气提塔数字模拟.docx
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气提塔数字模拟.docx
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气提塔数字模拟
Productionofacetone,butanolandethanolbyClostridiumbeijerinckiiBA101andinsiturecoverybygasstripping
Weexaminedtheeffectofgas-strippingontheinsituremovalofacetone,butanol,andethanol(ABE)frombatch
reactorfermentationbroth.Themutantstrain(ClostridiumbeijerinckiiBA101)wasnotaffectedadverselybygas
stripping.ThepresenceofcellsinthefermentationbrothaffectedtheselectivitiesofABE.Aconsiderable
improvementintheproductivityandyieldwasrecordedinthisworkincomparisonwiththenon-integratedprocess.
InanintegratedprocessofABEfermentation-recoveryusingC.beijerinckiiBA101,ABEproductivitiesandyield
wereimprovedupto200and118%,respectively,ascomparedtocontrolbatchfermentationdata.Inabatch
reactorC.beijerinckiiBA101utilized45.4gglucosel)1andproduced17.7gtotalABEl)1,whileintheintegrated
processitutilized161.7gglucosel)1andproducedtotalABEof75.9gl)1.Intheintegratedprocess,acidswere
completelyconvertedtosolventswhencomparedtothenon-integratedprocess(batchfermentation)which
containedresidualacidsattheendoffermentation.InsituremovalofABEbygasstrippinghasbeenreportedtobe
oneofthemostimportanttechniquesofsolventremoval.DuringthesestudieswewereabletomaintaintheABE
concentrationinthefermentationbrothbelowtoxiclevels.
Butanol,animportantindustrialchemical,iscurrently
usedasafeedstockintheplasticindustry.Itisalsoused
asafood-gradeextractantinthefoodandflavour
industry.Acetoneandbutanolwereusedassolventsand
intheproductionoftherubbermonomers,butadiene,
anddimethylbutadiene(Mollah&Stuckey1993).
Butanolcanalsobeusedasafuelextender,whichhas
severaladvantagesoverotherfermentativelyproduced
fuelsandtheseincludehighenergycontent,miscibility
withgasoline,anddieselfuel,andalowvapourpressure
(Qureshi&Blaschek1999a).Therenewalofinterestin
themicrobialproductionofbutanol[oftencalled
acetone,butanol,ethanol(ABEfermentation)]isbased
almostexclusivelyontheawarenessoffossilfuel
depletionandtheuncertaintyofpetroleumsupplies.
ClostridiumbeijerinckiiBA101isabletofermenta
varietyofcarbohydratestobutanol,includingapple
wasteandstarchpackingpeanuts(Jesseetal.2002).
Butanolhasgreatpotentialbecauseithasmany
characteristicswhichmakeitabetterfuelextenderthan
ethanol.Additionally,fermentation-derivedbutanolis
preferredinthefoodindustry(forfoodflavourextraction)
aspetroleum-derivedbutanolhasthepotentialfor
carcinogencarryover(Formaneketal.1997).Forthese
twoimportantreasons,bio-basedbutanolispreferred.
Productionofbutanolbyfermentationcanalsohelpthe
USwithrespecttoindependencefromforeignoil.
ThemainproblemassociatedwiththeABEfermentation
byC.acetobutylicumandC.beijerinckiiisbutanol
toxicity/inhibitiontotheculture.Tosolvethisproblem,
variousalternativeinsitu/onlinebutanolremovaltechniques
includingmembrane-basedsystems,suchas
pervaporation(Grootetal.1984;Qureshietal.1992,
1999,2001),perstraction(Qureshietal.1992),reverse
osmosis(Garciaetal.1986),adsorption(Ennisetal.
1987;Nielsonetal.1988),liquid–liquidextraction
(Evans&Wang1988)andgasstripping(Grootetal.
1989)havebeenexamined.Theapplicationofsomeof
theserecoverytechniquescanallowtheuseofconcentrated
sugarsolutionsinthefermentor(Maddoxetal.
1995;Qureshi&Blaschek2001)therebyreducing
volumesoftheprocessstreams.Insuchsystems,upto
100%utilizationofthesugaravailableinfeedhasbeen
demonstrated.Itshouldbenotedthatmembrane-based
systemsdemonstratehighselectivitiesforABEbutmay
sufferfromfoulingandclogging.Ontheotherhand,gas
strippingisatechniquewhichallowsfortheselective
removalofvolatilesfromfermentationbrothanduses
nomembrane.ThevolatilepropertiesoftheABEpermit
easyproductremovalbygasstripping.Gascanbe
spargedintothefermentorthrougharotatingfermentor
shaftandvolatilescanbecondensedandrecoveredfrom
thecondenser.Thegas-strippingprocesshasanumber
ofadvantagesoverotherremovalprocesses,forexample:
itissimpleandinexpensivetooperateanddoesnot
sufferfromfoulingorcloggingduetothepresenceof
biomass.Ennisetal.(1986)werethefirsttoinvestigate
theapplicationofgasstrippinginbutanolfermentation
usingC.acetobutylicum.Gasstrippinghasbeendescribed
asoneofthemostimportanttechniquesfor
removingbutanolfromthefermentationbroth(Maddox
1988;Grootetal.1992).
C.beijerinckiiBA101isageneticallymanipulated
strainwhichhasthepotentialforbecominganindustrial
microorganismforproducingbutanolfromcornand
othersubstrates.Ithasbeenreportedthatthisstrainis
abletoproduceupto32.6gl)1totalABE(Chen&
Blaschek1999)underoptimizedconditionsasopposed
to20gl)1byothersolventogeniccultures(Maddox
1988).WhileC.beijerinckiiBA101stopsgrowingabove
165gl)1(Qureshi&Blaschek1999a),C.acetobutylicum
P262cantolerateupto227gsugarl)1(Qureshi&
Maddox2003).Thisobservationisanimportantdifference
betweenC.acetobutylicumP262andC.beijerinckii
BA101.SinceC.beijerinckiiBA101isamutant(and
different)strain,butanolremovalstudiescarriedout
usingothersolventogenicstrainsmaynotbeapplicable
forC.beijerinckiiBA101.Forthisreason,thepresent
workisconsiderednovel.Theauthorsareawareofthe
gas-strippingworkperformedbyvariousinvestigators.
Itisalsoknownthatbutanol-producingculturesare
negativelyaffectedbymechanicalagitation.Insitugas
strippingcausesvigorousagitationofculturefluid
(dependingongasrecyclerate),andhencemaynegatively
affectC.beijerinckiiBA101.Additionalobjectives
weretostudytheeffectofcellconcentrationonABE
selectivityandtheuseofconcentratedsugarsolutions
forbutanolproduction.Inbatchreactors,asugar
concentrationmorethan60gl)1cannotbeutilized,due
toproductinhibitioncausedbybutanol.
Materialsandmethods
Organism,culturemaintenanceandfermentation
conditions
TheC.beijerinckiiBA101culturewasgeneratedusing
N-methyl-N¢-nitro-N-nitrosoguanidine(NTG)together
withselectiveenrichmentonthenon-metabolizable
glucoseanalog2-deoxyglucose(Annous&Blaschek
1991).LaboratorystocksofC.beijerinckiiBA101were
routinelymaintainedassporesuspensionsinsteriledoubledistilledwaterat4_C.C.beijerinckiiBA101
spores(200ll)wereheatshockedfor10minat80_C
followedbycoolingfor5minpriortoinoculation.Heat
shockedsporeswereinoculatedinto20mlTryptoneglucose-
yeastextract(TGY)medium(in50mlscrew
cappedPyrexbottle)whichwasincubatedanaerobically
intheanaerobicchamber(CoyLaboratoryProducts,
AnnArbor,MI)for15–16hat36±1_Cuntilactive
growthwasobserved.
Batchfermentation
A2-lbioreactor(NewBrunswickScientificCo.,New
Brunswick,NJ)wasusedthroughoutthesestudies.In
allexperiments,thetemperaturewasmaintainedat
35_CintheabsenceofagitationorpHcontrol.
Fermentorcontaining60gglucosel)1and1gyeast
extractl)1(1.6lreactionvolume)wassterilizedat
121_Cfor15min.Oncoolingto35_Cunderan
oxygen-freenitrogenatmosphere,filter-sterilizedP2
stocksolutions[(buffer:
KH2PO4,50gl)1;K2HPO4,
50gl)1;Ammoniumacetate,220gl)1),(vitamin:
Paraamino-
benzoicacid,0.1gl)1;thiamin,0.1gl)1;Biotin,
0.001gl)1),(mineral:
MgSO4?
7H2O,20gl)1;MnSO4Æ
H2O,1gl)1;FeSO4?
7H2O,1gl)1;NaCl,1gl)1)]
(Qureshi&Blaschek1999b)wereadded,followedbythe
inoculationofthebioreactorwith5%(vol/vol)highly
motilecellsofC.beijerinckiiBA101.Oxygen-free
nitrogengaswassweptovertheheadspaceofthe
fermentoruntilthecultureproduceditsowngases(CO2
andH2).Sampleswerewithdrawnatvariousintervals
foranalysis.
Gas-stripping
Themediumusedinthistechniquewasthesameasthat
usedforbatchfermentationexperimentsexceptthat
differentglucoseconcentrations(60–161.7gl)1)were
usedandthereactionvolumewas1.0luntilunless
otherwisestated.Batchfermentationswereallowedto
proceedfor15h(glucose60gl)1)and36h(glucose
161.7gl)1)whentheABEconcentrationsapproached
3–4gl)1,afterwhichgas-strippingwasapplied.During
gas-strippingstringentanaerobicconditionsweremaintained
usingoxygen-freenitrogengas.Thegases(CO2
andH2)wererecycled(3000mlmin)1,unlessotherwise
stated)throughthesystemusingatwin-headperistaltic
pump.TheABEvaporswerecondensedusingacooling
machine(GeneLine;BeckmanInstrumentsInc.,Palo
Alto,CA,USA)andacondenser(62·600mmand
coolingcoilsurfacearea1292cm2)to)2_C.Inorderto
cool,ethyleneglycol(50%v/v)wascirculatedataflow
rateof600mlmin)1throughthecondenser.The
strippedABEwerepumpedintothesolventcollector
usingaperistalticpump.Oxygen-freedistilledwaterwas
addedatintervalsintothereactortomaintaina
constantliquidlevel(tocompensateforwaterlossdue
togas-stripping)insidethereactor.Aschematicdiagram
oftheprocessisshowninFigure1.TemperatureintheButanolfermentationandrecovery
Results
Characterizationofthegas-strippingprocess
InordertofindratesofABEremovalunderfermentation
conditionsatvariousABEconcentrations,agasstripping
experimentwasconductedat35_Cusing
modelABEsolution.Nitrogengaswasusedtostrip
ABEatagasflowrateof4.6lmin)1.Theinitial
concentrationsof
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