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Module 1: Plant Cell Bioreactors

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Immobilization of Plant Cells

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So, one of the other strategy which can lead to productivity enhancements in plant celltechnology is Immobilization of plant cells.So, the use of high biomass levels for extended period, so which involves a number of cycles. So, would be one method to increase productivity and hence reduce the cost because, you canuse the same biomass for a number of processing cycles.So, this would intern reduce the cost , this can be achieved by immobilizing the cellsthere are different ways in which the cells can be immobilized.And these immobilized cells generally the kind of reactions which are carried out usingimmobilized cells involve your bio transformations or your bioconversions.Where the precursor will directly get converted to your product of interest or synthesis fromprecursor with the more number of more than one number of steps, were the precaution willreach the cells and being a repository of enzymes there will be said defined of reactionsleading to the product formation or De novo synthesis.Which means that as a result of the metabolism of the cell and the development of the cellthe substrate which is added, finally gets metabolized as we have been seeing in freecells into your desired product.So, this is how the immobilized beads look like.Now, what are the advantages of plant cell immobilization, continuous reutilization canbe done of the plant cells.Now here the key or the condition which has to be taken care is the viability of the cells,how many number of cycles they can remain viable.So, that you can use them or the cells do not leach out from the immobilized beads.Now, high biomass levels the immobilization of cells it will allow the use of higher biomasslevels for the bioconversion.Generally for free cells because we know there are certain limitations in in the scale ofa plant cell fomentations like the mass transfer limitation cells tends to aggregate.So, to get highest cell density for maximum productivity and then converting it for productproductivity is sometimes limited depending on the species on which we are working.Now, but in case of immobilized cells and if the production phase can be separated fromtheproduct production phase can be separated from the biomass production phase.Then if you have high cell density than you can use those cells immobilize them in theform of beads and then make it into a packed bed reactor and use the substrate of the productionor the production medium and continuously utilize that high biomass density therebyleading to higher productivity levels.So, separation of cells from the medium it is one of the advantages of plant cell immobilization,which will reduce what which can reduce your downstream processing cost or which can easeout the downstream processing.Now advantage other advantages it thereby makes your process continuous and therefore,increasing the productivity.Continuously you can have the product leached out from the cells in the effluent which isleaving the packed bed Decoupling of growth and product formation.So, actually it works for those very well for those fermentations where the productformation phase and the biomass formation phase is decoupled.Reduces what problems therefore, in comparison to free plant cell suspensions it can reducethe problem of aggregate formations, then foaming which can happen and the scale upto reach to high cell density the shear effects on the cells.Because of the moving parts of the reactor, so thereby on the viability and biosyntheticcapability of the cells, so susceptibility to mechanical damage can be avoided.So, what are the disadvantages now apart from all these advantages that are certain disadvantagesalso associated.Now what are those there can be cellular transport or artificially altered membrane permeability.Now what happens that you need to ensure that the secondary metabolites the process willbe successful only if the product comes out, so which needs the membrane to be permeablefor the product.So you need to alter the permeability of the membrane which may need to work around withthe kind of immobilization which you would use.So, that can incorporate cost or may sometime even if suppose leaching happens of the cellsbecause, you are altering the cell permeability oh sorry the membrane permeability then thecells may also leach out or the viability of the cells might get affected.Then the efficiency of the production process, now rather than depending on the conversionrate of the substrate to product it.Now is found to be rate limiting step is found to be the rate at which the product is leachedout from the and capsule or the capsule to the outside medium.So, the rate of release of the product rather than the actual rate of biosynthesis, nowbecomes the rate limiting step.The immobilization process may lead to reduce in the biosynthetic capacity, why do you thinkmay lead to reduced bio synthetic capacity?It depends on the micro environment which is produced around the biomass inside thatencapsulation, this may lead to maybe the material which has been used or there canbe inner gradients nutrine gradients gas transfer which may get affected, which may in turnaffect the biosynthetic capability of the immobilized cells.Now this is same whether it is plant cell or microbial cell.Products must be released from the cell into the medium that is the prerequisite, the producthas to come out then only the process is continuous higher productivity all that holds true.The micro environment favoring optimal production can be unfavorable for the release of secondarymetabolites, which may then cause what?If the rate of release is not able to balance out the rate of biosynthesis it may lead toproduct innovation, which may intern cause reduction in the biosynthesis or it will notbe able to drive the rate of biosynthesis forward.So, in this way that is the bottleneck, the product has to come out of the immobilizedbead.The prerequisites therefore, for successful immobilizations include what non growing cellsmust produce product, which means they the growth phase and the production phase haveto be decoupled it has to be a non growth associated product formation.Products must be released from the cell into the medium.Now when we say that it has to be non growth associated product formation, why it cannotbe growth associated product formation in the immobilized bead.Why am I saying decoupling is needed they will have a limitation, if they start growingthen the oxygen demand will keep on increasing.So, then the steps which involve mass transfer and oxygen transfer of the nutrients or thegas will start becoming your rate limiting steps.So, as I said the need for immobilization in plant cell fomentation is because of theirsensitivity to shear because of the large size, then the need for them it has been observedI told you that the biochemical capability is also dependent on cell to cell contact.So, sometimes immobilization gives them biochemical signals which improve the secondary metabolismin plant cells.Because, you attached to supports which may lead to differentiation those signals canbe provided, such that your synthesis of the secondary metabolite is induced or accelerated.Immobilization can overcome many of the limiting factors like easier separation of the biocatalyst from the product.So, that is one of the advantages which the immobilization can provide or they are moreplant cells are amenable for biotransformation of low value compounds to high value products.So, it can it is found to be very useful in case of immobilized plant cells, where biotransformationcan be easily done till the plant cells remain viable in the immobilized beads.So, what are the different ways in which immobilization is done, either directly binding to a naturalsubstance or using natural affinity.For example, adsorption adhesion agglutination, I hope you people know what is adsorptionadhesion and agglutination.Agglutination anyone can help chaithanya on this?This is surface phenomena is not it.So, involve surface energies in simple words agglutination means comingtogether and thereby.form any like bonds . There is no are there a chemical bonds formedin this.What is responsible for adsorption adhesion, adhesion is what?Attraction of one substance to.different To a similar kind of substance to or to another.to different different different different material.Fluidsa solid.in a solid Hmdifferent Where do you see adhesion everyday commonexample.what a mean surface, what a mean?Very good.what a mean . Cohesion is the attraction intermolecularforces and intra molecular forces.So, inter molecular forces is the interaction between same atoms and intra would be betweendifferent.So, when it is attracted adhesion is cohesion similar or adhesion similar.cohesion similar cohesion adhesion So agglutination is also a similar interactionwhich may lead to same kinds of molecules coming together . Now covalent coupling thisis where chemical bonds will form now covalent coupling with the inert matrices.So, using functional groups, for example this may the functional groups may be responsibleon the inert surface or the covalent coupling of the cells to the inert surface.Then connection via by or poly functional reagent which is called as cross linking,which means you can have a polymer matrix and yourselves.Now these polymer matrix it is these chains will join to each other to form a matrix usinga cross linker.Now this is like poly functional agents they will have different functional groups whichwill facilitate joining of these polymer chains, once these polymer chains interact then theywould form a matrix and if the cells are captured within that matrix then they will get encapsulated.Mixing with suitable materials which can change it is consistency with pH or temperature exampleexample.agar Agar gelatin physical retention within theframework of diverse pore size and permeability which is called as entrapment or microencapsulation,which you people must have done earlier in your lab classes which is like.sodium.Sodium alginate.Now the polymer material what is to be taken care is, the polymer material which is usedfor immobilization first must be available in large quantities easily available . Itshould be inert such that it does not affect the viability of the cells the biologicalmaterial, should be non toxic to the biological the plant cells and should be economical.It must be able to carry large quantities of biomass and it is fixing potential shouldbe high, the retention potential of the cells should be high.The immobilization process must not diminish the enzymatic activity of the biological catalystah.Let us take an example of enzyme immobilization; you must have already read that the kind ofimmobilization or the immobilizing matrix may sometimes impact the activity site ofthe enzyme., thereby disrupting the function of the enzyme.So, similarly the plant cells actually these are repository of enzymes.So, it should be taken care that the biological activity of the cells is not impacted by itis interaction with the cross linker or the inert matrix.If it is a chemical bonding or the cells should be easily available for biotransformationreaction , manipulation of the biological catalyst must be therefore as simple as possible.So, what are the different ways in which entrapment can be done gel entrapment by polymerization.A monomer what is it a monomer or a mixture of monomers is polymerized in the presenceof cell suspension, which is entrapped inside the lattice of the polymer, example polyacrylamidegels.Now it is said that the everybody knows what is polyacrylamide it is a polymer.So,it is based on what kind of polymerization happens to make it into a gel hm.It is called as free radical polymerization.Now in this care is taken that the polymerization process in the presence of cell suspensionis carried out in the absence of oxygen and at lower temperatures.So, because the free radical monomer which is responsible to form the free radical polymerare may interact with oxygen preferentially and thereby causing an oxygen radical andthen the polymer rate the polymer formation rate reduces drastically.Therefore, it is recommended to be carried out in the absence of oxygen.So, the free radical polymerization of acrylamide is conducted in an aqueous solution containingthe cells and the cross linking agent.Cross linking agent generally used is N, N' ethyl bisacrylamide.So, different polymer chains can be connected with the cross linkers so as to form a latticeor a matrix.Gel entrapment by ionic network formation, the most common method is entrapment in calciumalginate.So, what happens it is the non toxic process in which sodium alginate solution containingthe cell suspension is dropped in a mixture of counter ion.Such as calcium chloride a uniform spherical and high micro porous structures will resultso disruption of gel, now if I want to disrupt the gel . So, it can happen if something elsecan disrupt calcium.So, that is going to happen if you are using any other divalent ions like magnesium oreven people say one should avoid sulphates.So, you it may happens of which gives us what indication that even if you are using an immobilizedcalcium alginate bead for plant cell fermentation, the media composition in which the beads aregoing to be put in can disrupt the beads depending on the concentration of your magnesium isone of the major salts used in.Sulphates magnesium these are used in your media for plant cell fermentations.So, which they which means what it may impact the the bead structure , so care should betaken.Gel entrapment formation by precipitation , gels may be formed by precipitation of somenatural and synthetic polymers by changing one or more parameters in the solution.Like either you change the temperature or salinity or pH of the solution then the precipitationmight happen some disruption in viability is expected in this process.So, after doing this a check on the viability should be done.So, we have already studied about different methods to check the plant cell viabilityfree plant cell viability.What were those methods ?Schezwan is over.Schezwan over everything gone I have made my head blank.So, that now I can fit and rest of the material in it.And omit it when required or that will also happened two days before hm ok . So, entrapmentin preformed structures can also be done in which hollow fiber , an example is hollowfiber reactors they can be use to immobilize plant cells in by entrapment.So, generally they work on the principle of shell and tube bioreactors, there will bea number of thin very thin fibers through the fibers the media is send in and aroundthe fibers on the shell side plant cells are immobilized.Now to facilitate immobilization around these fibers evenagents like polyurethane foam areused.Now once the cells begin to divide and grow they will multiply and they will start growingaround these fibers.Now these fibers through which the media is flown in they are semi permeable membranes,through which the nutriens can flow from the inner tube to the outer lumen.And similarly the product which forms after the bioconversion can diffuse in in thesefibers and come out of the reactor in the effluent.So, they work on the principle of shell and tube reactors generally shell and tube isthis term is used in which cases.heat exchangers heat exchanger.Heat exchanger the cells are even added to performed polymerized structures such as polyurethaneto preformed polymerized structures which are such as polyurethane foam.So, why do you think the cells are able to stick to polyurethane foam.Have you seen polyurethane foam?There are different forms of polyurethane form the simplest everyday life those foamswhich are used for cleaning cars and all spongy material.So, there are lot of gaps you can see pores different size and shapes.So, polyurethane foam because of these pores which are there, the cells first enter thesepores and then as they divide the retention happens . So, when the cells actually we usethe polyurethane foam ohm for as a support for hairy root cultivations because, beingan inert matrix it was able to once you inoculate at the time of inoculation they will not stickto it.But in due course of cultivation time as they begin to multiply you will see that it willstick to the foam.It will enter the pores which are there the porous membrane and they will then stick thathow they are used as support materials for plant cells or even for immobilization .When cells in suspension are mixed with these materials, they are rapidly incorporated intothe network and subsequently grow into the cavities of the mesh and are entrapped byphysical retention physical restriction and attachment to the matrix material.There is no chemical interaction which is happening, no bonding, but it is the physicallyretention which happens as they grow . The mechanism involves first a mechanical entrapmentand later adsorption and adhesion and natural aggregation of plant cells .Surface immobilization, surface immobilization may occur on both natural and synthetic matriceslike natural would be cellulose synthetic like nylon.Immobilization by embedding this is another way, the temperature dependent solubilityof macromolecules like agarose agar carrageenan or different solubilities of the sodium andcalcium salts.Now, you will come across in literature different terms for similar methods.So but you need to understand what does it mean the concept behind the method.So, then you may whatever the terms may be given like for example embedding, embeddingcan be the cells have been embedded in a matrix.Now that can happen even by precipitation changing thenature of the matrix from liquidto solid by changing the temperature, like for example in case of agar agarose or carrageenanor by changing the pH or by using ionic interactions like calcium or sodium alginate interaction.So, insolubles are formed under cold conditions in aqueous calcium chloride solutions theirstructure is non uniform with different pore diameters at surface enact.So now, let s take an example of calcium alginate beads.So, how can you improve the efficiency of immobilization if given a chance of calciumalginate immobilization.How will you improve the efficiency in this process?What factors do you think you can play around with which will affect .molarity the efficiency.molarity of the solution calcium chloride solution strength.How is that going to affect.the ionic because the beads form because of ionic interactions.So, if we change it through it may interact it may form better beads.Better in what sense.width of Right what else what else can be done.size of the beads.The size of the beads can be changed very good .if the strength is low it it it is not that solid.That is what which will impact yeah what else.sodium alginate to the calcium chloride.So, how long we are keeping the calcium chloride solution.So, that will also the porosity also needs the time of sodium inside the gaseous howlong . So, all this is impacting what the qualityof.Like for example, the porosity will get affected maybe the cells will get leached out, thepermeability of the membrane will affected is not it.So, the viscosity of the solution concentration of alginate stirring speed and dropping aperturesize.Stirring speed how is that going to impact ?it will form smaller beads if the stirring is more.Why?surface area will increase because it will break down of .Beads will break down.forces Speed will affect what?mixing mixing Speed will affect what?what do you have what all things to you have in solution.sodium alginate Only calcium alginate and sodium alginatewe keep talking about only these two things what are you why are you doing all this?The cells uniform distribution of cells can also get impacted . So, we have forgottenabout viable by during all these.. So, I was talking about viability testing,once the beads are in place we would like to use them all the cells remain viable.How to find out how the cells or whether the cells are viable or not.So, if you remember we had spoken about fluorescein diacetate staining which in immobilized, thesesystems which are generally used it is not impacted by the membrane it is permeable tothe membrane.So, that it can be taken in and it can measure the viability of the cells.Now similarly phenosafranine it is another dye which stains dead cells.So, you can find out whether the cells present inside the beads again permeable to the membranepresent inside the beads are viable or nonviable.Then another way is looking at the respiration capability or their metabolic activity, metabolicactivity intern can be looked as the respiration ability.So, if you droppen the beads in a solution and there is a change in the specific oxygendemand or the dissolved oxygen levels, then you can assume that their viability is gettingaffected.Cell dry weight increases with known constant weight of the gel, generally because it isdecoupled you assume that not much multiplication or significant multiplication of cells ishappening.They are already now well grown and these well grown cells are immobilized .Immobilization can affect cell physiology and therefore, the production of secondarymetabolites.Process of immobilization reduces the rate of cell division and protein synthesis andthese effects are conducive for increase in secondary metabolite synthesis.So, you remember we were saying some of the advantages it can lead to also decouplingof growth and production phase.Immobilization reduces production of cell wall material which contains a substantialamount of bound phenolic compounds, which increases the availability of precursors forsecondary metabolism.So, which means what that if the cells are in division phase then a lot of phenolicsor the carbon flux has to go into the growth.But once you are immobilizing and driving the secondary metabolism the same carbon fluxcan now be utilized for secondary metabolite biosynthesis, thereby increasing the productivity.Process of immobilization causes plant cells to feel as biochemically differentiated, metabolicspecialization which controls the expression of specific enzymatic pathways for secondarymetabolite biosynthesis can be induced.mam why would immolation reduce production of cell wall material because it has cellhas material for cell wall production.Right, so why can somebody answer she is asking why would cell immobilization inhibit cellwall production.Why does cell wall synthesis or rejuvenation continuously keeps on happening and also dependenton the protection which is required for the cell to survive and also dependent on thecell division.hm hm So, when the cell division has been restricted,obviously the carbon flux flowing towards the cell wall material is also reduced.And generally because these are made of phenolics, so many a times phynolics hamper the productionof other secondary metabolites being toxic to the cells reduce variability.So, there it may improve the production on the flow of carbon flux towards the desiredother secondary metabolites.So, when the cells are in close contact they get such biochemical signals such that theyfeel differentiated, thereby leading to improvement in the secondary metabolite yield.There is a possibility that it can lead to improvement in secondary metabolite production. What are the different bioreactors which areused for plant cell immobilization Packed bed reactors.Now cells are immobilized either on the surface or throughout the support.So, even if you remember your immobilized enzymes the enzymes either can be embeddedinside the bead itself or they can be embedded on the surface.So, if they are embedded on the similarly the cells either can be attach to the surfaceor they can be embedded inside the bead.Now if they are inside the bead then if you remember your enzyme bio reactors or enzymekinetics then external mass transfer limitation and internal mass transfer limitation bothbecome crucial.But if they are placed only on the external surface then only external mass transfer limitationsare limiting.So, the cells can be immobilized either on the surface or throughout the support andthe fluid containing the substrate flows pass the support material.Now one thing is that mass transfer limit the substrate first has to diffuse to thesurface, from the surface till the inner core if they are embedded inside and similarlythe gas.A large number of cells per reactor volume can be accommodated.Low degree of mixing causes difficulty in mixing and gas transfer these are the limitations,high pressure requirements being a packed bed higher pressure drops . So, higher pumpingor suction pressure is needed , well mixed immobilized reactors is like suspension culturewith recycle or retention why?It is written well mixed immobilized reactors is like suspension free cell cultures withrecycle or retention and the cells are there only the.medium Spent medium is going out and the substrateis flowing in and it is a high cell density culture.So, better mass transfer and control of pH and temperature is facilitated; however, agitationcan cause particle collision and shear.Fluidized bed reactors they utilize energy of the flowing fluid liquid or gas to suspendthe particles.Now, in comparison to packed bed reactors fluid by a fluidized bed reactors are betterin what sense.In terms of.Mixing, energy requirement for fluidization increases with increase in particle size because,now more power is required to suspend the particles.Small immobilized particles are therefore often employed.For better mass transfer the fluid retention time in the reactor should be more.So, what is done if recirculation of the fluid can be done in the reactors.So, if the recirculation can be done you are increasing the retention time of the gas phaseor the liquid to interact with the cells for the bioconversion.Large gas volumes are used with low fluid rates, now if in order to see it is balancingbetween the merit and demerit.In order to keep the cell suspended you would like to have high flow rates.Now with high flow rats the retention time is less the time to interact with the cellsreduces.So, your productivity and your cost of production increases, because now all the substrate whichare feeding is not getting converted.So, you need to increase the retention time at the same time you need to keep it suspended.So, high gas flow rates are used, so as to create recirculation currents and therebyimproving the retention time before the fluid moves out.The shear and the particle collision may damage the beads and the complex fluid dynamic requirementsmake all though these kind of configuration scale up is difficult .Membrane bioreactors we were talking about hollow fibers, similarly spiral wound reactors.The cells are retained either within the tubes or in the outer region it can be shell sideor the tube side immobilization.The cells are retained ether within the tubes or in the outer region the inner portion ofthe thick cell layers are generally characterized by substrate deficiencies, because they keepaccumulating one on top of the other.So, this may lead to intern nutrien mass transfer and your gas limitations oxygen limitations.What else they the increase in the cell number may eventually clog the membrane pores, therebynot allowing the media exchange the nutrient and the product exchange to happen, so generallythey end up getting clogged the really.The membranes in these reactors can be re use.So, there are merits and demerits both are associated to every kind of reactor configurations.