Thedesignoffermenter

1.Introduction
2.Standardgeometryofastirredtankbioreactor
3.Headspacevolume
4.Basicfeaturesofastirredtankbioreactor
4.1.Agitationsystem
4.1.1Topentryandbottomentryimpellers
4.1.2Mechanicalseals
4.2Oxygendeliverysystem
4.2.1Compressor
4.2.2Airsterilizationsystem
4.2.3Positivepressure
4.2.4Sparger
4.2.5Effectofimpellerspeed
4.2.6Airflowrate

UNITII
FERMENTOR
R.KAVITHA,M.PHARM
LECTURER,
DEPARTMENTOFPHARMACUTICS
SRMCOLEGEOFPHARMACY
SRMUNIVERITY

Thestirredtankbioreactor
4.3Foamcontrol
4.4Temperaturecontrolsystem
4.5pHcontrolsystem
4.5.1Neutralizingagents
4.5.2Setpointanddeadband
4.6Cleaningandsterilizationfacilities
5.Agitatordesignandoperation
5.1Radialflowimpellers
5.1.1Rushtonturbine
5.2Axialflowimpellers
5.3Intermigimpeller

1.Introduction
Atypicalbioreactorusedformicrobialfermentationsisshowninthefollowing
figure:

Laboratory scale bioreactors with liquid volumes of less than 10 litres are
constructed out of Pyrex glass. For larger reactors,stainless steel is used.

2.Standardgeometryofastirredtankbioreactor
Astirredtankreactorwilleitherbeapproximatelycylindricalor
haveacurvedbase.Acurvedbaseassistsinthemixingofthe
reactorcontents.
Stirredtankbioreactorsaregenerallyconstructedtostandard
dimensions.
Thatis,theyareconstructedaccordingtorecognisedstandards
suchasthosepublishedbytheInternationalStandards
OrganisationandtheBritishStandardsInstitution.
Thesedimensionstakeintoaccountbothmixingeffectiveness
andstructuralconsiderations.

Standardgeometryofastirred
tankbioreactor
Amechanicallystirredtankbioreactorfittedwith
aspargerand
arushtonturbine
willtypicallyhavethefollowingrelative
dimensions:

Standardgeometryofastirred
tankbioreactor

3.Headspacevolume
A bioreactor is divided in a working volume and a headspace volume.
The working volume is the fraction of the total volume taken
up by the medium, microbes, and gas bubbles.
The remaining volume is called the headspace.
Typically, the working volume will be 70-80% of the total
fermenter volume.
This value will however depend on the rate of foam
formation during the reactor. If the medium or the
fermentation has a tendency to foam, then a larger
headspace and smaller working volume will need to be used.

Headspacevolume

4.Basicfeaturesofastirredtankbioreactor

Amodernmechanicallyagitatedbioreactorwillcontain:

Basicfeatures
*Anagitatorsystem
*Anoxygendeliverysystem
*Afoamcontrolsystem
*Atemperaturecontrolsystem
*ApHcontrolsystem
*Samplingports
*Acleaningandsterilizationsystem.
*Asumpanddumplineforemptyingofthereactor

4.1Basicfeaturesofastirredtankbioreactor
Agitationsystem
Thefunctionoftheagitationsystemisto
providegoodmixingandthusincreasemasstransferrates
throughthebulkliquidandbubbleboundarylayers.
providetheappropriateshearconditionsrequiredforthe
breakingupofbubbles.
Theagitationsystemconsistsoftheagitatorandthebaffles.
Thebafflesareusedtobreaktheliquidflowtoincreaseturbulenceand
mixingefficiency.Theroleofthebafflesisdiscussedindepthinalater
section.
Theagitatorconsistsofthecomponentsshowninthefollowingdiagram:

Agitationsystem

Theagitatorconsistsofthecomponentsshowninthefollowingdiagram:

Agitationsystem

Thenumberofimpellerswilldependontheheightoftheliquid
inthereactor.Eachimpellerwillhavebetween2and6blades.
MostmicrobialfermentationsuseaRushtonturbineimpeller.
Asinglephase(ie.240V)agitatordrivemotorcanbeusedwith
smallreactors.Howeverforlargereactors,a3phasemotor(ie
430V)shouldbeused.Thelatterwilltendtorequireless
currentandthereforegeneratelessheat.
Speedcontrolorspeedreductiondevicesareusedtocontrolthe
agitationspeed.

4.1.1Basicfeaturesofastirred
tankbioreactor;Agitationsystem
Topentryandbottomentry
impellers

Theimpellershaftcanenterfromthebottomofthetankorfromthetop.Atop
entryimpeller("overhungshaft")ismoreexpensivetoinstallasthemotorand
theshaftwillneedtobestructurallysupported:

Bottomentryimpellers

Areactorwithbottomentryimpellerhoweverwillneedhighermaintenanceduetodamageof
thesealbyparticulatesinthemediumandbymediumcomponentsthatcrystallizeintheseal
whenreactorisnotinuse:

Bottomentryagitatorstendtorequiremoremaintenancethantopentryimpellersduetothe
formationofcrystalsandothersolidsintheseals

4.1.2BasicfeaturesofaSTR
Agitationsystem Mechanicalseals
Themechanicalsealisusedpreventcontaminantsfromentering
thereactorandtopreventorganismsfromescapingthroughthe
shaft.
Thesealusesvapoursfromtheliquidforlubrication.
Itisthereforeimportantthatyoudonotturntheshaftwhenthe
tankisdrysoasnottodamagetheseal.

4.2Basicfeaturesofastirredtank
bioreactor Oxygendeliverysystem.
Theoxygendeliverysystemconsistsof
acompressor
inletairsterilizationsystem
anairsparger
exitairsterilizationsystem

4.2.1Basicfeaturesofastirredtankbioreactor;
Oxygendeliverysystem Compressor

Oxygendeliverysystem Compressor
Acompressorforcestheairintothereactor.Thecompressorwill
needtogeneratesufficientpressuretoforcetheairthroughthe
filter,spargerholesandintotheliquid.
Aircompressorsusedforlargescalebioreactorstypicallyproduce
airat250kPa.Theairshouldbedryandoilfreesoastonot
blocktheinletairfilterorcontaminatethemedium.
Notethatitisveryimportantthatan"instrumentair"
compressorisnotused.Instrumentairistypicallygeneratedat
higherpressuresbutisaspiratedwithoil.Instrumentair
compressorsareusedforpneumaticcontrol

4.2.2Basicfeaturesofastirredtankbioreactor;
Oxygendeliverysystem Airsterilizationsystem
Sterilizationoftheinletairisundertakentopreventcontaminating
organismsfromenteringthereactor.
Theexitairontheotherhandissterilizednotonlytokeep
contaminantsfromenteringbutalsotopreventorganismsinthe
reactorfromcontaminatingtheair.
Acommonmethodofsterilisingtheinletandexitairisfiltration.
Forsmallreactors(withvolumeslessthan5litres),diskshaped
hydrophobicTeflonmembraneshousedinapolypropylenehousing
isused.areused.Teflonistough,reusableanddoesnotreadily
block.

Sterilisationoftheair

For larger laboratory scale fermenters (up to 1000 litres),

pleated membrane filters housed in polypropylene
cartridges are used.

Sterilisationoftheair

Bypleatingthemembrane,itispossibletocreateacompactfilterwithavery
largesurfaceareaforairfiltration.Increasingthefiltrationareadecreasesthe
pressurerequiredtopassagivenvolumeofairthroughthefilter.

Sterilizationoftheinletandexitairinlargebioreactors(>10,000litres)can
presentamajordesignproblem.Largescalemembranefiltrationisavery
expensiveprocess.Thefiltersareexpensiveastheyaredifficulttomakeand
theenergyrequiredtopassairthroughafiltercanbequiteconsiderable.

Heatsterilizationisalternativeoption.Steamcanbeusedtosterilizetheair.
Witholderstylecompressors,itwaspossibletousetheheatgeneratedby
theaircompressionprocesstosterilizetheair.However,compressorsare
nowmultistagedeviceswhicharecooledateachstageanddisinfecting
temperaturesareneverreached.

Condenser
In small reactors, the exit air system will typically include a condenser.

Condenser

Thecondenserisasimpleheatexchangerthrough
whichcoolwaterispassed.
Volatilematerialsandwatervapourcondenseon
theinnercondensersurface.
Thisminimizeswaterevaporationandthelossof
volatiles.
Dryingtheairalsopreventsblockingoftheexitair
filterwithwater

4.2.3BasicfeaturesofaSTR
Oxygendeliverysystem
Airsterilisationsystem Positivepressure

Duringsterilisationtheconceptof"maintainingpositivepressure"willoftenbe
used.

Maintainingpositivepressuremeansthatduringsterilisation,coolingandfilling
andifappropriate,thefermentationprocess,airmustbepumpedintothe
reactor.

Inthiswaythereactorisalwayspressurisedandthusaerialcontaminantswill
notbe"sucked"intothereactor.

Itisveryimportantthatpositivepressureismaintainedwhenthebioreactoris
cooledfollowingsterilisation.Withoutairbeingcontinuouslypumpedintothe
reactor,avacuumwillformandcontaminantswilltendtobedrawnintothe
reactor.

Airsterilisationsystem
Positivepressure
Maintaining positive pressure at all stages of the fermentation setup and
operation is an important aspect of reducing the risk of contamination

Without aeration, a vacuum

forms as the reactor cools.

With aeration, positive pressure

is always maintained and
contaminants are pushed away
from the reactor

4.2.4Basicfeaturesofastirredtank
bioreactorOxygendeliverysystem Sparger
Theairspargerisusedtobreaktheincomingairintosmall
bubbles.
Althoughvariousdesignscanbeusedsuchasporousmaterials
madeofglassormetal,themostcommontypeoffilterusedin
modernbioreactorsisthespargering:

Oxygendeliverysystem
Sparger
Aspargeringconsistsofahollowtubeinwhichsmall
holeshavebeendrilled.Aspargeringiseasiertoclean
thanporousmaterialsandislesslikelytoblockduring
afermentation.
Thespargeringmustlocatedbelowtheagitatorand
willhaveapproximatelythesamediameterasthe
impeller.
Thus,thebubblesrisedirectlyintotheimpellerblades,
facilitatingbubblebreakup.

Oxygendeliverysystem
Sparger

During the emptying of a fermenter, it is important that the air

feed valve is closed. This will minimize the contamination of the
inlet air line

4.2.5BasicfeaturesofaSTR
Oxygendeliverysystem Effectofimpellerspeed

Asdiscussedinanotherlecture,theshearforcesthatanimpellergeneratesplay
amajorroleindeterminingbubblesize.Iftheimpellerspeedistoslowthenthe
bubbleswillnotbebrokendown.Inaddition,iftheimpellerspeedistooslow,
thenthebubbleswilltendtorisedirectlytothesurfaceduetotheirbuoyancy.

Slow impeller speed

The bubbles will not be sheared

into smaller bubbles and will
tend to rise directly towards the
surface

Fast impeller speed

Smaller bubbles will be generated and

these bubbles will move with
throughout the reactor increasing the
gas hold up and bubble residence time

Oxygendeliverysystem Effectofimpeller
speed
Anotherconsequenceoftooslowanimpellerspeedisaflooded
impeller.
Undertheseconditions,thebubbleswillaccumulateandcoalesce
undertheimpeller,leadingtotheformationoflargebubblesandpoor
oxygentransferrates.
Asimilarphenomenonwillhappenwhenaerationrateistoohigh.
Inthiscase,theoxygentransferefficiencywillbelow

4.2.6BasicfeaturesofaSTR
Oxygendeliverysystem Air
flowrates
Airflowratesaretypicallyreportedintermsof
volumepervolumeperminute
or
vvm
whichisdefinedas:

Airflowrates

Note the unit convention. The air flow rate and liquid volume
must have the same basal unit. The air flow rate must be
expressed in terms of volume per minute

4.3BasicfeaturesofaSTR
foamcontrolsystem

Foamcontrolisanessentialelementoftheoperationofaspargedbioreactor.
Thefollowingphotographshowstheaccumulationoffoamina2litre
laboratoryreactor.

Foamcontrolsystem
Excessivefoamformationcanleadtoblockedairexitfiltersandto
pressurebuildupinthereactor.
Thelattercanleadtoalossofmedium,damagetothereactorandeven
injurytooperatingpersonnel.
Foamistypicallycontrolledwithaidofantifoamingagentsbasedon
siliconeoronvegetableoils.
Excessiveantifoamadditioncanhoweverresultinpooroxygentransfer
rates.

 Theantifoamrequirementwilldependon
thenatureofthemedium.
Mediarichinproteinswilltendtofoammorereadilythansimplemedia.
theproductsproducedbythefermentation.
Secretedproteinsornucleicacidsreleasedasaresultofcelldeathandhydrolysishave
detergentlikeproperties.
theaerationrateandstirrerspeed.
Increasingtheaerationrateandstirrerspeedincreasesfoamingproblems.
theuseofmechanicalfoamcontroldevices
Foamcontroldevicessuchasmechanicalandultrasonicfoambreakershelptoreducethe
antifoamrequirement.
Theheadspacevolume
Thelargerheadspacevolume,thenthegreaterthetendencyforthefoamtocollapseunder
itsownweight.Forexample,forfermentationsinwhichhighlevelsoffoamisproduced,a
50%headspacevolumemayberequired.
Condensertemperature
Inlaboratoryscalereactors,acoldcondensertemperaturecanhelptocontrolthefoam.The
densityofthefoamincreaseswhenitmovesfromthewarmheadspacevolumetothecold
condenserregion.Thiscausesthefoamtocollapse.

Foamistypicallydetectedusingtwo
conductivityor"level"probes.

When the upper level probe is above the foam

level, no current will pass between the level
probes and the antifoam pump remains turned
off.

When the upper level probe is immersed

in the foam layer, a current is carried in
the foam. This causes the antifoam to
turn on.

Foamcontrolsystem

Oneprobeisimmersedinthefermentationliquidwhilethe
otherplacedabovetheliquidlevel.
Whenthefoamreachestheupperupperprobe,acurrentis
carriedthroughthefoam.
Thedetectionofacurrentbythefoamcontrollerresultsinthe
activationofapumpandtheantifoamisthenaddeduntilthe
foamsubsides.

4.4Basicfeaturesofastirredtank
bioreactor Temperaturecontrolsystem
Thetemperaturecontrolsystemconsistsof
temperatureprobes
heattransfersystem
Typicallytheheattransfersystemwillusea"jacket"totransfer
heatinoroutofthereactor.Thejacketisashellwhich
surroundspartofthereactor.Theliquidinthejacketdoesnot
comeindirectcontactwiththefermentationfluid.

Temperaturecontrolsystem

The jacket will typically be "dimpled" to encourage turbulence in the

jacket and thus increase the heat transfer efficiency.
An alternative to using jackets are coils. Coils have a much higher heat
transfer efficiency than jackets. However coils take up valuable reactor
volume and can be difficult to clean and sterilize.

Temperaturecontrol
system
The heating/cooling requirements are provided by the following methods:

Laboratory scale reactors

Pilot and production scale

reactors

Electric heaters

Steam generated in boilers

Cooling

Tap water or

Cooling water produced by

requirements

refrigerated water baths

cooling towers or refrigerants

Heating
requirements

such as ammonia.

Temperaturecontrolsystem
Inpilotandproductionscalereactors,heatingistypicallyonlyrequired
duringtheinitialstagesandfinalstagesofthefermentationasmost
processeswhichoccurduringafermentationprocess,including
thebiologicalreactions(eg.growth)
chemicalreactions
mixing
areexothermic.

4.5Basicfeaturesofastirredtank
bioreactor pHcontrolsystem

The pH control system consists of

a pH probe
alkali delivery system
acid delivery system
The pH probe is typically steam sterilizable

4.5.1Basicfeaturesofastirredtank
bioreactorpHcontrolsystem Neutralizing
agents
TheneutralizingagentsusedtocontrolpHshouldbenoncorrosive.
Theyshouldalsobenontoxictocellswhendilutedinthemedium.
PotassiumhydroxideispreferredtoNaOH,aspotassiumionstend
tobelesstoxictocellsthansodiumions.HoweverKOHismore
expensivethanNaOH.Sodiumcarbonateisalsocommonlyusedin
smallscalebioreactorsystems.
Hydrochloricacidshouldneverbeusedasitiscorrosiveevento
stainlesssteel.
Likewisesulphuricacidconcentrationsshouldnotbebetween10%
and80%asbetweenthisrange,sulphuricacidismostcorrosive.

Neutralizingagents

Forfermentationsthatproducelargeamountsofacids,forexamplelactic
acidsfermentationusingmediacontaininghighsugarconcentrations,high
concentrationsofalkali(4Mandabove)arepreferred.Thiswillprevent
dilutionofthemediumduetotheadditionofexcessiveadditionofthealkali
solution.

Forlaboratoryfermenters,aperistalticpumpisusedtoaddthepHadjusting
agents.Siliconetubingisoftenused.However,notethatsiliconetubingwill
decayinthepresenceofhighalkaliconcentrations.Thickwalledslicone
tubingshouldbeused.

AlternativelyTygonorNeoprenetubingcanbeused.Tygonisnot
autoclavablebutcanbesterilizedbypassingtheNaOHthroughthetubingfor
about1hour.Neopreneisautoclavablebutisnottransparentortranslucent
asisTygonorsilicone.

4.5.2.Basicfeaturesofastirredtankbioreactor
pHcontrolsystem Setpointanddeadband

Setpointanddeadband

ThepHcontrolsystem(andindeedallotherfermentercontrolsystems)are
designedtohaveadeadband.Adeadbandisusedtopreventexcessivealkali
andacidaddition.

ThepHcontroldeadbandisshowninthefollowingdiagram:

Setpointanddeadband

ThesetpointisthepHatwhichthefermenterisbeingattemptedtobecontrolled
at.Forexample,ifthefermentationistoberunataconstantpHof6.5,thenthe
setpointissetto6.50.

Ifforexample,a5%deadbandisused,thentheupperdeadbandlimitwillbe
1.05x6.5=6.83

andthelowerdeadbandlimitwillbe
0.95x6.5=6.18

Ifthedeadbandistoosmall,thenitispossiblethatpHwilloftenovershootand
undershootthedeadbandsleadingtoexcessivealkaliandacidaddition.Thetrade
offisthatawidedeadbandwillleadtolessprecisepHcontrol.

Asmanyfermentationstendtoproduceacidsratherthansubstancesthatincrease
thepH,acidadditionisoftennotrequired.Indeednotallfermentationsneed
continuouspHcontrol.

4.6.Basicfeaturesofastirredtankbioreactor
Cleaningandsterilizationfacilities.
Smallscalereactorsaretakenapartandthencleanedbeforebeing
reassembled,filledandthensterilizedinanautoclave.
However,reactorswithvolumesgreaterthan5litrescannotbe
placedinanautoclaveandsterilized.Thesereactorsmustbe
cleanedandsterilized"inplace".Thisprocessisreferredto"Clean
inPlace.
CIPinvolvesthecompletecleaningofnotonlythefermenterbut
alsoalllineslinkedtotheinternalcomponentsofthereactor.
Steam,cleaningandsterilizingchemicals,sprayballsandhigh
pressurepumpsareusedintheseprocesses.Theprocessisusually
automatedtominimizethepossibilityofhumanerror.

5.Agitatordesignandoperation
Agitators are classified as having radial flow or axial flow
characteristics.
With radial flow mixing, the liquid flow from the impeller is
initially directed towards the wall of the reactor; ie. along the
radius of the tank.
With axial flow mixing, the liquid flow from the impeller is
directed downwards towards the base of the reactor, ie. in the
direction of the axis of the tank.
Radial flow impellers are primarily used for gas-liquid
contacting (such as in the mixing of sparged bioreactors) and
blending processes.
Axial flow impellers provide more gentle but efficient mixing
and are used for reactions involving shear sensitive cells and
particles.

5.1.Agitatordesignandoperation
Radialflowimpellers

Radialflowimpellerscontaintwoormoreimpellerbladeswhichare
setataverticalpitch:

Agitatordesign

Theliquidflowfromthebladesisdirectedtowardsthewallsofthereactor;ie.
alongtheradiusofthetank.

Agitatordesign
Radialflowmixingisnotasefficientasaxialflowmixing.
Forradialflowimpellers,amuchhigherinputofenergyinputis
requiredtogenerateagivenlevelofflow.
Radialflowimpellersdoandaredesignedto,generatehigh
shearconditions.Thisisachievedbytheformationofvorticesin
thewakeoftheimpeller:

Agitatordesign

Thehighsheariseffectiveatbreakingupbubbles.Forthisreason,radialflow
impellersareusedforthecultureofaerobicbacteria.

Highshearcanalsodamageshearsensitivematerialssuchascrystalsand
precipitatesandshearsensitivecellssuchasfilamentousfungiandanimal
cells.

With radial flow impellers, vertical (or axial) mixing is achieved with the use of baffles

5.1.1Agitatordesignandoperation
Radialflowimpellers Rushton turbine
Themostcommonlyusedagitatorinmicrobialfermentationsis
theRushtonturbine.
Likeallradialflowimpellers,theRushtonturbineisdesignedto
providethehighshearconditionsrequiredforbreakingbubbles
andthusincreasingtheoxygentransferrate.
TheRushtonturbinehasa4or6bladeswhicharefixedontoa
disk.
ThediameteroftheRushtonturbineshouldbe1/3ofthetank
diameter.

Radialflowimpellers
ARushtonturbineisoftenreferredtoasadiskturbine.
Thediskdesignensuresthatmostofthemotorpoweris
consumedatthetipsoftheagitatorandthusmaximizingthe
energyusedforbubbleshearing.

Radialflowimpellers

5.2.Agitatordesignandoperation
Axialflowimpellers
Axial flow impeller blades are pitched at an angle and thus direct the liquid
flow towards the base of the tank. Examples of axial flow impellers are
marine impellers and hydrofoil impellers.

Axialflowimpellers

Theresultantflowpatternisthuspredominantlyvertical;ie.alongthetank
axis.

Axialflowimpellers

Axialflowmixingisconsiderablymoreenergyefficientthan
radialflowmixing.
Theyarealsomoreeffectiveatliftingsolidsfromthebaseof
thetank.
Axialflowimpellershavelowshearproperties.Theangled
pitchoftheagitatorscoupledwiththethintrailingedgesof
theimpellerbladesreducesformationofeddiesinthewake
ofthemovingblades.

Axialflowimpellers

Low shear conditions are achieved by pitching the impeller

blades at an angle and by making the edges of the impeller
blades thing and smooth.

Axialflowimpellers
Axialflowimpellersareusedformixingshearsensitive
processessuchascrystallizationandprecipitationreactions.
Theyarealsousedwidelyinthecultureofanimalcells.
Theirlowshearcharacteristicsgenerallymakesthemineffective
atbreakingupbubblesandthusunsuitableforuseinaerationof
bacterialfermentations

5.3.Agitatordesignandoperation
Axialflowimpellers Intermig Impeller

Intermigimpellerisaaxialflowwhichisusedformicrobialfermentations.

Theimpellerisshownbelow:

IntermigImpeller

Theagitationsystemhastwoimpellers.Thebottomimpellerhasa
largeaxialflowsection.Thetipsoftheimpellercontainfingerlike
extensionswhichcreateaturbulentwakeforbreakingbubbles.
Asthehighshearregionexistsonlyatthetip,theoverallshear
conditionsinthereactorarelowerthanwouldbegeneratedbya
radialflowimpellersuchasaRushtonTurbine.
Intermigimpellersareusedwidelyforagitationandaerationin
fungalfermentations.

Summary
Awareofstandardgeometryofastirredtankbioreactor
Knowthebasicfeaturesofastirredtankbioreactor
Understandworkingofthe agitationsystem
Agitatordesignandoperation
Componentsoftheoxygendeliverysystem
Foamcontrol
Temperaturecontrolsystem
pHcontrolsystem
Cleaningandsterilizationfacilities