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    17 views23 pages

    Large Scale Cultivation Using Bioreactors

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    akhil

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    © All Rights Reserved
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    Large Scale Cultivation of Plant Cells Using Bioreactors Se INTRODUCTION Te femestaton of microbial els to produce primary metabolites (eg amino acids and ofganic SG) amd scondary metabolites (antbiotis)has the long history, where asthe application oF Smentgciniues for the production of plant metabolites using plant tissue culture for use as Peemsretteal, flavors, colors of recent rig. Several new i to plant products have jut Teomdagemcommerial production levels (Table 20.1). Large-scale commertial production of SSzpdary compounds from plans in-vitro is recognized for Shikonn, Berberine, Ginseng saponins Tle 201. Commer and omega anionic esas sconay ett prosucton schemes for plant cells in vio. e; yi z Frode Cal ere sree Wioreairnarinan Vanes Frodo Frodhet Cll eure seureeBiorsactorinasimun volumes Prodwesr Berberine Thatciram minus, Batch & contionus Now; impeller Mitsui Petrochemical Cops jepontca”—_divens000 1. Shikoin Lithospermm Batch 750 a1 envtirerkison Ginseng saponins Panar ginseng Cll & ot cles 2,000 Vann anita plansoa impel dven reactor 721. Taxol Taxus revolt: sage sytem imple driven Teas 5p. and aii reactor sytem 2500 L7s000t Nippon Oil Company, pun, Samyang Genex Co, Lid, Korea Anthocyanins Euphorbia milli Rotary exture system Nippon pat Company, Japan Sanguinarine Paper sommiferam iif system 300 1 Vipon Reseach Las, USA, Digoxin Digualts lana Bocvinger, Gemany Rosmaviic eid Coleus lume) Nattermana, Germany ‘20 PEP ROT RP P-2-2 0.2 © oa a am om a. SPIT PEP FF NNN PUTT ELUDES LTO UYwe ee ev — Large Scalo Cutvation of Plant Colls Using Bioreactors 421 (One factor that is particularly important to note is that plant cell cultures are “totipotent”. I is, Doss to produce unorganized, dediferentiatd callus or suspension cultures from differentiated ‘hole plan material it also posible to regenrate whole plants from ths ddiffereniated plant ‘material, With animals terminally differentiated cells (c.g hepatocytes) can not be used to regenerate ‘ther cell types, The ability of plant cells tobe continually able to respond to development claesis an imporaat factor t consider in bioreactor stay. Related factors are the possibilities of cell-to-cell communication, Plant cells often grow inlarge ‘Aggregates and diffusional limitations may result in concentration gradients of key nutrients or metabolic by-products. Such gradients may result in microenvironments that foster cellular response to developmental lus, Further, the cells in a plant tse ae inferconneted With plasmodesmata ‘which are channels that interconnect the eytoplasm of plant cells ima tissue; the plasmodesmata allow the more rapid exchange of low molecular weight metabolites (<800 kDa) than would be Possible by simple diffusion (Table 20.2). Plant cells have a large central vacuole and typically sec- "eteproduts of interest wt such vce, hie anima els pall secret product of iret int the extacellular compartment. Tyble 292. Comparion tay cle ccs aman alma anne cues (Sher 's transformation pecesary to exh No No Yes ‘continous cell lines? y Conse inibiton? DGromth Monolayer Spiealy Monolayer for sonal els Cryopreservation Dita, Fairly ouine Fully rowine 's difereision of cls Inve? No Yer Yes Aszrepatefomation in Suspension? Yeu Unusual Unusual Lage ental vaso presen? Ye No No Catia Yes No No Interettcomectons Pasnodesnaia Gap,jontions ‘Sap junctions Si 15150jm 1020 10}m Doubling tine 24-1008 14268 1030 “Typical tine cel asin supension I0s0gdwh 2 2 yao Large Soele Cutivation of Plant Cols Using Bioreactors 423 i eon 4 ost (trot ee a cont le So ines — Sy tt Sobre Creating Fig. 20.10. Continuous farmenter(Wison et el. 1971). 20:33. Aeration and Agitation ‘The primary purpose of aeration shouldbe to provide plant cells/microbial cells in fermenter wath sulicient oxy for metabolic requirements, while agitation should ensure that a uniform suspension, ‘of microbial cells/plant cells is achieved ina homogenous nutrient medium, The type of aeration — ‘agitation system depends on characteristics of the fermentation proces. ‘Aeration and Agitation system contains 1, Theimpellerogiator) 2, Stier glands and bearings. 3. Baffles | The aeration system (sparse) 2033.1. Impelter ‘The functions ofthe impeller are: 1. To diminish the size of si bubbles to give a bigger interfacial area of oxygen transfer and to secrease the diffusion path. —_—_ 2. Tomaintan uniform environment throughout the vessel contents, Impellers maybe classified as disc turbines, vaneddises and open turbines of variable pitch and properties (Fig 20.12). The most commonly used necessary to consider the size ofthe imept er and Thhere to postion iin the vessel In all vessels more than one impeller isncededifadgquateseration {sitaton Isobe oblained. Leal the impeller shouldbe one third to one-half ofthe vessel diameter above the baso-of the vessel. 480 Modicinal Plant Biotochnology ‘Aspe ‘eclaton Pe abe b+ —+4 Fig. 20.11. Typical design of bioreactor. / 2033.2. Stirrer Glands and Bearings The satisfactory sealing of the stirer shaft assembly has been one of the most difficult problems to -vercome in the contruction of fermentation equipment, which can be operated aseptically for long. >eriods. A numberof different designs have been developed to obtain aseptic seals. The stirrer shaft ‘an enter the vessel from the top, side (Richards, 1968) o bottom of the vessel. Top entry is most commonly used but bottom entry may be advantageous if more space is needed on the top plate for tntry ports, and the shorter shaft permits highly stirrer speeds to be used by eliminating the problem of the shaft whipping at high speeds. In general, bottom entry timers would be undesirable, as the Searing would be submerged, although Cain etal (1952) has successfully operated vessels ofthis ‘ype. The vatious aseptic seals used for this purpose are packed gland seal, bush seal, mechanical seal and magnetic drives. 203.33, Batfles Four baffles are normally incorporated into agitated vessels ofall sizes to prevent a vartex and to improve aeration efficiency. They are metal stripes roughly one-tenth ofthe vessel diameter and ‘wall. Walker and Holdsworth (1958) recommended that bales should be attached radially to installed so that a gap existed between them and the vessel wall, s0 that there wasa scouring action round and behind the baffles thus minimizing microbial growth dn the baffles and the fermenter walls, 20.3.3.4. The aeration system (sparger) ‘Asparger may be defined as a device for introducing ar into the liquid ina fermenter. tis important OUR eeeanan. . ~ - dur 2d qT =PreP Ps ‘ , b , » , > , > a > > , » > NS Large Scale Cutivation of Plant Calls Using Bioroaciors 431 =] Fig. 20.12. Types of Impeller. a) disc turbine, b) Vaned dis, ¢) Open turbine d) Marine propek {Solomens, 1969) gH ae ae gh {o know whether the sparger isto be used in its own or with mechanical agitation as this can influence equipment design to determine intial bubble size. Three basic types of sparger have been used and may be described asthe porous sparge, the orifice sprager (a perforated pipe) and the nozzle sparget (an open or patally clase pipe). A combined sparge agitator may be used in lsboratory fermenter, 20.4, THE ACHIEVEMENT AND MAINTENANCE OF ASEPTIC CONDITIONS The following operations may have to be performed to achieve nd maintain aseptic conditions during fermentation. 204.1. Sterilization ofthe fermenter ‘The fermenter should be so designed that it may be steam sterilized under pressure. The medium ‘may be seized inthe vesel or separately, and subsequently added asepialy Ifthe eho ce 482 Mesicinal Plant Botctnoogy strlized inst its temperature shoud be ra fomtion of large amount of condense. ss Allpipes shoul be constructed as smply a posible and slope towards drainage points oma surest reaches al part ofthe equipment an is at echt by sponsor poke of condense ptior tothe injection of ive steam to prevent the 2042. Sterilization ofthe sir supply S Sterile air will be required in very large volumes in many aerobic fermentation processes. thou ‘there are a numberof ways of sterilizing air, only three have found permanent application. These are hea filration through fbrous material and ilraton through granular material. 20.43. Aeration and Agitation (s09 deta on20:3.3) ‘ 20.44. The addition of inoculum, nutrients and other supplements To prevent contamination during an addition itis essential that both the addition vessel and the fermenter are maintained unde a postive presure andthe adition ports equipped witha stam 20.45. Sampling ‘Sampling por is keptin 40 % formalin ora suitable substitute for aseptic conditions. 2048, Foam contol Jn any fermentation tt very important to minnie fang. When foming becomes excessive thee a danger tha fiers become et euing in contamination. Tee a5 the possibly of siphoning developing leading to the loss a all or part of the contents to the fermenter. [tis common practieto add antifoasoafementer when theca bing aboveacran pede Feel. Ves atch to ements nd anly equiped fo cooing the ow of gus and gos Ball valves Ned vals pounales ce 20.5. IMPORTANT FACTORS FOR BIOREACTORS 20.5.1. Gos-Liquid Mass Tranter Most bioreactors are rated in tems oftheir oxygen mus tansfer coefficient. K, sale - up of ‘many bioprocessesis based on mantinng constant K, The gol for culturing plsitcellisoeno ‘maximize the oxygen ransfx coefficient. Tiss achive by using mechanical agitators at high ional speeds andby spaying ge vue of a (eg 1 volume of gs per minute per volume of liquid) Since plant cells have lower esprton rates. The oxygen transfer requirements are considerably les. fells whic respi ata rae of 02 m ml/h are to grown to 10g/ without Sllowing the dissolved oxygen concentration ofall below 20% af saturation The equation suggest that te Ky, should be _QomaxX _ _ O2mmel _ (19.97) /(0.25 mmol ~02 (0.25 mmol] - 10k" ke ee a ee (02s: Plant cll bioreactors are typically operated st K,, values of 10:30 br’. Operation at higher, ‘aluestas often Been observed forest in pore plait ell growth (Tanaka 198, 1987, Smart and Fowler 1984) or product synthesis (YamaKewa et al, 1983). This poor performance at high K, ‘Values may be due to either increased shea associated withthe high-K,, conditions orto enhanced Carbondoxde stripping from the liquid (Hegarty ea, 1986) Se a (TTT TT eee oC beer LET) Large Scale Cutvation of Plant Cols Using Bioreactors 433, 20.5.2. Shear Sensitivity and Rheology Because of ther large size, extensive vacuole, and rigid cell wall plant cells have been regarded as sensitive to shear stress. It is difficult to define what is meant by shear. Shear refers to forces ‘exerted on the surface of body in a direction parallel tothe surface. Ths s in contrast o the normal Trees, which are exerted on a surface but perpendicular to the surface. ‘Shear is described by the equation T=ne : Where T = shear stress (a force per unit surface area) hear rte (a change in velocity across a distance) 11 Viscosity (a coefficient which describes the resistance to flow) ‘Todescribe the shear damage to eukaryotic cells in mechanically agitated systems shear damage resulted from “highly localized velocity gradien's (Midler and Finn 1966) and these localized gradients or the maximum shear rates could be related tothe speed with which the outer edges ofthe impeller blade traveled (Oldshue 1966). Impeller tip speed is given by the equation Tip speed = x Nai ‘Where N is the Impeller speed (rpm) isthe impeller diameter In adition to hydrodynamic shear, recent studies reveal that cell damage may result fom ell cell and ce-impeller collisions (Chery and Papoutsakis 1988, Prokop and Rosenberg 1989). Shear an also results from gs sparging even inthe absence of mechanical agitation. ‘The’ theological behaviour of certain plant cell eutures have been determined, for example “Morinda irifolia howe shear inning and thixorophic behavior wheres Cudrania tricuspidata, Vinca rosea and Agrostonma gthago were non-Newtonian and pseudoplastc. In these studies the ‘heologital properties of thecatures could be estimated using normal rotational vscometers such as the cup-and-bob system. However, plant cell suspensions ae in general highly aggreated. As a ‘consequence ofthis particulate nature, the normal methods of measuring viscosity, i, the cone- and-plant or cup-and-bob viscometer ar sully inadequate, because the agaregats sete rapidly or become trapped in the naow gaps inthe viscometer. Alfemative designs of viscometer using either anchor an turbine impellers that clminate the problems ofseting have been used but they 40 not give a direct measurement of viscosity because ofthe turbulent flow produced. Using both alternative types of viscometer, plant cell suspensions have been shown fo be non-Newtonian and pseudoplastic having alow vscsiyin the order of I-10 pas. 20.5.3. Mixing Mixing refers tothe convective transport of matter (e.g transfer of solutes associated with bulk fluid motion) The mixing of dissolved nutien's e.salls and sugars) has generally not been a problem in suspension culture. There are thre important mixing problems in suspension culture systerm. 1. Because of the large size of plant cells and especially cell aggregates which lead to setting atthe bottom of the bioreactor. These cells can then setled into dead zones or unmixed regions of the bioreactor. These dead zones.can become depleted of key nutrients (eg, dissolved oxygen). 2. Attachment of cells onto surface above the level ofthe liquid. Siice these cells are not bathed in the liquid mediurn, they can become deprived of nutrients 3. Because of the desire to culture cells at high concentrations. With moderate cell concentration (packed cll vlumes less then 50%), bringing cells in contact with the nutrient-containing Liquid 494 Medicinal Plant Betecnology seuct.ota problem, However, incomplete mixing can bea serious problem when plant cel is ‘cultured of high concentrations. i ce re 206. TYPE OFBIOREACTORS. eee in culturing plant cel, whether in hake flask orn bioreactors to provide conditions {ee culture which allow it perfor ina repoduibe and optimal manner. Various measures OF pang tal maximum cell concentrations, Isecondary metabolites are desired, then an appropriate Pemance measure would be the overall poductiviy, othe amount of product produced pe tal

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