Acetone butanol production

Bioprocess Engineering and Biosafety

1. ACETONE –BUTANOL
PRODUCTION
Dr. Esther Shoba R
Assistant Professor
Kristu Jayanti College
Bangalore

2. INTRODUCTION
•Acetoneandbutanolareproducedthroughanaerobicfermentationby
speciesofClostridiumbutyricum.
•
•Theproductionofbutanolbybutyricacidbacteriawasfirstobservedby
LouisPasteurinthe19
th
century.
•BeforeWorldWar-Iprocessesinvolvingmicroorganismsweredeveloped
fortheproductionofbutadienewhichisrequiredfortheproductionof
syntheticrubber.
•LateronWeizmannreportedthatClostridiumacetobutylicumiscapable
ofproducingacetone,butanolandethanolinaneconomicallyfeasible
quantities.

3. •DuringWorldWar-I,acetonewasingreatdemandtomanufacturethe
explosivetrinitrotoluene(TNT).
•Hence,theacetone-butanolfermentationrapidlyexpanded.
•Butafterwar,thedemandforacetonedecreasedandbutanol
increased,asitwasrequiredasasolventfortherapiddryingof
nitrocellulosepaintsinautomobileindustry.
•Thus,thecommercialprocessofacetone-butanolsurvivedevenaftera
lackofdemandofacetoneafterWorldWar-I.

4. ACETONE AND BUTANOL STRUCTURE

5. FLOW CHART
•1. Clostridium acetobutylicum–1st organism –industrial production of
acetone from starch.
•2. Clostridium saccharoacetobutylicum–convert molasses into acetone &
butanol.
•• submerged cultures
•• Substrate: sterile diluted molasses or cooked corn meal.
•• pH : 7.2
•• Type of fermentation: anaerobic
•• By-products : CO₂( preparation of dry ice) & H (fuel)
•• Product recovery : fractional distillation

6. •Largebioreactors–200000-1000000Lr•
•CO2wasbubbledthroughtheculturetoensurethatO2wasexcluded.
•Fermentation–biphasicAcidogenesisSolventogenenis
•Acidogenesis–formingacetate,butyrate,hydrogen,andCO2.
•Solventogenesis–formingbutanol,acetone,andethanol.
•After40-60hrs–12-20gm/Lofsolvent(6B:3A:E1).
•Thesolventswasremovedbydistillation.
•Theremainingmicrobialdriedsolidswereusedashighnutrient
animalfeed.

7. Fermentation Process of Acetone-Butanol
•Acetone-butanol fermentation process can be described under the
following phases:
•(i) Production of inoculum
•(ii) Preparation of medium
•(iii) Fermentation process
•(iv) Harvest and recovery

8. MICROORGANISMS INVOLVED
•ClostridiumacetobutylicumandCl.saccharo-acetobutylicumarethe
speciesinvolved.
•Cl.saccharo-acetobutylicumrequirescornmediumandClostridium
acetobutylicummolassesmediumforthegrowth.
•Ingeneral,inoculumgrowthandfermentativeproductionofthesolvents
arecarriedoutat31°to32°CforCl.saccharoacetobutylicumandat
approximately37°CforCl.acetobutylicum.
•EntericBacteriae.g.Escherichiacoliarealsoused

9. •Clostridiaarerod-shaped,spore-forminggrampositivebacteriaand
typicallystrictanaerobes.
•Solventogenic(solventproducing)clostridiacanutilizealargevariety
ofsubstratesfrommonosaccharidesincludingmanypentosesand
hexosestopolysaccharides.

11. PREPARATION OF THE INOCULUM -Cl. Saccharoacetobutylicum
•InoculumofCl.Saccharoacetobutylicumisdevelopedemploying
molasses,calciumcarbonate,ammoniumsulphateorphosphateand
sometimescorn-steepliquor.
•Clostridia,beingsporeformers,areeasilymaintainedassoilstocksin
contrasttothevegetativecells,thesporesarenotverysensitiveto
oxygen.

12. •Sporesfromsoilstocksareinitiallyaddedtodeeptubesofsemisolid
potato-glucosemediumformolassescultures.
•Asthesporesareaddedtothebottomofthesetubestheyalongwith
soilparticlessinktothebottomofthetubesandbecomesubmerged.
•Thesubmergedlocationofthesporesandhighreducingpowerofthe
mediumcanprotectthevegetativecellsfromoxygenafter
germinationofspores/vegetativecells.

13. •Inoculatedtubesareheatshockedandrapidlycooledtoincubation
temperaturetoselectheatresistantspores.
•Thetubesarethenincubatedat31°to32°Cfor20hours.
•Thegrowththatoccursinthetubesareusedasinoculumforlarger
batchofmolassesmediumpresentininoculumtanks.
•Further,increasedvolumesofinoculumareproducedbysuccessive
transfersofapproximately2%to4%inoculumbyvolumetolarger
mediawithincubationperiodof20to24hoursateachtransfer.

14. PREPARATION OF THE INOCULUM -Cl. acetobutylicum
•Stages of inoculum preparation of this type of Clostridium are generally similar to
the one described above for Cl. Saccharo-acetobutylicumexcept the following
items:
•1. Spores of soil stock are added to the deep tubes containing corn medium with
about 5% corn meal in water.
•2. The tubes are incubated at 37°C for 20 hours.
•3. The concentration of corn in the corn mash is adjusted to 6½ % during
repeated inoculum transfers.

15. PRODUCTION MEDIUM
•MOLASSESANDCORNMEDIUM
•MOLASSES:Molasseswhichisformedasaby-productinthesugar
industry,isusedasarawmaterialforthepreparationofthemedium.
Sugarcontentintheformofsucroseismaintainedat6%.Forthiseither
blackstraporhigh-testmolassesisused.Nitrogensourceisaddedinthe
formofammoniumsulphate.
•Inadditioncalciumcarbonate,superphosphateandsometimescorn-
steepliquorarealsoadded.
•Calciumcarbonateisaddedtopreventdevelopmentofgrossacidityin
themedium.
•Ammoniumsulphateisaddedat18to24hoursoffermentation.

16. •Cornmealispreparedbypassingcornthroughamagneticfieldto
removedustandmetallicdebrisfollowedbydegermingthecorn.
•Cornoilisremovedfromthesproutedgerm.
•Thedegermedcornisthengroundtoafinepowderinrolleror
hammermill.
•Topreparecorn-mealproductionmedium8%to10%cornmealis
addedtowaterwithorwithoutstillage,thatis,residuefromthe
precedingfermentation.
•Itisthenheatedfor20minutesat65°Ctogelatinizethestarchbefore
sterilizationofthemedium.

17. •Thetwomediadescribedabovearethensterilizedandemployedin
fermentationdependinguponthetypeofClostridiumspeciesusedin
thefermentation.
•Sometimesstillage,thatis,residueformedintheprevious
fermentationisaddedtothemediumapproximatelyat30%to40%.
•Itresultsintheadditionofcertainnutrientslikeproteins,
carbohydratesandmineralstothefreshlypreparedmedium.

18. FERMENTATION PROCESS
•Fermentation is carried out under anaerobic conditions.
•Production tanks of the capacity of 50,000 to 2.5 lakhs gallons are
used in the fermentation.
•The incubation period is 2 to 2 ½ days. If the freshly steamed
molasses medium is employed, approximately 2 to 4% of inoculum is
needed, while for freshly steamed corn medium slightly less inoculum
is employed.
•The inoculum is added first to the production fermenter followed by
the addition of medium.

19. Fermentation generally passes through three phases:
•Inthisphaserapidgrowthofthebacteriumandformationofaceticacid
andbutyricacidinlargeamountsalongwiththeproductionoflarge
quantitiesofcarbondioxideandhydrogengases.
•ThepHofthemediumwhichwasinitially5.0to6.5forcornmediumand
5.5to6.5formolassesmedium,decreasesandthenremainsconstantfor
therestofthefermentationprocess.
•Thisphase,lastsforapproximately13to17hoursofincubation.
•Thetitratableacidityincreasestoamaximumandadaptiveenzymesare
producedwhichconvertacidstoneutralsolvents.

20. •Asharpdecreaseinthetitratableacidityduetoconversionofmore
acidsintoacetoneandbutanol.
•Thisprocessiscalledasacidbreak,whichgetsdelayedifthereis
contamination.
•Therateofgasformationreachesmaximumafteracidbreak.

21. •Therateofgasformationdecreasessubstantiallyalongwith
decreasedrateofsolventproduction.
•ThetitratableacidityslowlyincreasesleadingtoapHof4.2to4.4in
thecornmediumand5.2to6.2inthemolassesmedium.
•Manycellsundergoautolysisatthispointresultinginthereleaseof
riboflavinintothemedium.

24. YEILD
•Theratioofyieldofacetone,butanolandethanoldifferslightlydepending
onthefermentationmedium.
•But,generallytheyieldis2%byweightofthebroth,whichisapproximately
equalto30%conversionofcarbohydratetosolvents.
•Inacornmediumtheratioofbutanol,acetoneandethanolare6:3:1
respectively,butinmolassesmediumtheratiosare6:5:3.
•Apartfromthesesolvents,3partsofcarbondioxideand2partsof
hydrogenbyvolumearealsoproducedasbyproductsofthefermentation.
•Theacetone-butanolfermentationyieldsseveralproductsinadditiontothe
gasesdescribedabove.Theyincludeisopropanol,formicacid,aceticacid,
butyricacid,acetylmethylcarbinolandyellowoil,whichisamixtureof
higheralcoholsandacids,whichareindustriallyveryimportant.

25. RECOVERY
•Abeerstillisusedfortherecoveryoftheproductsfromthefermentationbroth.
Thebeerstillisatallverticalandcontinuousstillconsistingofabout30perforated
plates.
•Therecoveryprocessconsistsofthefollowingsteps:
•1.Thefermentationbrothisallowedtoenterthestillfromtop.Itdescendsthe
stillpassingthroughperforatedplates.
•2.Acontinuousflowofsteamisallowedintothestillfromitsbottom.Itmovesup
thestillinadirectionoppositetothedirectionoffermentationbroth.
•3.Acetoneandbutanolvaporizesduetotheeffectofsteam.

26. •4.Thesteamandsolventsarethencollectedandcondensedbycoolingtogeta
solutionwhichcontainsapproximately40%byweightofsolventmixture.
•5.Theindividualsolventspresentinthesolventmixtureareseparatedby
fractionaldistillation.
•6.Acetoneandbutanolarecollectedinseparatefractions.
•7.Ethanolandisopropanolarealsocollectedasasinglefractionandsoldasa
generalsolvent.
•8.TheresiduecontainsriboflavinandotherBvitaminsaswellasconsiderable
quantityofbacterialcells.Theresidueisconcentratedanddriedandusedas
vitaminfeedsupplement.

28. Uses of Acetone-Butanol:
•1.Atpresentbutanolisextensivelyusedinbrakefluidantibiotic
recoveryprocedures,urea,formaldehyderesins,aminesforgasoline
additivesandasesterintheprotectivecoatingindustry.
•2.Butanolisalsousedforthesynthesisofbutadienewhichisusedin
thepreparationofsyntheticrubber.
•3.Acetoneisusedasauniversalorganicsolventandalsointhe
preparationofexplosivesliketrinitrotoluene.

29. •Butanol is a valuable solvent for the production of lacquer(used to
prepare the finish of automobiles),latex,enamels….
•• Used as biofuel(it has similar physical properties to gasoline).
•• Used as an additive to plastics to keep them flexible.
•• Used as a solvent in the manufacture of antibiotics , vitamins , and
hormones.
•• Acetone is important in making cordite.