralredadditionenhancedNADHproduction,followedwithstrengtheningthemetabolicfluxesoforganicacidsformation/re-assimilationpathways,resultedinunchangedinbutanol/acetoneratio.
Conclusions:Afurtherincreaseinbutanol/acetoneratiocouldberealizedwhenNADHregenerationwasenhancedandthemetabolicfluxesinorganicacidsformation/reutilizationrouteswerecontrolledatsuitablylowlevelssimultaneously.
Keywords:ABEfermentation;Butanol/acetoneratio;Cassava;Metabolicflux;TranscriptionalanalysisBackgroundClostridiumacetobutylicum,aGram-positive,spore-form-ing,andobligateanaerobe,hastheabilitytoproducesol-ventswithrenewablebiomassesincludingacetone,butanol,andethanol[1].
Inacetone-butanol-ethanol(ABE)fermen-tationbyC.
acetobutylicumusingcorn-basedsubstrate,thesolventsaregenerallyproducedataratioof3:6:1(A/B/E,w/w).
Amongthesesolvents,butanolhasthemostattrac-tionsinceithasbeenconsideredasahigh-performancebiofuel,aswellasanimportantplatformchemical.
How-ever,highsubstrate(suchascorn)priceandtoomuchpurificationcostduetoverylowsolventconcentrationsarethetwomajorfactorsimpactingoneconomicsofbutanolproduction[2].
AcostsheetfromanABEfermentationplantusingcornindicatedthatthesubstratepriceaccountedforupto79%oftheoverallproductioncost,whileenergycostconsumedinproductdistillationalmostcontributedtherest14%oftheentirecost[3].
Therefore,seekingcheaperfeedstockandincreasingbutanolratiointotalsolventshavebecomethemajorchallengesfortheeconomicviabilityofABEfermentation.
Somelow-pricedagricultureresiduesforexamplecornfiberandwheatbranhavebeenusedinABEfermentationbyclostridia,butbutanolconcentrationandproductivityinthesefermentationsaremuchlowerthanthoseincorn-*Correspondence:lixin084@163.
comKeyLaboratoryofIndustrialBiotechnology,MinistryofEducation,JiangnanUniversity,Wuxi214122,People'sRepublicofChina2014Lietal.
;licenseeSpringer.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Lietal.
BioresourcesandBioprocessing2014,1:13http://www.
bioresourcesbioprocessing.
com/content/1/1/13basedfermentation[4-7].
Cassava,anon-grainandhighstarchcontentcrop,isrecognizedasaneconomicalandpracticalsubstrateforindustrialfermentation.
Insomepre-viousstudies,cassavawassuccessfullyusedinsteadofcornassubstrateinABEfermentationbyC.
acetobutylicum,andhigherbutanol/acetoneratioswereobservedwhenferment-ingoncassavaascomparedwiththesameproceduresusingcorn[8,9].
Inthesecases,theonlinemonitoredpa-rameters(pH,ORP,H2/CO2ratio,etc.
)andorganicacidformation/reassimilationpatternswerequitedifferentfromthoseoncorn-basedsubstrate.
Butthemechanismofhigherbutanolratiosincassava-basedfermentationhasstillnotbeenillustratedclearly,fortheactivitiesofkeyenzymesinvivoweredifficultorimpossibletobemeasured,causedbyhardlyseparatingcellsfrommixedsolidresiduesofsubstrate.
Someeffortshaveinvestigatedthespecialfeaturespre-sentedinABEfermentationbyC.
acetobutylicum,usingeithermetabolicfluxanalysisortranscriptionalanalysis[10-12].
Metabolicfluxanalysisisasystematicapproachdevelopedtoevaluateeachindividualreactionratewithinametabolicnetwork.
Investigationongenetictrans-criptionallevelsdirectlycorrelatestheactivitiesofrelevantenzymes.
Tosolvetheproblemsmentionedabove,themethodsofmetabolicfluxanalysiswascombinedwithgenes(keyenzymes)transcriptionalmeasurementstoexplorethemechanismofhigherbutanol/acetoneratiofeatureincassava-basedfermentation.
TraditionalbatchprocessisstillthemostcommonlyusedoperationmodeinindustrialABEfermentations.
However,itissufferedwithseverebutanolend-inhibitionleadingtoashortfermentationperiod,sothatinterpretingmanyattractivephenomenabecomesdifficult.
Bycontrast,in-situextract-ivefermentationcouldrelievebutanolinhibitoryeffecttoimprovefermentationproductivityandtoprolongfer-mentationtime[13-15].
Thein-situextractivefermen-tationtechniqueisnotwidelyusedinindustrialABEfermentationbecauseofthehighextractantcostandoper-ationcomplexity.
However,itcouldbeusedasanimport-antprototypeforinvestigatingvariouscharacteristicsofABEfermentation,andguidingtheoptimaloperationwaysofABEtraditionalfermentation.
Amongvariousin-situfermentationextractantsforbutanol,oleylalcoholhasbeenrecognizedasthebestonebecauseofitsnon-toxicitytocellgrowthandhighbutanolextractionco-efficient[16].
Inthisstudy,ABEextractivefermentationsbyC.
acetobutylicumATCC824wereconductedina7-Lanaerobicfermentator,undertheconditionsofusingdif-ferentbiomasssubstrate(cornorcassava).
Combinationalanalysisofmetabolicfluxdistributionandgenetranscrip-tionallevelswerecarriedouttofindoutthevariationsinintracellularcarbondistributionsandtranscriptionallevelswhenusingcorn-orcassava-basedsubstrate.
Alltheseef-fortsaimedtoclarifythemechanismofhigherbutanol/acetoneratioobtainedwhenusingcassava-basedmediumandexploretheoptimaloperationwayfortraditionalABEfermentationcharacterizedwithhighbutanol/acetoneratio.
MethodsMicroorganismC.
acetobutylicumATCC824wasusedinthisstudy.
Thestrainwasmaintainedassporesuspensionin5%cornmealmediumat4°C.
Themethodsofinoculationandpre-culturefollowedthosedescribedintheliteratures[17,18].
Substrate(media)preparationandin-situfermentativeextractantpretreatmentThecornflour(rawstarchcontentabout50%w/w)wasob-tainedatlocalmarketandcassavapowder(rawstarchcon-tentabout65%to70%w/w)wasprovidedbyHenanTianguanFuelEthanolCo.
Ltd.
,Nanyang,China.
Themediawerepretreatedbyaddingcertainamountofα-amylase(8U/g-cornorcassava,heatedinboilingwaterbathfor45min)andthenglucoamylase(120U/g-cornorcassava,heatedat62°Cfor60min).
Subsequently,theviscosity-reducedmediawereautoclavedat121°Cfor20min.
Oleylalcohol(TokyoKaseiCo.
Ltd.
,Tokyo,Japan)wasusedastheextractantforin-situextractivefermenta-tion.
Oleylalcoholwaseithersterilizedat121°Cfor20minordirectlyusedwithoutsterilization,andthenaddedintothefermentor.
Whenusingcassavaassubstrate,thecon-centratedyeastextractsolutionwassterilizedat115°Cfor30min,andthenpumpedintothebrothuponrequirementsinceithasbeenrevealedinthepreviousstudythatyeastextractcouldpromotethephaseshiftinABEfermentationwithcassavasubstrate[9].
Neutralredwasdissolvedinster-ilizedwaterandpumpedintothebrothat60h.
FermentationmethodandconditionSeedculturewascarriedoutin100-mLanaerobicfermen-tationbottlesusingcornasthesubstrate.
Theinitialcornorcassavamealcontentforextractivefermentationswas30%or25%(w/v).
Thefermentationswereconductedina7-Lstaticfermentor(BaoxingBioengineeringCo.
,Shanghai,China)equippedwithpHandORP(oxidative-reductivepo-tential)electrodesandamanualadjustedpressureunit.
Atemperature-controllablewaterbath(MP-10,ShanghaiPer-manentScienceandTechnology,Co.
,Shanghai,China)wasusedtocirculatehotwaterintothecoilpipessettledinsidethefermentortomaintainbrothtemperatureat37°C.
Thefermentationmediumloadingvolumerangedfrom1.
8to2.
5L,andequivalentvolumeofoleylalcoholwasaddedtoensurea1:1oil/brothvolumetricratio.
N2wasspargedintotheextractantreservoirfor10mintoremoveresidualoxy-gen.
10%(v/v)inoculumwastransferredintothefermentorandthenN2wasalsospargedintobrothfor10min.
Theoxygen-freeoleylalcoholwaspouredintothefermentorusingaperistalticpumpafterinoculation.
TheinitialLietal.
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com/content/1/1/13pressureinsidethefermentorwascontrolledatabout0.
02MPa(N2)tostrictlymaintaintheanaerobiccondition.
Thepressuregraduallyincreasedsincefermentationstartedandself-generatedgasbegantoevolve.
Thepressurewasthencontrolledinarangeof0.
030to0.
055MPathrough-outfermentation.
Agitationwasoccasionallyadoptedforashorttime(5min,400rpm)topromotebutanoldiffusionfromaqueousphaseintoextractantphase.
AnalyticalmethodsThemeasurementsofconcentrationsofsolvents,organicacidsandreducingsugar(glucose)werethesameasthosedescribedinourpreviousreports[17,18].
Onaccountofthevolumeratioofaqueoustoorganicphasebeingjust1:1inextractivefermentation,thetotalconcentrationofbuta-nol(oracetone)wasthesumofbutanol(oracetone)inbrothandinextractant.
H2/CO2ratioinexhaustgaswasdeterminedusingthesamemethodreportedinourprevi-ouswork[9].
Theconcentrationofimportantintermediate,butyraldehydeinbrothwasdeterminedbyagaschroma-tography(ShimadzuGC-2010,Kyoto,Japan)withflameionizationdetectorandDB-23capillarycolumn(60m*0.
25mmID*0.
32μm,Agilent,Sta.
Clara,CA,USA).
Theconditionwasdescribedasfollows:nitrogenwasusedasthecarriergasatavelocityof1.
2mL/min;thehydrogenandairflowrateswere47and400mL/min,respectively;injectortemperaturewasoperatedat200°C,anddetectortemperatureat250°C;theinitialtemperaturestayedat40°Cfor5min,andthenraisedatthevelocityof10°Cperminuntilarrivingto180°C,andfinallystayedforanother5min.
MetabolicfluxanalysisMetabolicfluxanalysisinvolvesthecalculation(orestima-tion)ofinvivofluxesfromsubstrateandproductdata,byusingasystemoflinearequationsdevelopedfromreactionstoichiometry[19].
Forthepurposeofmetabolicfluxdistri-butionanalysis,asimplifiedmetabolicreactionmodel(MR)wasdevelopedforbutanolsynthesisbyC.
acetobutylicumATCC824.
Thissimplifiedmodel(Figure1)coveredthebasicreactionsandoccurredintheglycolysispathway,or-ganicacidsformation/reutilizationroutes,solventsynthe-sisbranches,andtheelectrontransportshuttlesystem.
CellgrowthandATPsynthesiswerenotincludedinthemodel,sincethemetabolicfluxdistributionanalysiswasconductedinthesolventogenicphasewherecellgrowthalmostceasedandATPdemandwasless[20].
AsshowninFigure1andtheAppendix,theMRmodelcontained19metabolicreactionrates(k=19).
Amongtherates,sevenextracellularsubstanceratesweremeasurable(m=7)includingratesofglucoseconsumption,organicacidformationorre-assimilation,solventssynthesis,andhydrogenevolution.
AsshownintheAppendix,therewereatotalof13substances(n=13)coveringsubstrates,prod-uctsandintermediatemetabolites,andthus13massbalanceequationswereavailable.
Thus,thisMRmodelisanoverdeterminedsystem(n=13>k-m=12).
Alloftheunknownreactionratescouldbeoptimallydeterminedusingthemeasurableratedataandthestoichiometricco-efficientsofthemetabolicreactionmatrixwiththeaidofthecalculationpackageembeddedinMatlab(Ver.
R2010b,MathWorksInc.
,Natick,MA,USA)[21].
Thefollowingtreatmentswereappliedinthenetworkmodelcalculation:(1)glucosewascalculatedinthemodelassin-glecarbonresourcesinceitwasthemostpreferredforstrainandhighestpercentageinthesecomplexmediums;(2)glucoseconsumptionrateswerenormalizedas100mmol/(Lh),andtheothermeasurablerateswererecalculatedusingtheabove(glucose)normalizationcoefficient;(3)pseudo-steadystateassumptionwasadoptedforintracellularintermediatemetabolites.
RNApurification,cDNAsynthesis,andreal-timefluorescencequantitativePCRanalysisTotalbacterialRNAwasextractedusingTrizolPlusRNAPurificationKit(Invitrogen,LifeTechnologiesCorp.
,GrandIsland,NY,USA).
BeforestartingRNAextraction,allthesampleswerepercolatedthroughfilterpapertore-movecassava/cornresidues.
1mLTrizolReagentwasaddedtowetcellforlysingcellanddisassociatingproteinfromnucleicacid.
Then,0.
2mLofchloroformwassup-pliedintothehomogenateliquidfollowingwithavigorousshaking,inordertoextracttheproteinoutofaqueousphase.
Aftertransferringtheaqueousphasetoanewtube,100%isopropanol(0.
5mL)wasaddedtoseparateRNAoutoftheaqueousphase.
Itmustbenotedthatusingtheap-propriateprecautionstoavoidRNasecontaminationwhenpreparingandhandlingRNA.
TotalRNAwasusedasthetemplatetosynthesizecDNA,andthencDNAproductswereamplifiedbythemethodofreal-timefluorescencequantitativePCRwiththeprimerslistedinTable1.
Thefol-lowingPCRconditionswereadopted:aninitialdenatur-ationstepat95°Cfor10min,followedbyanamplificationandquantificationprogramrepeatingfor40cycles(95°Cfor10s,60°Cfor60swithasinglefluorescencemeasure-ment),andameltingcurveprogram(acontinuousfluores-cencemeasurementraisingtemperaturefrom60°Cto95°Cwithaslowheatingunit).
ResultsFermentationperformancescomparisonwhenusingcornorcassavaassubstrateFigure2showstheextractivefermentationperformances(witholeylalcoholasextractant)whenusingeithercornorcassavaassubstrate.
ThechangepatternsofpHandtotalgasproductionyieldsnosignificantdistinction(Figure2a).
However,ORPandH2/CO2ratioforthetwocases(Figure2b)arequitedifferent.
ORPincassava-basedbrothreached520mVinthesolventogenicLietal.
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com/content/1/1/13phaseoffermentation,whichwasvisiblylowerthanthatofcorn-basedbroth(470mV).
H2/CO2ratiostayedatalowlevelof0.
2to0.
7whenculturingwithcassava-basedsubstrate,whilefluctuatingintherangeof0.
6to2.
5incorn-basedfermentation.
Themaximumbutyrateaccumulationincassavabrothwas1.
2g/L,onlyhalfofwhichincornbroth.
Furthermore,afterenteringsolventogenicphase,acetateandbutyratere-assimilationratesweresignificantlyslowerincassava-basedfermentation.
Thefinalsolventconcentrations(includingbutanolandacetoneinbothextractiveandaqueousphase.
ethanolwasnotaccountedforduetolowaccumulation)reachedatotallevelof50g/Linbothcases,inwhichbuta-nol/acetoneratiowas2.
87usingcassava-basedsubstrateand1.
75withcorn.
Allthesedistinctions,particularlybu-tanol/acetoneandH2/CO2ratios,wereimportantinun-derstandingtheoptimalregulationforABEfermentation.
Theirmechanismsshouldbeproperlyinterpreted.
MetabolicfluxanalysisinABEextractivefermentationswithcornandcassavaHighbutanol/acetoneratioisdesirableforABEfermen-tation.
Itreachednearly2.
9whenusingcassava-basedsubstrate,a64%enhancementcomparedtocorn.
Toun-derstandthisresult,metabolicfluxanalysiswasconductedtoexplorethemechanisms.
AsshowninFigure1,themetabolicfluxesofbutanolproductionbyC.
actobutylicumATCC824mainlyinclu-desthemetabolicreactionratesofrAc-CoAandrBy-CoAincentralbutanolsynthesisrouteafteracetyl-CoAnode,rAcHandrByHinacetate/butyrateformationroutes,rACE-AcandrACE-Byinacetonesynthesisbranch,rBtOHinbuta-nolproductionbranch,rEtOHinethanolformationbranchandrNADH(NADHgenerationrate)intheelectrontrans-portshuttlesystem.
Themetabolicfluxesduringthesol-ventogenicphase(40to100h)aredepictedinFigure3.
Asshowninthefigure,whenfermentingoncorn-basedsubstrate,acetateandethanolformationfluxes(rAcHandrEtOH)aremuchhigherthanthoseoncassava-basedsub-stratethroughoutthesolventogenicphase,resultinginaTable1Primersequencesusedinthereal-timefluorescencequantitativePCRGenePrimesequencesFragmentsize(bp)16SRNAF:5′CTGGACTGTAACTGACGCTGA3′80R:5′CGTTTACGGCGTGGACTAC3′ctfABF:5′CAGAAAACGGAATAGTTGGAATG3′151R:5′TGACCACCACGGATTAGTGAA3′adhEF:5′GTTTTGGCTATGTATGAGGCTGA3′241R:5′CAAGCGTGAAAGAAGGTGGTAT3′bdhBF:5′ACGCTTCTGCCATTCTATCC3′175R:5′ATTGCGGCACATCCAGATA3′askF:5′GTATGGGATTTACTCCTCTTGG3′63R:5′CTGGGTCCATATCTCCACTTC3′bukF:5′TCCGCCTTTGCCGTTTA3′194R:5′ACATGGGTGGAGGTACTTCAGT3′Figure1ThebasicmetabolicpathwayofacetoneandbutanolbiosynthesisbyClostridiumacetobutylicumATCC824.
Lietal.
BioresourcesandBioprocessing2014,1:13Page4of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13lesscarbonfluxinthecentralbutanolroutes(rAc-CoA).
Moreover,butyrateformationfluxofrByHwhenferment-ingcorn-basedsubstrateisalsosignificantlyhigher.
Thebutyrateformation(rByH)fluxisinverselyassociatedwiththefluxinbutanolproductionbranch(rBtOH)becausetheycompeteforcarbonresourceatbutyryl-CoAnode.
Therefore,ahigherrByH(inthecaseoffermentingcorn-basedsubstrate)ledtoalowerfluxinbutanolsynthesisbranch(rBtOH).
SinceacetoneformationbranchfluxisthesumofrACE-AcandrACE-By(shownintheAppendix),whenfermentingcassava-basedsubstrate,bothrACE-AcandrACE-Bydeclinedsharplyafter55h,resultinginaloweracetonesynthesisflux.
Inaddition,thereductivepowerNADH,anextremelyimportantcofactorinbutanolsynthesis,alsomayhavecontributiontotheextentofbutanol/acetoneratio.
NADHismainlyproducedfromtwopathways,theglycolysisandtheelectrontransportshuttlesystem.
Inthisstudy,theglucoseconsumptionamountandrateusingtwodifferentsubstrateshadnearlythesamechangepatterns(showninFigure2c),whichmeantthattheNADHproducedfromtheglycolysiswereequivalentbetweenfermentationsoncornandcassava.
However,theNADHgeneratedfromtheelectrontrans-portshuttlesystemhadvisiblydifferentproductionratesintwocases.
AsshowninFigure3,rNADHstaysathighlevelsduringmostperiodofthesolventogenicphasewhenusingcassava-basedsubstrate.
Correspondently,fluxesintheNADH-dependentpathwaysofbutyryl-CoAandbuta-nolformations(rBy-CoAandrBtOH)arelargelyenhancedinthecaseoffermentingcassava.
InordertoverifytheNADHbalance,thedifferencevaluesbetweenNADHpro-duction(2*rgly+rNADH)andutilization(2*rEtOH+2*rBy-CoA+2*rBtOH)werecalculated.
Theresultindicatedthatthevalueswerealmostintherangefrom5to5,accountingforlessthan5%ofNADHproduction(orutilization),whichconfirmedthereliabilityofmetabolic01020304050607080901001104.
04.
55.
05.
56.
0pH(-)Fermentationtime(h)a0102030405060708090100110-560-540-520-500-480-460-440bORP(mV)Fermentationtime(h)0102030405060708090100110050100150200cConcentrationofglucose(g/L)Fermentationtime(h)012345Concentrationsoforganicacids(g/L)01020304050607080901001100510152025303540dConcentrationsofsolventproducts(g/L)Fermentationtime(h)0.
00.
51.
01.
52.
02.
53.
0Concentrationofbutyraldehyde(mg/L)0102030405060708090Gasproduction(L/L-broth)0.
00.
51.
01.
52.
02.
53.
0H2/CO2(-)Figure2Performancecomparisonsofbutanolfermentationsusingcorn-(opensymbolsandbrokenlines)andcassava-basedsubstrates(filledsymbolsandsolidlines).
(a)pH(line)andgasproduction(circle).
(b)ORP(line)andH2/CO2ratio(square).
(c)Acetate(square),butyrate(circle),andglucoseconcentrations(triangle).
(d)Totalbutanolconcentration(square),totalacetoneconcentration(circle),andbutyraldehydeconcentrationinbroth(triangle).
Lietal.
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Allofthemetabolicfluxanalysisresultsupportsthefeatureofhighbutanol/acetoneratiowhenadoptingcassavasubstrate.
GenetranscriptionalanalysisforABEextractivefermentationswithcornandcassavaThereal-timefluorescencequantitativePCRwasalsoconductedtofurtherinterpretthemechanismofhighbutanol/acetoneratioincassava-basedfermentation.
Ac-cordingtobutanolsynthesismapshowninFigure1,acetatekinaseandbutyratekinaseresponsibleforacetateandbutyratesynthesisrespectivelyareencodedbythegenesofaskandbuk;CoA-transferaseencodedbyctfABisinchargeofacidreutilizationcouplingwithacetoneformation;butyraldehydedehydrogenaseandbutanolde-hydrogenaseencodedbyadhEandbdhBarethekeyenzymesinthefinaltwostepsofbutanolproduction.
TheresultsofgenetranscriptionalanalysisaredepictedinFigure4.
Variationinthetranscriptionallevelofaskduringthesolventogenicphasewaslimitedregardlessofthesub-stratetypes.
However,thetranscriptionallevelofbukde-creasedsignificantlyinfermentationusingcassava-basedsubstrate,whilestayingratherhighincorn-basedfermen-tation.
TranscriptionallevelofctfABreachedthemaximallevelat79hwhenusingcorn-basedsubstrate,whichwasapproximatelysevenfoldhigherthanthatincassava-basedfermentation.
Likewise,themaximalvaluesofadhEandbdhBappearedat79hwhenfermentingoncorn,whichwererespectivelyeightfoldandtwofoldhigherthanthoseoncassava.
Attemptoffurtherbutanol/acetoneratioenhancementbyaddingneutralredinABEfermentationwithcassavaNeutralred,anelectroncarrier,couldforceelectronflowdirectionchange.
Addingatinyamountofneutralredcouldweakenhydrogenformationwhichoriginatedfromtheelectronshuttletransportsystem,leadingtoanenhancementinNADHproduction.
Inordertofurther4050607080901001100510152025303540rEtOH(-)40506070809010011051015202530rAcH(-)4050607080901001101012141618202224262830rACE-Ac(-)405060708090100110051015202530354045rACE-By(-)4050607080901001105101520253035rByH(-)405060708090100110160170180190200210220rAc-CoA(-)4050607080901001103035404550556065707580rBy-CoA(-)40506070809010011030405060708090100rBtOH(-)Fermentationtime(h)405060708090100110020406080100rNADH(-)Figure3MetabolicfluxanalysisofClostridiumacetobutylicumATCC824whenusingcorn-andcassava-basedsubstrates.
Corn-basedsubstrates(opensymbolsandbrokenlines);cassava-basedsubstrates(filledsymbolsandsolidlines).
Lietal.
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350.
400.
450.
50c0.
00000.
00050.
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00150.
00200.
0025bFermentationtime(h)TranscriptionallevelofbdhB(-)TranscriptionallevelofadhE(-)Transcriptionallevelofbuk(-)Transcriptionallevelofask(-)264468792644687926446879a0.
0000.
0020.
0040.
0060.
0080.
0100.
0000.
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0020.
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18TranscriptionallevelofctfAB(-)Figure4Changesintranscriptionallevelsofkeygenesincorn-andcassava-basedfermentations.
Corn-basedfermentation(whiteandparallelshadowbars);cassava-basedfermentation(blackandslashedshadowbars).
(a)Transcriptionallevelsofask(blackandwhite)andbuk(shadow).
(b)TranscriptionallevelofctfAB.
(c)TranscriptionallevelsofadhE(blackandwhite)andbdhB(shadow).
Lietal.
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bioresourcesbioprocessing.
com/content/1/1/13increasethebutanol/acetoneratio,neutralred(0.
1%w/v-broth)wasaddedintocassavabrothat60h,whenbutanolproductionwasinarelativelyhighrate.
Asexpected,rNADHwasvisiblyelevatedandthensustainedathigherlevelsafterneutralredaddition(Figure5d).
Moreover,anincrementofbutyraldehydeconcentrationwasobserved,whichsuggeststhatbutanolsynthesispathwaywasenhanced.
Infact,butanolproductionwastrulyincreasedandreached43g/L(Figure5c).
However,butanol/acetoneratiowasnotimprovedbyaddingneutralred.
Metabolicfluxanalysisindicatedthattheratesofor-ganicacidformationandre-assimilationwerepromotedafterneutralredaddition,althoughtheconcentrationsofacidsinbrothhadnotanapparentchange.
Acetonepro-ductionwasconsequentlypromoted.
Therefore,simultan-eousincreaseinbothbutanolandacetoneproductionledtoanunchangedbutanol/acetoneratiointhiscase.
Table2summarizesthefermentationperformanceunderdif-ferentruns.
Figure6comparesthetranscriptionallevelsofgenesen-codingthekeyenzymesinbutanolsynthesiswith/withoutneutralredsupplement.
Afterneutralredaddition,thetranscriptionallevelsofctfABandadhE,responsibleforacetoneandbutanolsynthesisrespectively,wereappar-entlyenhanced.
Thisresultwasbasicallyinaccordancewiththemetabolicfluxanalysisresultsmentionedabove.
DiscussionThebutanol/acetoneratioreachedamuchhigherlevelof2.
87inextractivefermentationwithcassavasubstrate,a64%incrementascomparedtousingcornsubstrate(Table2).
Itwasclosetothereportedratioobtainedinfer-mentationsbyahyper-butanolstrainofC.
acetobutylicumEA2018withcorn-basedmedium[22].
ThepreliminaryanalysistofermentationperformancesshowedthatORPandH2/CO2ratiowerebothatlowlevelswhenusingcas-sava(Figure2).
ORPcouldbeconsideredasacompre-hensiveindexreflectingpH,dissolvedoxygen,reductivepotentialofcompoundsdissolvedinmedium[23].
TheABEfermentationrequiredananaerobicenvironmentsothatdissolvedoxygeninbrothcouldbeignored.
ChangepatternsofpHwerebasicallysimilarwhenusingdifferentsubstrates.
Thus,thelowerORPsuggestedthatcassava-basedmediumwasrichinreductivesubstances.
Ontheotherhand,H2andCO2arethetwomajorcomponentsintheexhaustgasemittedbyclostridia.
H2isgeneratedfromtheelectrontransportshuttlesystemviathereactionof2H++2e→H2catalyzedbyhydrogenase[24].
CO2ismainlyproducedinreactionofPyruvate→Acetyl-CoAassociatedwithformationofreductiveferredoxin,theelectrondonorforhydrogenorNADHgeneration[25].
Therefore,lowerH2/CO2ratioimpliedthatmoreelectronflowsweredistributedtoNADHproductionintheelec-trontransportshuttlesystem.
Basedonthepreliminaryanalysisresults,metabolicfluxandgenetranscriptionalanalysiswereconductedtoverifytheassumptionandtoelucidatethemechanismabouthighbutanol/acetoneratioincassava-basedfer-mentation.
ThemetabolicfluxanalysisrevealedthatNADHwastrulygeneratedmoreunderthecassava-basedenvironment(Figure3),whichwasconsistentwiththepreliminaryanalysisresults.
Itshouldbeaddressedthatthegenes(adhEandbdhB)regulatingbutanolsyn-thesishadhighertranscriptionallevelsundercorn-basedenvironment,butmorebutanolproductionwasobtainedincassava-basedfermentation.
ThisfactdemonstratedthatNADHgenerationrateisoneofdominatedfactorscontrollingbutanol/acetoneratioinABEfermentationsbyC.
acetobutylicum.
Besides,metabolicfluxofbutyrateclosed-loop(rByHandrACE-By)incassava-basedfermen-tationwaslargelyweakenedaftershiftingintosolvento-genicphase.
Correspondently,transcriptionallevelsofbukandctfAB(responsibleforbutyrateformationandre-assimilationrespectively)weremuchlowerthanthoseincorn-basedfermentation.
InmetabolicpathwayofC.
acetobutylicum,butyrateformationandre-assimilationreactionconstituteaclosed-loop(butyrateloop)atbutyryl-CoAnode(Figure1).
Thebutyrateloopnotonlycompeteswithbutanolsynthesisrouteforcarbonresource,butalsorelatestoacetoneformation.
Therefore,highbuta-nol/acetoneratioincassava-basedfermentationwasalsoattributedtoweakenedmetabolicstrengthofbutyrateclosed-loop.
ItcouldbeconcludedthathigherNADHgenerationrateandlowermetabolicfluxinbutyrateclosed-loopworkedjointly,leadingtothehighbutanol/acetoneratiofeatureinfermentationwithcassava-basedsubstrate.
ThealterationofredoxbalancetopromoteNADHgenerationhasbeenreportedinmanystudiesusingcornorglucoseassubstrate.
Themethodsofprovisionofartificialelectroncarrierssuchasneutralred[26,18]andmethylviologen[27-29],ORPregulation[30],andinhibitionofhydrogenasebysprayingcarbonmonoxide[31,32],allofthemhaveeffectonenhancingbutanol/acet-oneratio.
Amongtheseapproaches,addingneutralredseemstobethemostappropriateoneoflow-costandeasytooperate.
Inthepreviousworks,ithasbeendemon-stratedthataddingneutralredcouldacquirea63%incre-mentinbutanol/acetoneratiowithcorn-basedsubstrate[26,18].
Therefore,neutralredwasaddedintocassavabrothat60hwhenbutanolproductionratewasinarelativelyhighlevel,inordertofurtherenhancebuta-nol/acetoneratio.
Theresultsindicatedthatfinalbutanolconcentrationcouldslightlyincrease,duetotheenhance-mentsinrNADHandadhEtranscriptionallevelafterneu-tralredaddition.
However,themetabolicfluxesoforganicacidformation/re-assimilationpathwayandctfABtran-scriptionlevelwerealsoenhancedaftersupplementingLietal.
BioresourcesandBioprocessing2014,1:13Page8of13http://www.
bioresourcesbioprocessing.
com/content/1/1/134050607080901001100510152025303540arAcH(-)d40506070809010011005101520253035rACE-Ac(-)405060708090100110051015202530354045rACE-By(-)40506070809010011005101520253035rByH(-)405060708090100110020406080100120140rNADH(-)4050607080901001100102030405060708090100rBtOH(-)Fermentationtime(h)010203040506070809010011001234Concentrationsoforganicacids(g/L)0102030405060708090100110020406080100120140160180200Concentrationofglucose(g/L)0102030405060708090100110012345678Concentrationofbutyraldehyde(mg/L)Fermentationtime(h)0246810121416Concentrationofacetone(g/L)051015202530354045cbConcentrationofbutanol(g/L)Figure5FermentationperformancesandmetabolicfluxanalysisofClostridiumacetobutylicumATCC824whenusingcassava-basedsubstrate.
Thesubstrateiseitherwith(filledsymbolsandsolidlines)orwithoutneutralredaddition(opensymbolsandbrokenlines).
(a)Acetate(square)andbutyrateconcentrations(circle).
(b)Glucose(square)andtotalacetoneconcentrations(circle).
(c)Butyraldehydeconcentrationinbroth(square)andtotalbutanolconcentration(circle).
(d)Metabolicfluxesincellsupplementedwithorwithoutneutralred.
Table2FermentationperformanceundervariousoperationmodeswithcornandcassavasubstratesFermentationcharacteristicsSubstrateCornCassavaCassava+NeutralredNumberofexperiments222Fermentationtime(h)100100100Totalbutanol(g/L)33.
26±2.
6137.
48±3.
1142.
60±2.
02Totalacetone(g/L)18.
98±1.
3913.
06±0.
9314.
59±0.
60Butanol/acetoneratio()1.
75±0.
012.
87±0.
032.
92±0.
02Acetate(g/L)Max.
3.
20±0.
602.
94±0.
272.
80±0.
03Final1.
40±0.
221.
24±0.
341.
60±0.
08Butyrate(g/L)Max.
2.
58±0.
341.
27±0.
161.
44±0.
28Final0.
39±0.
060.
41±0.
120.
51±0.
15Gasproduction(L/L-broth)80.
00±6.
1372.
37±5.
3764.
86±0.
11Butanolproductivity(g/L/h)0.
33±0.
020.
37±0.
030.
42±0.
02Lietal.
BioresourcesandBioprocessing2014,1:13Page9of13http://www.
bioresourcesbioprocessing.
com/content/1/1/130.
00000.
00050.
00100.
00150.
0020Transcriptionallevelofask(-)0.
0000.
0010.
0020.
0030.
0040.
0050.
006Transcriptionallevelofbuk(-)0.
000.
030.
060.
090.
120.
150.
180.
210.
24TranscriptionallevelofadhE(-)0.
0000.
0010.
0020.
0030.
0040.
0050.
006aTranscriptionallevelofbdhB(-)cbFermentationtime(h)0.
000.
010.
020.
030.
040.
050.
060.
07264468792644687926446879TranscriptionallevelofctfAB(-)Figure6Changesintranscriptionallevelsofkeygenesinfermentationsusingcassava-basedsubstrate.
Thesubstrateiseitherwith(blackandslashedshadowbars)orwithoutneutralredaddition(whiteandparallelshadowbars).
(a)Transcriptionallevelsofask(blackandwhite)andbuk(shadow).
(b)TranscriptionallevelofctfAB.
(c)TranscriptionallevelsofadhE(blackandwhite)andbdhB(shadow).
Lietal.
BioresourcesandBioprocessing2014,1:13Page10of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13neutralred(Figures5and6),whichwasactuallybeneficialforacetoneformation.
Thesimultaneousenhancementofbothbutanolandacetonesynthesisrouteledtoanun-changedbutanol/acetoneratioincassava-basedfermen-tationwithneutralredaddition.
Itwasspeculatedthatunderthereductivecompound-enrichedenvironmentusingcassava-basedsubstrate,reductivepowerNADHmighthavebeenexcessivelyproduced/consumedleadingtoaburdenoncellularmetabolism[33].
TomatchuprelativelylowfluxesoftheacidloopswithenhancedNADHregenerationinanappropriatewaywillbethekeyissueinobtainingfurtherhighbutanol/acetoneratiowhilemaintainingcomparablyhighbutanolproductivity.
Inadditiontostrengthofthereductivepower,manyotherapproacheshavebeenadoptedtoacquirehighbutanol/acetoneratio.
Amongtheseefforts,somemodifi-cationstometabolicpathwayswereobtainedgoodeffectonincreasingbutanol/acetoneratio.
Harrisetal.
usedmetabolicengineeringtoolstorestrainbuk(encodingbutyratekinase)expressionandresultedinasignificantincreaseinbutanol/acetoneratio(3.
8)[34].
Jangetal.
furtherstrengthenedthebutanolsynthesispathwaybyup-regulatingadhEexpression,onthebasisoftheweakenedacidformationpathway,whichachievedaratherhighbutanol/acetoneratio(8.
8)[35].
However,someotherattemptstoregulatepathwayshadtotallydifferentresults.
Lehmannetal.
soughttoconstructapta/ptbdoubleknockoutstrainofC.
acetobutylicumbutfailedtofindanypositiveclones[36].
TummalaattemptedtodownregulatetheexpressionofctfAB,butobservedhighorganicacidaccumulationinbrothwithlowsolventproduction[37].
Itcouldbefoundthatalltheseeffortstoextendthebuta-nol/acetoneratiowereinvolvedinthemodificationsofacidformationpathways.
Itisknownthatacetateandbutyrateformationpathwaysarethemajorenergysub-stance(ATP)productionrouteinC.
acetobutylicum.
Asobligateanaerobes,clostridiaareratherinefficientinen-ergyproduction.
Soanirreversiblechangeonthemajorenergysubstance(ATP)productionroutewouldinevitablybringadverseimpactoncellgrowthandsolventproduc-tion.
Inthisstudy,a64%incrementinbutanol/acetoneratiowasobtainedbychangingcorntoacheaperfeedstockcassava,withoutirreversibledamagestocellorcostin-crease(theadditionalamountofyeastextractwasratherless,andthecostofcassava-addedyeastextractwasevenmuchlowerthanthatofcorn).
Moreover,mostofthemechanismanalysisresearchesinthepastwerebasedonconcisecultureenvironmentbyusingadefinedmedium.
Therefore,thismechanismresearchbasedonbiomasssubstratewasnecessaryforachievinghighbutanol/acetoneratiounderindustrialABEfermentationcondition.
Inaddition,itwasnoteworthythatwhyhigherNADHgenerationrateandlowermetabolicfluxofbutyrateclosed-loopappearedincassava-basedcultureenvironmentnotcornWhatspecialchemicalcompositionsofcassavare-sultedinthesephenomenaAimingtoillustratetheabovequestions,anotherresearchisnowcarriedoutbyus.
Cur-rently,itisdiscoveredthatwhencarbon/nitrogenratio(C/Nratio)inthesubstrateisincreasedfrom46.
7to186.
7mol/mol,acidformationisvisiblyrestrainedinthesolventogenicphase,leadingtoa28%reductioninacet-oneproduction,butnoadverseimpactonbutanolpro-duction.
Moreover,H2generationdroppedoffrapidly,associatedwithC/Nratioincrease,whichimpliedthathighC/NratiocouldlimittheelectrondirectingtoH2synthesisandcontributetoenhanceNADHproduction.
TheseresultsindicatedthathigherC/Nratiocontainedincassava(118.
8mol/mol)comparedtocorn(21.
9mol/mol)maybeoneofthefactorsleadingtohigherbutanol/acet-oneratio.
Besides,therearestillotherpotentialfactorsneededtobefurtherconfirmed.
Thetoxiccompoundscontainedincassava,whichmayhavecausedtherestraintofsomeenzymeactivitiessuchasCoA-transferorbutyr-atekinase,isanotableaspectandneedstobeexploreddeeply.
ConclusionsAhigherbutanol/acetoneratioofapproximately2.
9:1wasobservedinextractivefermentationoncassava-basedsubstrate,whichhada64%incrementcomparedtothatoncorn-basedsubstrate.
Theresultsfrommeta-bolicfluxandtranscriptionalanalysisindicatedthattheweakenedmetabolicfluxesinorganicacids(especiallybutyrate)formation/re-assimilationpathways,aswellastheenhancementofNADHgeneration,contributedtothishigherbutanol/acetoneratiofeatureinfermentationoncassava.
Moreover,neutralredaddedincassavabrothcouldnotfurtherincreasebutanol/acetoneratio,whichdemonstratedthatafurtherhigherbutanol/acetoneratiocouldberealizedonlywhenNADHregenerationisen-hancedandthemetabolicfluxesinorganicacidforma-tion/reutilizationroutesarecontrolledatsuitablylowlevelssimultaneously.
AppendixMetabolicreactionsinABEfermentationbyClostridiumacetobutylicumATCC824rGlu(r1):Glucose→2Pyruvate+2NADH+2ATPrPyr(r2):Pyruvate→Acetyl-CoA+CO2+ReducedferredoxinrEtOH(r3):Acetyl-CoA+2NADH→EthanolrAcH(r4):Acetyl-CoA→Acetate+ATPrAc-CoA(r5):2Acetyl-CoA→Acetoacetyl-CoArACE-Ac(r6):Acetoacetyl-CoA+Acetate→Acetone+CO2+Acetyl-CoArACE-By(r7):Acetoacetyl-CoA+Butyrate→Acetone+CO2+Butyryl-CoALietal.
BioresourcesandBioprocessing2014,1:13Page11of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13rBy-CoA(r8):Acetoacetyl-CoA+2NADH→Butyryl-CoArByH(r9):Butyryl-CoA→Butyrate+ATPrBtOH(r10):Butyryl-CoA+2NADH→ButanolrH2(r11):Reducedferredoxin→H2rNADH(r12):Reducedferredoxin→NADHIntercelluarenzymesinC.
acetobutylicumATCC824E1,pyruvate-ferredoxinoxidoreductase;E2,ferredoxin-NADreductase;E3,hydrogenase;E4,phosphotransacety-lase;E5,acetatekinase(encodedbyask);E6,acetaldehydedehydrogenase;E7,ethanoldehydrogenase;E8.
thiolase;E9,CoA-transferase(encodedbyctfAB);E10,acetoacetatedecarboxylase;E11,β-hydroxybutyryl-CoAdehydroge-nase;E12,crotonase;E13,butyryl-CoAdehydrogenase;E14,phosphotransbutyrylase;E15,butyratekinase(encodedbybuk);E16,butyraldehydedehydrogenase(encodedbyadhE);E17,butanoldehydrogenase(encodedbybdhB).
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsXLcarriedoutthefermentationexperiment,performedthestatisticalanalysis,anddraftedthemanuscript.
Z-GLcarriedoutthefermentationexperiment.
Z-PSconceivedofthestudyandparticipatedinitsdesignandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThestudywassupportedbytheNationalNaturalScienceFoundationProgram(#20976072)andMajorStateBasicResearchDevelopmentProgram(#2007CB714303)ofChina.
TheauthorsalsoappreciatedtheassistancefromMr.
KevinDinginrefiningtheEnglishofthemanuscript.
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1186/s40643-014-0013-9Citethisarticleas:Lietal.
:Metabolicfluxandtranscriptionalanalysiselucidatehigherbutanol/acetoneratiofeatureinABEextractivefermentationbyClostridiumacetobutylicumusingcassavasubstrate.
BioresourcesandBioprocessing20141:13.
Submityourmanuscripttoajournalandbenetfrom:7Convenientonlinesubmission7Rigorouspeerreview7Immediatepublicationonacceptance7Openaccess:articlesfreelyavailableonline7Highvisibilitywithintheeld7RetainingthecopyrighttoyourarticleSubmityournextmanuscriptat7springeropen.
comLietal.
BioresourcesandBioprocessing2014,1:13Page13of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13
Results:Ahigherbutanol/acetoneratioof2.
9:1wasfoundwhencassavawasusedasthesubstrateofanin-situextractivefermentationbyC.
acetobutylicum.
Thisratiohada64%incrementcomparedtothatoncorn-basedsubstrate.
Themetabolicfluxand(keyenzymes)genestranscriptionalanalysisindicatedthatweakenedmetabolicfluxesinorganicacids(especiallybutyrate)formationandre-assimilationpathways,whichassociatedwithlowerbukandctfABtranscriptionallevels,contributedtohigherbutanolandloweracetoneproductionrateinfermentationsoncassava.
Moreover,NADHgenerationwasenhancedundertheenrichedreductiveenvironmentofusingcassava-basedsubstrate,whichconvertedmorecarbonfluxtobutanolsynthesispathway,alsoleadingtoahigherratioofbutanol/acetone.
Tofurtherincreasebutanol/acetoneratio,tinyamountofelectroncarrier,neutralredwassupplementedintocassava-basedsubstrateat60hwhenbutonalproductionratereachedmaximallevel.
However,neutralredadditionenhancedNADHproduction,followedwithstrengtheningthemetabolicfluxesoforganicacidsformation/re-assimilationpathways,resultedinunchangedinbutanol/acetoneratio.
Conclusions:Afurtherincreaseinbutanol/acetoneratiocouldberealizedwhenNADHregenerationwasenhancedandthemetabolicfluxesinorganicacidsformation/reutilizationrouteswerecontrolledatsuitablylowlevelssimultaneously.
Keywords:ABEfermentation;Butanol/acetoneratio;Cassava;Metabolicflux;TranscriptionalanalysisBackgroundClostridiumacetobutylicum,aGram-positive,spore-form-ing,andobligateanaerobe,hastheabilitytoproducesol-ventswithrenewablebiomassesincludingacetone,butanol,andethanol[1].
Inacetone-butanol-ethanol(ABE)fermen-tationbyC.
acetobutylicumusingcorn-basedsubstrate,thesolventsaregenerallyproducedataratioof3:6:1(A/B/E,w/w).
Amongthesesolvents,butanolhasthemostattrac-tionsinceithasbeenconsideredasahigh-performancebiofuel,aswellasanimportantplatformchemical.
How-ever,highsubstrate(suchascorn)priceandtoomuchpurificationcostduetoverylowsolventconcentrationsarethetwomajorfactorsimpactingoneconomicsofbutanolproduction[2].
AcostsheetfromanABEfermentationplantusingcornindicatedthatthesubstratepriceaccountedforupto79%oftheoverallproductioncost,whileenergycostconsumedinproductdistillationalmostcontributedtherest14%oftheentirecost[3].
Therefore,seekingcheaperfeedstockandincreasingbutanolratiointotalsolventshavebecomethemajorchallengesfortheeconomicviabilityofABEfermentation.
Somelow-pricedagricultureresiduesforexamplecornfiberandwheatbranhavebeenusedinABEfermentationbyclostridia,butbutanolconcentrationandproductivityinthesefermentationsaremuchlowerthanthoseincorn-*Correspondence:lixin084@163.
comKeyLaboratoryofIndustrialBiotechnology,MinistryofEducation,JiangnanUniversity,Wuxi214122,People'sRepublicofChina2014Lietal.
;licenseeSpringer.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
Lietal.
BioresourcesandBioprocessing2014,1:13http://www.
bioresourcesbioprocessing.
com/content/1/1/13basedfermentation[4-7].
Cassava,anon-grainandhighstarchcontentcrop,isrecognizedasaneconomicalandpracticalsubstrateforindustrialfermentation.
Insomepre-viousstudies,cassavawassuccessfullyusedinsteadofcornassubstrateinABEfermentationbyC.
acetobutylicum,andhigherbutanol/acetoneratioswereobservedwhenferment-ingoncassavaascomparedwiththesameproceduresusingcorn[8,9].
Inthesecases,theonlinemonitoredpa-rameters(pH,ORP,H2/CO2ratio,etc.
)andorganicacidformation/reassimilationpatternswerequitedifferentfromthoseoncorn-basedsubstrate.
Butthemechanismofhigherbutanolratiosincassava-basedfermentationhasstillnotbeenillustratedclearly,fortheactivitiesofkeyenzymesinvivoweredifficultorimpossibletobemeasured,causedbyhardlyseparatingcellsfrommixedsolidresiduesofsubstrate.
Someeffortshaveinvestigatedthespecialfeaturespre-sentedinABEfermentationbyC.
acetobutylicum,usingeithermetabolicfluxanalysisortranscriptionalanalysis[10-12].
Metabolicfluxanalysisisasystematicapproachdevelopedtoevaluateeachindividualreactionratewithinametabolicnetwork.
Investigationongenetictrans-criptionallevelsdirectlycorrelatestheactivitiesofrelevantenzymes.
Tosolvetheproblemsmentionedabove,themethodsofmetabolicfluxanalysiswascombinedwithgenes(keyenzymes)transcriptionalmeasurementstoexplorethemechanismofhigherbutanol/acetoneratiofeatureincassava-basedfermentation.
TraditionalbatchprocessisstillthemostcommonlyusedoperationmodeinindustrialABEfermentations.
However,itissufferedwithseverebutanolend-inhibitionleadingtoashortfermentationperiod,sothatinterpretingmanyattractivephenomenabecomesdifficult.
Bycontrast,in-situextract-ivefermentationcouldrelievebutanolinhibitoryeffecttoimprovefermentationproductivityandtoprolongfer-mentationtime[13-15].
Thein-situextractivefermen-tationtechniqueisnotwidelyusedinindustrialABEfermentationbecauseofthehighextractantcostandoper-ationcomplexity.
However,itcouldbeusedasanimport-antprototypeforinvestigatingvariouscharacteristicsofABEfermentation,andguidingtheoptimaloperationwaysofABEtraditionalfermentation.
Amongvariousin-situfermentationextractantsforbutanol,oleylalcoholhasbeenrecognizedasthebestonebecauseofitsnon-toxicitytocellgrowthandhighbutanolextractionco-efficient[16].
Inthisstudy,ABEextractivefermentationsbyC.
acetobutylicumATCC824wereconductedina7-Lanaerobicfermentator,undertheconditionsofusingdif-ferentbiomasssubstrate(cornorcassava).
Combinationalanalysisofmetabolicfluxdistributionandgenetranscrip-tionallevelswerecarriedouttofindoutthevariationsinintracellularcarbondistributionsandtranscriptionallevelswhenusingcorn-orcassava-basedsubstrate.
Alltheseef-fortsaimedtoclarifythemechanismofhigherbutanol/acetoneratioobtainedwhenusingcassava-basedmediumandexploretheoptimaloperationwayfortraditionalABEfermentationcharacterizedwithhighbutanol/acetoneratio.
MethodsMicroorganismC.
acetobutylicumATCC824wasusedinthisstudy.
Thestrainwasmaintainedassporesuspensionin5%cornmealmediumat4°C.
Themethodsofinoculationandpre-culturefollowedthosedescribedintheliteratures[17,18].
Substrate(media)preparationandin-situfermentativeextractantpretreatmentThecornflour(rawstarchcontentabout50%w/w)wasob-tainedatlocalmarketandcassavapowder(rawstarchcon-tentabout65%to70%w/w)wasprovidedbyHenanTianguanFuelEthanolCo.
Ltd.
,Nanyang,China.
Themediawerepretreatedbyaddingcertainamountofα-amylase(8U/g-cornorcassava,heatedinboilingwaterbathfor45min)andthenglucoamylase(120U/g-cornorcassava,heatedat62°Cfor60min).
Subsequently,theviscosity-reducedmediawereautoclavedat121°Cfor20min.
Oleylalcohol(TokyoKaseiCo.
Ltd.
,Tokyo,Japan)wasusedastheextractantforin-situextractivefermenta-tion.
Oleylalcoholwaseithersterilizedat121°Cfor20minordirectlyusedwithoutsterilization,andthenaddedintothefermentor.
Whenusingcassavaassubstrate,thecon-centratedyeastextractsolutionwassterilizedat115°Cfor30min,andthenpumpedintothebrothuponrequirementsinceithasbeenrevealedinthepreviousstudythatyeastextractcouldpromotethephaseshiftinABEfermentationwithcassavasubstrate[9].
Neutralredwasdissolvedinster-ilizedwaterandpumpedintothebrothat60h.
FermentationmethodandconditionSeedculturewascarriedoutin100-mLanaerobicfermen-tationbottlesusingcornasthesubstrate.
Theinitialcornorcassavamealcontentforextractivefermentationswas30%or25%(w/v).
Thefermentationswereconductedina7-Lstaticfermentor(BaoxingBioengineeringCo.
,Shanghai,China)equippedwithpHandORP(oxidative-reductivepo-tential)electrodesandamanualadjustedpressureunit.
Atemperature-controllablewaterbath(MP-10,ShanghaiPer-manentScienceandTechnology,Co.
,Shanghai,China)wasusedtocirculatehotwaterintothecoilpipessettledinsidethefermentortomaintainbrothtemperatureat37°C.
Thefermentationmediumloadingvolumerangedfrom1.
8to2.
5L,andequivalentvolumeofoleylalcoholwasaddedtoensurea1:1oil/brothvolumetricratio.
N2wasspargedintotheextractantreservoirfor10mintoremoveresidualoxy-gen.
10%(v/v)inoculumwastransferredintothefermentorandthenN2wasalsospargedintobrothfor10min.
Theoxygen-freeoleylalcoholwaspouredintothefermentorusingaperistalticpumpafterinoculation.
TheinitialLietal.
BioresourcesandBioprocessing2014,1:13Page2of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13pressureinsidethefermentorwascontrolledatabout0.
02MPa(N2)tostrictlymaintaintheanaerobiccondition.
Thepressuregraduallyincreasedsincefermentationstartedandself-generatedgasbegantoevolve.
Thepressurewasthencontrolledinarangeof0.
030to0.
055MPathrough-outfermentation.
Agitationwasoccasionallyadoptedforashorttime(5min,400rpm)topromotebutanoldiffusionfromaqueousphaseintoextractantphase.
AnalyticalmethodsThemeasurementsofconcentrationsofsolvents,organicacidsandreducingsugar(glucose)werethesameasthosedescribedinourpreviousreports[17,18].
Onaccountofthevolumeratioofaqueoustoorganicphasebeingjust1:1inextractivefermentation,thetotalconcentrationofbuta-nol(oracetone)wasthesumofbutanol(oracetone)inbrothandinextractant.
H2/CO2ratioinexhaustgaswasdeterminedusingthesamemethodreportedinourprevi-ouswork[9].
Theconcentrationofimportantintermediate,butyraldehydeinbrothwasdeterminedbyagaschroma-tography(ShimadzuGC-2010,Kyoto,Japan)withflameionizationdetectorandDB-23capillarycolumn(60m*0.
25mmID*0.
32μm,Agilent,Sta.
Clara,CA,USA).
Theconditionwasdescribedasfollows:nitrogenwasusedasthecarriergasatavelocityof1.
2mL/min;thehydrogenandairflowrateswere47and400mL/min,respectively;injectortemperaturewasoperatedat200°C,anddetectortemperatureat250°C;theinitialtemperaturestayedat40°Cfor5min,andthenraisedatthevelocityof10°Cperminuntilarrivingto180°C,andfinallystayedforanother5min.
MetabolicfluxanalysisMetabolicfluxanalysisinvolvesthecalculation(orestima-tion)ofinvivofluxesfromsubstrateandproductdata,byusingasystemoflinearequationsdevelopedfromreactionstoichiometry[19].
Forthepurposeofmetabolicfluxdistri-butionanalysis,asimplifiedmetabolicreactionmodel(MR)wasdevelopedforbutanolsynthesisbyC.
acetobutylicumATCC824.
Thissimplifiedmodel(Figure1)coveredthebasicreactionsandoccurredintheglycolysispathway,or-ganicacidsformation/reutilizationroutes,solventsynthe-sisbranches,andtheelectrontransportshuttlesystem.
CellgrowthandATPsynthesiswerenotincludedinthemodel,sincethemetabolicfluxdistributionanalysiswasconductedinthesolventogenicphasewherecellgrowthalmostceasedandATPdemandwasless[20].
AsshowninFigure1andtheAppendix,theMRmodelcontained19metabolicreactionrates(k=19).
Amongtherates,sevenextracellularsubstanceratesweremeasurable(m=7)includingratesofglucoseconsumption,organicacidformationorre-assimilation,solventssynthesis,andhydrogenevolution.
AsshownintheAppendix,therewereatotalof13substances(n=13)coveringsubstrates,prod-uctsandintermediatemetabolites,andthus13massbalanceequationswereavailable.
Thus,thisMRmodelisanoverdeterminedsystem(n=13>k-m=12).
Alloftheunknownreactionratescouldbeoptimallydeterminedusingthemeasurableratedataandthestoichiometricco-efficientsofthemetabolicreactionmatrixwiththeaidofthecalculationpackageembeddedinMatlab(Ver.
R2010b,MathWorksInc.
,Natick,MA,USA)[21].
Thefollowingtreatmentswereappliedinthenetworkmodelcalculation:(1)glucosewascalculatedinthemodelassin-glecarbonresourcesinceitwasthemostpreferredforstrainandhighestpercentageinthesecomplexmediums;(2)glucoseconsumptionrateswerenormalizedas100mmol/(Lh),andtheothermeasurablerateswererecalculatedusingtheabove(glucose)normalizationcoefficient;(3)pseudo-steadystateassumptionwasadoptedforintracellularintermediatemetabolites.
RNApurification,cDNAsynthesis,andreal-timefluorescencequantitativePCRanalysisTotalbacterialRNAwasextractedusingTrizolPlusRNAPurificationKit(Invitrogen,LifeTechnologiesCorp.
,GrandIsland,NY,USA).
BeforestartingRNAextraction,allthesampleswerepercolatedthroughfilterpapertore-movecassava/cornresidues.
1mLTrizolReagentwasaddedtowetcellforlysingcellanddisassociatingproteinfromnucleicacid.
Then,0.
2mLofchloroformwassup-pliedintothehomogenateliquidfollowingwithavigorousshaking,inordertoextracttheproteinoutofaqueousphase.
Aftertransferringtheaqueousphasetoanewtube,100%isopropanol(0.
5mL)wasaddedtoseparateRNAoutoftheaqueousphase.
Itmustbenotedthatusingtheap-propriateprecautionstoavoidRNasecontaminationwhenpreparingandhandlingRNA.
TotalRNAwasusedasthetemplatetosynthesizecDNA,andthencDNAproductswereamplifiedbythemethodofreal-timefluorescencequantitativePCRwiththeprimerslistedinTable1.
Thefol-lowingPCRconditionswereadopted:aninitialdenatur-ationstepat95°Cfor10min,followedbyanamplificationandquantificationprogramrepeatingfor40cycles(95°Cfor10s,60°Cfor60swithasinglefluorescencemeasure-ment),andameltingcurveprogram(acontinuousfluores-cencemeasurementraisingtemperaturefrom60°Cto95°Cwithaslowheatingunit).
ResultsFermentationperformancescomparisonwhenusingcornorcassavaassubstrateFigure2showstheextractivefermentationperformances(witholeylalcoholasextractant)whenusingeithercornorcassavaassubstrate.
ThechangepatternsofpHandtotalgasproductionyieldsnosignificantdistinction(Figure2a).
However,ORPandH2/CO2ratioforthetwocases(Figure2b)arequitedifferent.
ORPincassava-basedbrothreached520mVinthesolventogenicLietal.
BioresourcesandBioprocessing2014,1:13Page3of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13phaseoffermentation,whichwasvisiblylowerthanthatofcorn-basedbroth(470mV).
H2/CO2ratiostayedatalowlevelof0.
2to0.
7whenculturingwithcassava-basedsubstrate,whilefluctuatingintherangeof0.
6to2.
5incorn-basedfermentation.
Themaximumbutyrateaccumulationincassavabrothwas1.
2g/L,onlyhalfofwhichincornbroth.
Furthermore,afterenteringsolventogenicphase,acetateandbutyratere-assimilationratesweresignificantlyslowerincassava-basedfermentation.
Thefinalsolventconcentrations(includingbutanolandacetoneinbothextractiveandaqueousphase.
ethanolwasnotaccountedforduetolowaccumulation)reachedatotallevelof50g/Linbothcases,inwhichbuta-nol/acetoneratiowas2.
87usingcassava-basedsubstrateand1.
75withcorn.
Allthesedistinctions,particularlybu-tanol/acetoneandH2/CO2ratios,wereimportantinun-derstandingtheoptimalregulationforABEfermentation.
Theirmechanismsshouldbeproperlyinterpreted.
MetabolicfluxanalysisinABEextractivefermentationswithcornandcassavaHighbutanol/acetoneratioisdesirableforABEfermen-tation.
Itreachednearly2.
9whenusingcassava-basedsubstrate,a64%enhancementcomparedtocorn.
Toun-derstandthisresult,metabolicfluxanalysiswasconductedtoexplorethemechanisms.
AsshowninFigure1,themetabolicfluxesofbutanolproductionbyC.
actobutylicumATCC824mainlyinclu-desthemetabolicreactionratesofrAc-CoAandrBy-CoAincentralbutanolsynthesisrouteafteracetyl-CoAnode,rAcHandrByHinacetate/butyrateformationroutes,rACE-AcandrACE-Byinacetonesynthesisbranch,rBtOHinbuta-nolproductionbranch,rEtOHinethanolformationbranchandrNADH(NADHgenerationrate)intheelectrontrans-portshuttlesystem.
Themetabolicfluxesduringthesol-ventogenicphase(40to100h)aredepictedinFigure3.
Asshowninthefigure,whenfermentingoncorn-basedsubstrate,acetateandethanolformationfluxes(rAcHandrEtOH)aremuchhigherthanthoseoncassava-basedsub-stratethroughoutthesolventogenicphase,resultinginaTable1Primersequencesusedinthereal-timefluorescencequantitativePCRGenePrimesequencesFragmentsize(bp)16SRNAF:5′CTGGACTGTAACTGACGCTGA3′80R:5′CGTTTACGGCGTGGACTAC3′ctfABF:5′CAGAAAACGGAATAGTTGGAATG3′151R:5′TGACCACCACGGATTAGTGAA3′adhEF:5′GTTTTGGCTATGTATGAGGCTGA3′241R:5′CAAGCGTGAAAGAAGGTGGTAT3′bdhBF:5′ACGCTTCTGCCATTCTATCC3′175R:5′ATTGCGGCACATCCAGATA3′askF:5′GTATGGGATTTACTCCTCTTGG3′63R:5′CTGGGTCCATATCTCCACTTC3′bukF:5′TCCGCCTTTGCCGTTTA3′194R:5′ACATGGGTGGAGGTACTTCAGT3′Figure1ThebasicmetabolicpathwayofacetoneandbutanolbiosynthesisbyClostridiumacetobutylicumATCC824.
Lietal.
BioresourcesandBioprocessing2014,1:13Page4of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13lesscarbonfluxinthecentralbutanolroutes(rAc-CoA).
Moreover,butyrateformationfluxofrByHwhenferment-ingcorn-basedsubstrateisalsosignificantlyhigher.
Thebutyrateformation(rByH)fluxisinverselyassociatedwiththefluxinbutanolproductionbranch(rBtOH)becausetheycompeteforcarbonresourceatbutyryl-CoAnode.
Therefore,ahigherrByH(inthecaseoffermentingcorn-basedsubstrate)ledtoalowerfluxinbutanolsynthesisbranch(rBtOH).
SinceacetoneformationbranchfluxisthesumofrACE-AcandrACE-By(shownintheAppendix),whenfermentingcassava-basedsubstrate,bothrACE-AcandrACE-Bydeclinedsharplyafter55h,resultinginaloweracetonesynthesisflux.
Inaddition,thereductivepowerNADH,anextremelyimportantcofactorinbutanolsynthesis,alsomayhavecontributiontotheextentofbutanol/acetoneratio.
NADHismainlyproducedfromtwopathways,theglycolysisandtheelectrontransportshuttlesystem.
Inthisstudy,theglucoseconsumptionamountandrateusingtwodifferentsubstrateshadnearlythesamechangepatterns(showninFigure2c),whichmeantthattheNADHproducedfromtheglycolysiswereequivalentbetweenfermentationsoncornandcassava.
However,theNADHgeneratedfromtheelectrontrans-portshuttlesystemhadvisiblydifferentproductionratesintwocases.
AsshowninFigure3,rNADHstaysathighlevelsduringmostperiodofthesolventogenicphasewhenusingcassava-basedsubstrate.
Correspondently,fluxesintheNADH-dependentpathwaysofbutyryl-CoAandbuta-nolformations(rBy-CoAandrBtOH)arelargelyenhancedinthecaseoffermentingcassava.
InordertoverifytheNADHbalance,thedifferencevaluesbetweenNADHpro-duction(2*rgly+rNADH)andutilization(2*rEtOH+2*rBy-CoA+2*rBtOH)werecalculated.
Theresultindicatedthatthevalueswerealmostintherangefrom5to5,accountingforlessthan5%ofNADHproduction(orutilization),whichconfirmedthereliabilityofmetabolic01020304050607080901001104.
04.
55.
05.
56.
0pH(-)Fermentationtime(h)a0102030405060708090100110-560-540-520-500-480-460-440bORP(mV)Fermentationtime(h)0102030405060708090100110050100150200cConcentrationofglucose(g/L)Fermentationtime(h)012345Concentrationsoforganicacids(g/L)01020304050607080901001100510152025303540dConcentrationsofsolventproducts(g/L)Fermentationtime(h)0.
00.
51.
01.
52.
02.
53.
0Concentrationofbutyraldehyde(mg/L)0102030405060708090Gasproduction(L/L-broth)0.
00.
51.
01.
52.
02.
53.
0H2/CO2(-)Figure2Performancecomparisonsofbutanolfermentationsusingcorn-(opensymbolsandbrokenlines)andcassava-basedsubstrates(filledsymbolsandsolidlines).
(a)pH(line)andgasproduction(circle).
(b)ORP(line)andH2/CO2ratio(square).
(c)Acetate(square),butyrate(circle),andglucoseconcentrations(triangle).
(d)Totalbutanolconcentration(square),totalacetoneconcentration(circle),andbutyraldehydeconcentrationinbroth(triangle).
Lietal.
BioresourcesandBioprocessing2014,1:13Page5of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13fluxanalysis.
Allofthemetabolicfluxanalysisresultsupportsthefeatureofhighbutanol/acetoneratiowhenadoptingcassavasubstrate.
GenetranscriptionalanalysisforABEextractivefermentationswithcornandcassavaThereal-timefluorescencequantitativePCRwasalsoconductedtofurtherinterpretthemechanismofhighbutanol/acetoneratioincassava-basedfermentation.
Ac-cordingtobutanolsynthesismapshowninFigure1,acetatekinaseandbutyratekinaseresponsibleforacetateandbutyratesynthesisrespectivelyareencodedbythegenesofaskandbuk;CoA-transferaseencodedbyctfABisinchargeofacidreutilizationcouplingwithacetoneformation;butyraldehydedehydrogenaseandbutanolde-hydrogenaseencodedbyadhEandbdhBarethekeyenzymesinthefinaltwostepsofbutanolproduction.
TheresultsofgenetranscriptionalanalysisaredepictedinFigure4.
Variationinthetranscriptionallevelofaskduringthesolventogenicphasewaslimitedregardlessofthesub-stratetypes.
However,thetranscriptionallevelofbukde-creasedsignificantlyinfermentationusingcassava-basedsubstrate,whilestayingratherhighincorn-basedfermen-tation.
TranscriptionallevelofctfABreachedthemaximallevelat79hwhenusingcorn-basedsubstrate,whichwasapproximatelysevenfoldhigherthanthatincassava-basedfermentation.
Likewise,themaximalvaluesofadhEandbdhBappearedat79hwhenfermentingoncorn,whichwererespectivelyeightfoldandtwofoldhigherthanthoseoncassava.
Attemptoffurtherbutanol/acetoneratioenhancementbyaddingneutralredinABEfermentationwithcassavaNeutralred,anelectroncarrier,couldforceelectronflowdirectionchange.
Addingatinyamountofneutralredcouldweakenhydrogenformationwhichoriginatedfromtheelectronshuttletransportsystem,leadingtoanenhancementinNADHproduction.
Inordertofurther4050607080901001100510152025303540rEtOH(-)40506070809010011051015202530rAcH(-)4050607080901001101012141618202224262830rACE-Ac(-)405060708090100110051015202530354045rACE-By(-)4050607080901001105101520253035rByH(-)405060708090100110160170180190200210220rAc-CoA(-)4050607080901001103035404550556065707580rBy-CoA(-)40506070809010011030405060708090100rBtOH(-)Fermentationtime(h)405060708090100110020406080100rNADH(-)Figure3MetabolicfluxanalysisofClostridiumacetobutylicumATCC824whenusingcorn-andcassava-basedsubstrates.
Corn-basedsubstrates(opensymbolsandbrokenlines);cassava-basedsubstrates(filledsymbolsandsolidlines).
Lietal.
BioresourcesandBioprocessing2014,1:13Page6of13http://www.
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com/content/1/1/130.
000.
050.
100.
150.
200.
250.
300.
350.
400.
450.
50c0.
00000.
00050.
00100.
00150.
00200.
0025bFermentationtime(h)TranscriptionallevelofbdhB(-)TranscriptionallevelofadhE(-)Transcriptionallevelofbuk(-)Transcriptionallevelofask(-)264468792644687926446879a0.
0000.
0020.
0040.
0060.
0080.
0100.
0000.
0010.
0020.
0030.
0040.
0050.
0060.
0070.
0080.
0090.
0100.
000.
020.
040.
060.
080.
100.
120.
140.
160.
18TranscriptionallevelofctfAB(-)Figure4Changesintranscriptionallevelsofkeygenesincorn-andcassava-basedfermentations.
Corn-basedfermentation(whiteandparallelshadowbars);cassava-basedfermentation(blackandslashedshadowbars).
(a)Transcriptionallevelsofask(blackandwhite)andbuk(shadow).
(b)TranscriptionallevelofctfAB.
(c)TranscriptionallevelsofadhE(blackandwhite)andbdhB(shadow).
Lietal.
BioresourcesandBioprocessing2014,1:13Page7of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13increasethebutanol/acetoneratio,neutralred(0.
1%w/v-broth)wasaddedintocassavabrothat60h,whenbutanolproductionwasinarelativelyhighrate.
Asexpected,rNADHwasvisiblyelevatedandthensustainedathigherlevelsafterneutralredaddition(Figure5d).
Moreover,anincrementofbutyraldehydeconcentrationwasobserved,whichsuggeststhatbutanolsynthesispathwaywasenhanced.
Infact,butanolproductionwastrulyincreasedandreached43g/L(Figure5c).
However,butanol/acetoneratiowasnotimprovedbyaddingneutralred.
Metabolicfluxanalysisindicatedthattheratesofor-ganicacidformationandre-assimilationwerepromotedafterneutralredaddition,althoughtheconcentrationsofacidsinbrothhadnotanapparentchange.
Acetonepro-ductionwasconsequentlypromoted.
Therefore,simultan-eousincreaseinbothbutanolandacetoneproductionledtoanunchangedbutanol/acetoneratiointhiscase.
Table2summarizesthefermentationperformanceunderdif-ferentruns.
Figure6comparesthetranscriptionallevelsofgenesen-codingthekeyenzymesinbutanolsynthesiswith/withoutneutralredsupplement.
Afterneutralredaddition,thetranscriptionallevelsofctfABandadhE,responsibleforacetoneandbutanolsynthesisrespectively,wereappar-entlyenhanced.
Thisresultwasbasicallyinaccordancewiththemetabolicfluxanalysisresultsmentionedabove.
DiscussionThebutanol/acetoneratioreachedamuchhigherlevelof2.
87inextractivefermentationwithcassavasubstrate,a64%incrementascomparedtousingcornsubstrate(Table2).
Itwasclosetothereportedratioobtainedinfer-mentationsbyahyper-butanolstrainofC.
acetobutylicumEA2018withcorn-basedmedium[22].
ThepreliminaryanalysistofermentationperformancesshowedthatORPandH2/CO2ratiowerebothatlowlevelswhenusingcas-sava(Figure2).
ORPcouldbeconsideredasacompre-hensiveindexreflectingpH,dissolvedoxygen,reductivepotentialofcompoundsdissolvedinmedium[23].
TheABEfermentationrequiredananaerobicenvironmentsothatdissolvedoxygeninbrothcouldbeignored.
ChangepatternsofpHwerebasicallysimilarwhenusingdifferentsubstrates.
Thus,thelowerORPsuggestedthatcassava-basedmediumwasrichinreductivesubstances.
Ontheotherhand,H2andCO2arethetwomajorcomponentsintheexhaustgasemittedbyclostridia.
H2isgeneratedfromtheelectrontransportshuttlesystemviathereactionof2H++2e→H2catalyzedbyhydrogenase[24].
CO2ismainlyproducedinreactionofPyruvate→Acetyl-CoAassociatedwithformationofreductiveferredoxin,theelectrondonorforhydrogenorNADHgeneration[25].
Therefore,lowerH2/CO2ratioimpliedthatmoreelectronflowsweredistributedtoNADHproductionintheelec-trontransportshuttlesystem.
Basedonthepreliminaryanalysisresults,metabolicfluxandgenetranscriptionalanalysiswereconductedtoverifytheassumptionandtoelucidatethemechanismabouthighbutanol/acetoneratioincassava-basedfer-mentation.
ThemetabolicfluxanalysisrevealedthatNADHwastrulygeneratedmoreunderthecassava-basedenvironment(Figure3),whichwasconsistentwiththepreliminaryanalysisresults.
Itshouldbeaddressedthatthegenes(adhEandbdhB)regulatingbutanolsyn-thesishadhighertranscriptionallevelsundercorn-basedenvironment,butmorebutanolproductionwasobtainedincassava-basedfermentation.
ThisfactdemonstratedthatNADHgenerationrateisoneofdominatedfactorscontrollingbutanol/acetoneratioinABEfermentationsbyC.
acetobutylicum.
Besides,metabolicfluxofbutyrateclosed-loop(rByHandrACE-By)incassava-basedfermen-tationwaslargelyweakenedaftershiftingintosolvento-genicphase.
Correspondently,transcriptionallevelsofbukandctfAB(responsibleforbutyrateformationandre-assimilationrespectively)weremuchlowerthanthoseincorn-basedfermentation.
InmetabolicpathwayofC.
acetobutylicum,butyrateformationandre-assimilationreactionconstituteaclosed-loop(butyrateloop)atbutyryl-CoAnode(Figure1).
Thebutyrateloopnotonlycompeteswithbutanolsynthesisrouteforcarbonresource,butalsorelatestoacetoneformation.
Therefore,highbuta-nol/acetoneratioincassava-basedfermentationwasalsoattributedtoweakenedmetabolicstrengthofbutyrateclosed-loop.
ItcouldbeconcludedthathigherNADHgenerationrateandlowermetabolicfluxinbutyrateclosed-loopworkedjointly,leadingtothehighbutanol/acetoneratiofeatureinfermentationwithcassava-basedsubstrate.
ThealterationofredoxbalancetopromoteNADHgenerationhasbeenreportedinmanystudiesusingcornorglucoseassubstrate.
Themethodsofprovisionofartificialelectroncarrierssuchasneutralred[26,18]andmethylviologen[27-29],ORPregulation[30],andinhibitionofhydrogenasebysprayingcarbonmonoxide[31,32],allofthemhaveeffectonenhancingbutanol/acet-oneratio.
Amongtheseapproaches,addingneutralredseemstobethemostappropriateoneoflow-costandeasytooperate.
Inthepreviousworks,ithasbeendemon-stratedthataddingneutralredcouldacquirea63%incre-mentinbutanol/acetoneratiowithcorn-basedsubstrate[26,18].
Therefore,neutralredwasaddedintocassavabrothat60hwhenbutanolproductionratewasinarelativelyhighlevel,inordertofurtherenhancebuta-nol/acetoneratio.
Theresultsindicatedthatfinalbutanolconcentrationcouldslightlyincrease,duetotheenhance-mentsinrNADHandadhEtranscriptionallevelafterneu-tralredaddition.
However,themetabolicfluxesoforganicacidformation/re-assimilationpathwayandctfABtran-scriptionlevelwerealsoenhancedaftersupplementingLietal.
BioresourcesandBioprocessing2014,1:13Page8of13http://www.
bioresourcesbioprocessing.
com/content/1/1/134050607080901001100510152025303540arAcH(-)d40506070809010011005101520253035rACE-Ac(-)405060708090100110051015202530354045rACE-By(-)40506070809010011005101520253035rByH(-)405060708090100110020406080100120140rNADH(-)4050607080901001100102030405060708090100rBtOH(-)Fermentationtime(h)010203040506070809010011001234Concentrationsoforganicacids(g/L)0102030405060708090100110020406080100120140160180200Concentrationofglucose(g/L)0102030405060708090100110012345678Concentrationofbutyraldehyde(mg/L)Fermentationtime(h)0246810121416Concentrationofacetone(g/L)051015202530354045cbConcentrationofbutanol(g/L)Figure5FermentationperformancesandmetabolicfluxanalysisofClostridiumacetobutylicumATCC824whenusingcassava-basedsubstrate.
Thesubstrateiseitherwith(filledsymbolsandsolidlines)orwithoutneutralredaddition(opensymbolsandbrokenlines).
(a)Acetate(square)andbutyrateconcentrations(circle).
(b)Glucose(square)andtotalacetoneconcentrations(circle).
(c)Butyraldehydeconcentrationinbroth(square)andtotalbutanolconcentration(circle).
(d)Metabolicfluxesincellsupplementedwithorwithoutneutralred.
Table2FermentationperformanceundervariousoperationmodeswithcornandcassavasubstratesFermentationcharacteristicsSubstrateCornCassavaCassava+NeutralredNumberofexperiments222Fermentationtime(h)100100100Totalbutanol(g/L)33.
26±2.
6137.
48±3.
1142.
60±2.
02Totalacetone(g/L)18.
98±1.
3913.
06±0.
9314.
59±0.
60Butanol/acetoneratio()1.
75±0.
012.
87±0.
032.
92±0.
02Acetate(g/L)Max.
3.
20±0.
602.
94±0.
272.
80±0.
03Final1.
40±0.
221.
24±0.
341.
60±0.
08Butyrate(g/L)Max.
2.
58±0.
341.
27±0.
161.
44±0.
28Final0.
39±0.
060.
41±0.
120.
51±0.
15Gasproduction(L/L-broth)80.
00±6.
1372.
37±5.
3764.
86±0.
11Butanolproductivity(g/L/h)0.
33±0.
020.
37±0.
030.
42±0.
02Lietal.
BioresourcesandBioprocessing2014,1:13Page9of13http://www.
bioresourcesbioprocessing.
com/content/1/1/130.
00000.
00050.
00100.
00150.
0020Transcriptionallevelofask(-)0.
0000.
0010.
0020.
0030.
0040.
0050.
006Transcriptionallevelofbuk(-)0.
000.
030.
060.
090.
120.
150.
180.
210.
24TranscriptionallevelofadhE(-)0.
0000.
0010.
0020.
0030.
0040.
0050.
006aTranscriptionallevelofbdhB(-)cbFermentationtime(h)0.
000.
010.
020.
030.
040.
050.
060.
07264468792644687926446879TranscriptionallevelofctfAB(-)Figure6Changesintranscriptionallevelsofkeygenesinfermentationsusingcassava-basedsubstrate.
Thesubstrateiseitherwith(blackandslashedshadowbars)orwithoutneutralredaddition(whiteandparallelshadowbars).
(a)Transcriptionallevelsofask(blackandwhite)andbuk(shadow).
(b)TranscriptionallevelofctfAB.
(c)TranscriptionallevelsofadhE(blackandwhite)andbdhB(shadow).
Lietal.
BioresourcesandBioprocessing2014,1:13Page10of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13neutralred(Figures5and6),whichwasactuallybeneficialforacetoneformation.
Thesimultaneousenhancementofbothbutanolandacetonesynthesisrouteledtoanun-changedbutanol/acetoneratioincassava-basedfermen-tationwithneutralredaddition.
Itwasspeculatedthatunderthereductivecompound-enrichedenvironmentusingcassava-basedsubstrate,reductivepowerNADHmighthavebeenexcessivelyproduced/consumedleadingtoaburdenoncellularmetabolism[33].
TomatchuprelativelylowfluxesoftheacidloopswithenhancedNADHregenerationinanappropriatewaywillbethekeyissueinobtainingfurtherhighbutanol/acetoneratiowhilemaintainingcomparablyhighbutanolproductivity.
Inadditiontostrengthofthereductivepower,manyotherapproacheshavebeenadoptedtoacquirehighbutanol/acetoneratio.
Amongtheseefforts,somemodifi-cationstometabolicpathwayswereobtainedgoodeffectonincreasingbutanol/acetoneratio.
Harrisetal.
usedmetabolicengineeringtoolstorestrainbuk(encodingbutyratekinase)expressionandresultedinasignificantincreaseinbutanol/acetoneratio(3.
8)[34].
Jangetal.
furtherstrengthenedthebutanolsynthesispathwaybyup-regulatingadhEexpression,onthebasisoftheweakenedacidformationpathway,whichachievedaratherhighbutanol/acetoneratio(8.
8)[35].
However,someotherattemptstoregulatepathwayshadtotallydifferentresults.
Lehmannetal.
soughttoconstructapta/ptbdoubleknockoutstrainofC.
acetobutylicumbutfailedtofindanypositiveclones[36].
TummalaattemptedtodownregulatetheexpressionofctfAB,butobservedhighorganicacidaccumulationinbrothwithlowsolventproduction[37].
Itcouldbefoundthatalltheseeffortstoextendthebuta-nol/acetoneratiowereinvolvedinthemodificationsofacidformationpathways.
Itisknownthatacetateandbutyrateformationpathwaysarethemajorenergysub-stance(ATP)productionrouteinC.
acetobutylicum.
Asobligateanaerobes,clostridiaareratherinefficientinen-ergyproduction.
Soanirreversiblechangeonthemajorenergysubstance(ATP)productionroutewouldinevitablybringadverseimpactoncellgrowthandsolventproduc-tion.
Inthisstudy,a64%incrementinbutanol/acetoneratiowasobtainedbychangingcorntoacheaperfeedstockcassava,withoutirreversibledamagestocellorcostin-crease(theadditionalamountofyeastextractwasratherless,andthecostofcassava-addedyeastextractwasevenmuchlowerthanthatofcorn).
Moreover,mostofthemechanismanalysisresearchesinthepastwerebasedonconcisecultureenvironmentbyusingadefinedmedium.
Therefore,thismechanismresearchbasedonbiomasssubstratewasnecessaryforachievinghighbutanol/acetoneratiounderindustrialABEfermentationcondition.
Inaddition,itwasnoteworthythatwhyhigherNADHgenerationrateandlowermetabolicfluxofbutyrateclosed-loopappearedincassava-basedcultureenvironmentnotcornWhatspecialchemicalcompositionsofcassavare-sultedinthesephenomenaAimingtoillustratetheabovequestions,anotherresearchisnowcarriedoutbyus.
Cur-rently,itisdiscoveredthatwhencarbon/nitrogenratio(C/Nratio)inthesubstrateisincreasedfrom46.
7to186.
7mol/mol,acidformationisvisiblyrestrainedinthesolventogenicphase,leadingtoa28%reductioninacet-oneproduction,butnoadverseimpactonbutanolpro-duction.
Moreover,H2generationdroppedoffrapidly,associatedwithC/Nratioincrease,whichimpliedthathighC/NratiocouldlimittheelectrondirectingtoH2synthesisandcontributetoenhanceNADHproduction.
TheseresultsindicatedthathigherC/Nratiocontainedincassava(118.
8mol/mol)comparedtocorn(21.
9mol/mol)maybeoneofthefactorsleadingtohigherbutanol/acet-oneratio.
Besides,therearestillotherpotentialfactorsneededtobefurtherconfirmed.
Thetoxiccompoundscontainedincassava,whichmayhavecausedtherestraintofsomeenzymeactivitiessuchasCoA-transferorbutyr-atekinase,isanotableaspectandneedstobeexploreddeeply.
ConclusionsAhigherbutanol/acetoneratioofapproximately2.
9:1wasobservedinextractivefermentationoncassava-basedsubstrate,whichhada64%incrementcomparedtothatoncorn-basedsubstrate.
Theresultsfrommeta-bolicfluxandtranscriptionalanalysisindicatedthattheweakenedmetabolicfluxesinorganicacids(especiallybutyrate)formation/re-assimilationpathways,aswellastheenhancementofNADHgeneration,contributedtothishigherbutanol/acetoneratiofeatureinfermentationoncassava.
Moreover,neutralredaddedincassavabrothcouldnotfurtherincreasebutanol/acetoneratio,whichdemonstratedthatafurtherhigherbutanol/acetoneratiocouldberealizedonlywhenNADHregenerationisen-hancedandthemetabolicfluxesinorganicacidforma-tion/reutilizationroutesarecontrolledatsuitablylowlevelssimultaneously.
AppendixMetabolicreactionsinABEfermentationbyClostridiumacetobutylicumATCC824rGlu(r1):Glucose→2Pyruvate+2NADH+2ATPrPyr(r2):Pyruvate→Acetyl-CoA+CO2+ReducedferredoxinrEtOH(r3):Acetyl-CoA+2NADH→EthanolrAcH(r4):Acetyl-CoA→Acetate+ATPrAc-CoA(r5):2Acetyl-CoA→Acetoacetyl-CoArACE-Ac(r6):Acetoacetyl-CoA+Acetate→Acetone+CO2+Acetyl-CoArACE-By(r7):Acetoacetyl-CoA+Butyrate→Acetone+CO2+Butyryl-CoALietal.
BioresourcesandBioprocessing2014,1:13Page11of13http://www.
bioresourcesbioprocessing.
com/content/1/1/13rBy-CoA(r8):Acetoacetyl-CoA+2NADH→Butyryl-CoArByH(r9):Butyryl-CoA→Butyrate+ATPrBtOH(r10):Butyryl-CoA+2NADH→ButanolrH2(r11):Reducedferredoxin→H2rNADH(r12):Reducedferredoxin→NADHIntercelluarenzymesinC.
acetobutylicumATCC824E1,pyruvate-ferredoxinoxidoreductase;E2,ferredoxin-NADreductase;E3,hydrogenase;E4,phosphotransacety-lase;E5,acetatekinase(encodedbyask);E6,acetaldehydedehydrogenase;E7,ethanoldehydrogenase;E8.
thiolase;E9,CoA-transferase(encodedbyctfAB);E10,acetoacetatedecarboxylase;E11,β-hydroxybutyryl-CoAdehydroge-nase;E12,crotonase;E13,butyryl-CoAdehydrogenase;E14,phosphotransbutyrylase;E15,butyratekinase(encodedbybuk);E16,butyraldehydedehydrogenase(encodedbyadhE);E17,butanoldehydrogenase(encodedbybdhB).
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsXLcarriedoutthefermentationexperiment,performedthestatisticalanalysis,anddraftedthemanuscript.
Z-GLcarriedoutthefermentationexperiment.
Z-PSconceivedofthestudyandparticipatedinitsdesignandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThestudywassupportedbytheNationalNaturalScienceFoundationProgram(#20976072)andMajorStateBasicResearchDevelopmentProgram(#2007CB714303)ofChina.
TheauthorsalsoappreciatedtheassistancefromMr.
KevinDinginrefiningtheEnglishofthemanuscript.
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Submityourmanuscripttoajournalandbenetfrom:7Convenientonlinesubmission7Rigorouspeerreview7Immediatepublicationonacceptance7Openaccess:articlesfreelyavailableonline7Highvisibilitywithintheeld7RetainingthecopyrighttoyourarticleSubmityournextmanuscriptat7springeropen.
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