PRESENTED BY:Gurpreet KaurL-2011-BS-239-MINDUSTRIAL BIOREACTORSDESIGN AND FUNCTIONALCHARACTERISTICS
BIOREACTOR Vessel for the growth of microorganismswhich, while not permitting contamination,enables the provision of conditions necessaryfor the maximal production of the desiredproducts.
FERMENTATION Fermentation processes utilize microorganisms toconvert solid or liquid substrates into variousproducts. Commonly consumed fermented products includebread, cheese, beer, wine, citric acid, vinegar etc.• Submerged culture citric acid production byAspergillus niger• Solid state koji fermenationAEROBICFERMENTATION• Submerged culture yoghurt production byStreptococcus thermophilus andLactobacillus bulgaris.• Solid state anaerobic fermentation by acidformingbacteria, particularly, Lactobacillus, Pediococcus, Micrococcus sp. produce fermentedmeat products like pepperoni .ANAEROBICFERMENTATION
• A batch of culture medium in a bioreactor isinoculated with starter culture.• Fermentation proceeds for a certain durationand the product is harvested.BATCHFERMENTATION• Sterile culture medium is added periodically tothe inoculated fermentation broth.• Bioreactor is harvested after the batch time.FED BATCHFERMENTATION• Sterile medium is fed continuously intobioreactor.• Fermented product is continuously drawn.CONTINUOUSFERMENTATION
SUBMERGEDFERMENTATION• Utilizes free flowingliquidsubstrates, such asmolasses andbroths.• Substrates areutilized quiterapidly, need to bereplaced constantly.• Best suited forbacteria that requirehigh moisturecontent.SOLID-STATEFERMENTATION• Utilizes solidsubstrates like bran,bagasse and paperpulp.• Substrates areutilized very slowly,need not to bereplaced.• Best suited for fungithat require lessmoisture content.
ASEPTIC OPERATION AND CONTAINMENTASEPTIC OPERATION• Protection against contaminationCONTAINMENT• Prevention of escape of viable cells from thebioreactor.o Bioreactor must be sterilized prior to operation.o Sterile air must be introduced into bioreactor.o Inoculation and sampling must be done underaseptic conditions.o Ports and nozzles must be sealed properly.
VESSEL: MATERIALS OF CONSTRUCTION Industrial scale vessels are constructed ofstainless steel: SS304, SS304L, SS316, SS316L SS304: lower value, unlicensed products. SS316L: high value, licensed products. AISI grade 316 steels which contain 18%chromium, 10% nickel and 2.5% molybdenum arecommonly used for industrial operations. AISI grade 317 (stainless steel with 3-4%molybdenum) is used in citric acid fermentation(pH 1-2) to prevent leaching of heavy metals fromthe steel which would interfere with fermentation. AISI grade 304 (18.5% chromium & 10% nickel) isused for brewery equipment.
AERATION AND AGITATIONAERATION• Provide sufficient oxygen for metabolic requirements ofmicroorganismsAGITATION• Make uniform suspension of microbial cells in homogeneousnutrient medium.AGITATOR (IMPELLER)BAFFLESAERATION SYSTEM (SPARGER)
AGITATOR (IMPELLER) Mounted on the shaft at a specific distance off thetank bottom Bulk fluid and gas phase mixing Oxygen transfer Heat transfer Suspension of solid particles Maintain a uniform environment throughout thevessel contentsRushton turbine of 1/3rd thebioreactor diameter is consideredthe optimum design for use inmany fermentation processes.
SCABA6SRGT• Handle high airflow rate• Radial flowagitator• Better for bulkblending thanrushton turbinePROCHEMMAXFLO• 4,5 or 6 hydrofoilblades set at acritical angle on acentral hollowhub• Agitator to vesselratio is greaterthan 0.4• Maximum powerrequirement is66% of that withrushton turbine.EKATOINTERMIG• More complex indesign• Agitator to vesseldiameter ratio is0.6-0.7• Less loss inpower than withrushton turbine
BAFFLES To minimize fluidswirling and vortexformation Metal strips roughly1/10th vessel diameterand attached radiallyto the wall 6-8 baffles are usedin industrial scalebioreactors. Baffling tends toincrease transmittablepower, to improvemixing and aeration
AERATION SYSTEM (SPARGER)POROUS SPARGER• Produce small bubble size, difficult to cleanORIFICE SPARGER• Perforated pipe arranged below impeller in the form of ringsNOZZLE SPARGER• Single open or partially closed pipe, does not get blockedCOMBINED SPARGER AGITATOR• Air introduced via hollow shaft and emitted through holes drilled in the diskbladesDYNAMIC SPARGER• Gas pumped through porous metal tube directed into pipeline, one of thecheapest and most efficient spargerINTRUSIVE SPARGER• Sparger element located in pipeline
CONTROL AND MONITORING Allows better process modelling and closer processcontrol. Highly selective in-situ methods are developed forprocess monitoring. An essential development in process analysis inbioreactor is the development of miniaturized sensorsfor in-situ measurement of pH, dissolved oxygen (DO),foam etc.PARAMETERS MONITORED ANDCONTROLLED IN BIOREACTORS TEMPERATURE DISSOLVED OXYGEN PRESSURE pH FOAM
TEMPERATURE Cultivation temperature is normallymonitored with an accuracy of not lessthan ±0.5˚C. Stainless steel Pt100 sensors areused Other temperature measurementdevices:GLASS THERMOMETERSBIMETALLIC THERMOMETERSPRESSURE BULB THERMOMETERSTHERMOCOUPLESMETAL RESISTANT THERMOMETERS (THERMISTORS)
TEMPERATURE CONTROLWater jackets or pipe coils within the bioreactorare used as means of temperature control.Temperature is measured by the sensor andsignal is sent to temperature controllerThe set point is entered in the controller whichis compared with the measured valueEither heating or cooling finger of bioreactor isactivated to slowly decrease the error andbring the measured value close to set point
DISSOLVED OXYGEN Measured by DO probe. DO electrodes measure partial pressure of dissolved oxygen. In the event of low oxygen tension in broth, more oxygen ispurged in bioreactor and/or agitator speed is increased. Polarographic electrodes Phase fluorometric oxygen sensorpH Only sterilizable electrodes are used. Combined glass reference electrode: silver/silver chlorideelectrodes with KCl as electrolyte. The control of pH values is ensured with the help of peristalticpumps, correspondingly metering out acid/alkali.
PRESSURE Industrial bioreactors are designed to withstand aspecific working pressure. Pressure measurements are required as a factor ofsafety. It is important to fit the equipment with devices thatsense, indicate and control pressure. Pressure measuring sensors: Bourdon tube pressure gauge Diaphragm gauge Piezoelectric transducer The correct pressure is maintained by regulatory valvescontrolled by associated pressure gauges.
FOAM The appearance of foam is very undesirablephenomenon, since, there is a risk to lose an essential part offermentation broth. During foaming, it is not possible to perform high qualityanalysis and measurements. ELIMINATION OF FOAM: Additional metering of antifoam based on sensor: Probe is inserted through top of bioreactor: stainlesssteel rod set at a defined level above the broth surface. When foam rises and touches the probe tip, pump isactivated and antifoam is released into bioreactor. Mechanical metering of foam: Mechanical antifoam devices: discs, propellers, brushesor hollow cones attached to agitator shaft above the brothsurface. Foam is broken down when it is thrown against the wallsof the bioreactor.
Category Example ChemicalnatureRemarksSilicones Antifoam A (dowcorning ltd.)Polymers ofpolydimethyl-siloxane fluidsVery active, inert,highly dispersable,low toxicity,expensivePolyethers P400, P1200, P2000(dow chemical co.)Polymers of ethyleneoxide & propyleneoxideActive, but varieswith fermentationNatural oils and fats Peanut oil, soyabeanoilEsters of glyceroland long chainmono-basic acidsNot very efficient,used as carriers forother antifoams, maybe metabolizedAlcohols Sorbitan alcohol Mainly alcohols with8-12 carbon atomsNot very efficient,may be toxic ormaybe metabolized
PRODUCT MICROORGANISMBIOREACTOR VOLUME(Litres)REFERENCEOrganic acids Escherichia coli 22000 Enfors et al, 2001Glutamic acid Corynebacteriumglutamicum24000 Hermann, 2003Lysine Corynebacteriumglutamicum10000 Pfefferle et al,2003Xanthan Xanthomonascampestris3000 Herbot, 2004Ethanol Kluyveromycesmarxianus1200 Singh et al, 2002REPORTS ON THE INDUSTRIAL USE OFSTIRRED TANK BIOREACTOR
AIRLIFTBIOREACTORo The content is agitated bya stream of air.o Gas stream facilitatesexchange of materialbetween gas phase andthe mediumo Oxygen is transferred toliquid and reactionproducts are removedthrough exchange withgas phase.
SECTIONS OF AIRLIFT BIOREACTOR4 sections with different flowcharacteristics RISER: the gas is injectedat bottom of this section andflow of gas and liquid isupward. DOWNCOMER: this sectionis connected to riser atbottom and top. The flow ofgas and liquid is downward. BASE: the bottomconnection zone betweenriser and downcomer isbase. GAS SEPARATER: thissection at the top ofbioreactor connects riser todowncomer, facilitatingliquid recirculation.
COMPARISON WITH STIRREDTANK BIOREACTORS Higher efficiency in mass transfer Easy to scale-up Require less energy to operate As stirred tank bioreactors grow in size, theirmixing quality suffers. On the other hand, themixing time is not compromised with airliftbioreactors.
COMMERCIAL AIRLIFTBIOREACTOR DESIGNS USED ININDUSTRIESCONCENTRICDRAFT TUBEAIRLIFTBIOREACTORTOWERLOOP AIRLIFTBIOREACTOR
CONCENTRIC DRAFT TUBE AIRLIFTBIOREACTOR Most industrial airliftbioreactors are of this type. Draft tube functions asaerated section Air sparged liquid rises upthe draft tube, is partiallydegassed and flows downthe annulus.
TOWER LOOP AIRLIFTBIOREACTOR Air sparger riser columnphysically separated fromdowncomer. 2 vertical columns ofdifferent diametersconnected at the top withdegassing zone and atbottom with liquid return line. Luttmann et al (1982)developed a steady statemodel for mass productionof bacteria and yeast.
CASE STUDY Chang (2010) investigated the production ofethanol by Antrodia cinnamomea in 500Lairlift bioreactor. 17µg/ml ethanol extractswere produced after 28 days of cultivation. Liu et al (2003) studied the productionchitinase by Verticillum lecanii in 600 L airliftbioreactor. At the aeration rate of0.9vvm, 19.9mU/ml chitinase activity wasobserved.
BUBBLE COLUMN BIOREACTOR Gas in the form ofbubbles come incontact with liquid.Purpose is mixingthe liquid andtransfer ofsubstances fromone phase to other. Cylindrical vesselwith gas distributorat bottom. Gas is fed into thecolumn at thebottom and rises inthe liquid, escapingfrom it at the uppersurface.
REPORTS ON INDUSTRIAL USE OFBUBBLE COLUMN BIOREACTORSPRODUCT MICROORGANISM REFERENCEEthanol fermentation SaccharomycescerevisiaeOgbonna et al, 2001Organic acids(acetic, butyric acid)Eubacterium limosum Chang et al, 2001Thienamycin Streptomyces cattleya Arcuri et al, 2002Acetic acid Acetobacter aceti Sun et al, 1998Glucoamylase AureobasidiumpullulansFederici et al, 2000
SOLID STATE BIOREACTORS Simple technology Product yields are usually higher Lower chance of contamination due to low moisture levels Easy product separation Oxygen is typically freely available at the surface of the particles. Energy efficiency Resembles natural environment for microorganisms. Use of waste materials as substrates No foam generation Lower capital operating costsSOLID STATEBIOREACTORSTRAYBIOREACTORPACKED BEDBIOREACTORROTATINGDRUMBIOREACTORAGITATED ANDFORCEFULLYAERATEDBIOREACTOR
The top of tray is opened and bottom & sidesmay be perforated for aeration. Temperature is regulated by circulatingwarm/cool water as required. Relative humidity is controlled by passingsaturated or dry air through the chamber. Height of substrate in tray ranges from 5-15cm. Scale up is achieved by increasing the areaand number of trays. Large scale processes use a large number oftrays of same size that are used in laboratory.
PACKED BED BIOREACTORS Operated under conditions of forced aeration,in which air is blown through a sieve, but thesubstrate bed is not mixed.
On the basis of heat removal considerations, the column maybe covered with water jacket that would be called aTRADITIONAL PACKED BED BIOREACTOR, or use heattransfer plate inserted into the bed, which is called ZYMOTISPACKED BED BIOREACTOR. In traditional packed bed bioreactors, there is a problem ofheat removal.
ZYMOTIS PACKED BED BIOREACTOR Best suited forindustrial operations. Packed bedbioreactors withinternal cooling platesfor heat transfer. Small spacingsbetween plates areused in order toachieve highproductivity Cooling water isvaried duringfermentation inresponse to bedtemperature
CASE STUDY Roussos et al (1993) studied the design andevaluation of zymotis bioreactor at differentcapacities for cellulase production byTrichoderma harzianum , which gave similarperformance as in the parallel fermentationunder optimized parameters in columnfermenter of high efficiency.CAPACITY (kg) CELLULASEPRODUCTION INZYMOTIS (IU/g)CELLULASEPRODUCTION INCOLUMN (IU/g)4 133.54 131.368 135.26 131.6410 128.03 125.8112 74.16 71.85
ROTATING DRUM BIOREACTOR Bed of bioreactor is mixed either continuouslyor intermittently and air is circulated throughhead space of the bed.
Consist of a cylindrical drumlying horizontally Drum is partially filled with abed of substrate and air isblown through headspace. The drum rotates around thecentral axis to mix the bed. Intermittent mixing bioreactoroperates like a traybioreactor during staticperiod and like a continuousrotating bioreactor duringperiod of rotation. It is necessary to limit theheight of substrate bed inorder to achieve good O₂ andCO₂ Might include the use of baffles
CASE STUDY Kaloris et al (2003) studied the production ofcellulases and hemicellulases byThermoascus aurantiacus in an intermittentagitation rotating drum bioreactor. The effectof initial moisture content, temperature andairflow were studied to find the optimumconditions for industrial production. Mitchell et al (2002) studied the growth ofAspergillus oryzae in rotating drum bioreactor.It was found that the initial velocity of rotationneeded for 24L bioreactor is 0.0023m/s andfor 2200L bioreactor is 0.4m/s.
AGITATED AND FORCEFULLYAERATED BIOREACTORS The bed of bioreactors is agitated and air isblown forcefully through the bed Combination of agitation and forced aerationhelps in avoiding temperature and moisturegradients in the bed.CONTINUOUS MIXING, FORCEFULLYAERATED BIOREACTORSINTERMITTENT MIXING, FORCEFULLYAERATED BIOREACTORS
CONTINUOUSMIXING, FORCEFULLY AERATEDBIOREACTORSCONTINUOUSLYSTIRRED AERATEDBEDROCKING DRUMBIOREACTORGAS-SOLIDFLUIDIZED BED
CONTINUOUSLY STIRREDAERATED BED: Used for ethanol production Not used for fungi because ofdamage due to continuousmixing.GAS-SOLID FLUIDIZED BED: Gas is blown upwardsthrough perforated baseplate to fluidize the substratebed. The gas flow rate is highenough to give good heatand mass transfer betweenthe substrate particles andgas-phase.
ROCKING DRUM BIOREACTOR Consist of substrate held between two perforateddrums encased in an unperforated outerbioreactor shell. The outer two drums are rotated backwards andforth in relation to the inner drum at 0.2 rpm.
INTERMITTENT MIXING, FORCEFULLYAERATED BIOREACTOR Similar to packed bed, except that the bed containsan agitator. INRA STIRRED BED DESIGN: large scaleintermittent mixing, forcefully aerated bioreactor. Agitators are mounted across the width on a movabletrolley, which moves up and down the bioreactor. The speed of movement of trolley affects the intensityof mixing. Used for enzyme production and biopesticideproduction on large scale.
REPORTS OF THE USE OF VARIOUS SOLID STATEBIOREACTORSBioreactor Key processes and bioreactor features ReferenceTray Alkaline protease production by Aspergillus flavus on 30 kg steamedwheat bran in perforated steel trays in koji roomMalathi et al,2001Zymotispacked-bedCellulase production by Trichoderma harzianum on 40 kg sugarcanebagasse and wheat bran mixtureRoussos et al,2003Continuouslyrotated drumKinetic study with Rhizopus oligosporus on steamed wheat bran, in astainless steel rotating drum with detectable bafflesFung andMitchell, 2005Intermittentlymixed aeratedbedProtein enrichment by Aspergillus tamari on 25 ton moist sugar beetpulp in a 50 m3 stirred packed-bedXue et al, 2002Continuouslymixed bedEthanol production by Saccharomyces cerevisiae on cooked corn grits ina continuously stirred bioreactorSato et al, 2008Air-solidfluidized bedEnzyme production by Aspergillus sojae on 500 kg dry wheat bran in an8 m3 bioreactor.Matsuno et al,2003
PLAFRACTOR™ The bioreactor is modular in natureand carries out all the processes offermentation in a single containedenvironment. Constructed by stacking individualmodules and the base containsmultiple channels to deliver fluidsinto modules and to extractproducts from modules. The interior of each module has amixing arm that revolves aroundcentral axis of module.Mycophenolic acid: PenicilliumarenicolaCyclosporin A: Fusariumsolani
CONCLUSION Bioreactors, the core of bioprocess, are ofsubmerged and solid-state type. In submerged type, stirred tank bioreactors arethe most commonly used in fermentationindustries. Solid-state bioreactors have gained widerattention from industries due to simpletechnology and higher yields. The parameters controlling the fermentation arestrictly monitored in all the bioreactor designs inorder to ensure maximum productivity.