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Module 1: Plant Cell Culture

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Plant Cell Bioreactors

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So,most of the reactors I was saying in industry are working with configurations which havealready been standardized with microbial fermentations.So, what you will observe is the same reactors are modified for plant cell cultivations,so, but one has to be clear how plants and cultivations would differ from microbial formulations.So, in terms of size, size microbial cells are pretty small, 2 to 10 micron size whileplant cell suspensions the aggregate size there can be cell clumps which can go as highas 50 to 100 cells together and the size of the plant cell itself is much larger whichis around 20 to 40 microns.So, you can imagine there is a big difference in the entire aggregated cell, the size ,whilemicrobial cell suspensions will be uniform; they do not, they are not self and mobilizingin nature.Then homogeneous suspension, that is what it is.So, microbial are homogeneous, but in plant cell cultures aggregate formation would takeplace because of which there will be eventually with high cell density you will see non-homogeneityarising in the reactors.Then growth rates in microbial fermentations it is quite rapid the doubling time is inhours while in plant cell fermentation the doubling time is in days, around 2 to 5 days.So, which also means that the reactor cultivation time would be much longer than the microbialfermentation.So, keeping a septic environment, maintaining it the sterility maintenance also becomesone of the critical factors here.Shear stress tolerance it is not that plant cells are are highly shear sensitive, in comparisonto microbial cells they areless shear tolerant.The reason being because they have vacuoles, so the cell contents are stretched onto thecell membrane.Majorly this would be the scenario when you are working with matured cells or if the productionphase is happening with the matured cells.So, there is one large vacuoles.So, the cytoplasm and the cell contents are stretched on to the cell membrane.So, there is and there is cell wall, so more rigid structure, so more brittle in nature.Thenshear stress tolerance.So, because there is presence of cellulose based cell wall which is of high tensile strengthmakes them more sensitive to shear and less flexible.Aeration requirements all those specific oxygen demand of plant cells is much less than microbialcells.So, what will happen?Initially you would not like to have as much aeration as you would see in microbial fermentations.So, because the specific oxygen demand is less, but as the cells would grow and therewill be high cell density, aggregate formation will take place.So, which will lead to what?Which will lead to mass transfer limitations.Now, in order for the oxygen transport rate to increase such that the oxygen reaches tillthe inner core of the aggregates OTR has to be manipulated.Now, because oxygen transport rate has to be manipulated the only controls in our handare aeration rates, then increase in rpm, so this is what is generally done.But then balancing between the shear forces which will get generated over the oxygen transport,right which is required has to be done.Then, so, despite the fact why it is important?Despite the fact that specific oxygen demand is less, but the oxygen requirements for plantsand foundations will keep on varying during the cultivation.So, cultivation timewith continuous fermentation in microbes you will see it can go up to days,2 to 10 days or so, but in plant cell fermentation if you go to continuous cultivation then itcan reach up to a month or so.Product accumulation that microbial fermentations it is generally extracellular, but the plantcell fermentations you will find it, it is often intracellular.So, which means what?The impact on productivity is much higher because anything which impacts the cell withproduct forming inside can lead to change in the yield of the product.Then also, the if you want to make the process continuous downstream is affected becauseagain there is an extra step of disrupting the cell wall and taking the product out ifthe cost is involved.Now, what are the implications on bioreactor design with these factors?The differences between the two the microbial fermentation and plant cell cultivations.Lower oxygen demand than microbial cultures due to low metabolic rate that was one.Specific oxygen demand is low during exponential growth, but high oxygen transport rates arerequired at high biomass concentration.What does that mean?I just spoke about this.Specific oxygen demand is low during exponential growth, specific oxygen demand is low thatis fine.What is specific oxygen demand?The amount of oxygen per unit time per unit biomass is low because the metabolism is slowerin comparison to microbes.But what is high?High oxygen transport rates are required as the biomass concentration is increasing.What does that mean?The two are different specific oxygen demand, which determines what?The overall.. Oxygen uptake rate.So, with increasing biomass concentration the oxygen uptake rate is increasing, so oxygentransport rate has to be increased and moreover with cell aggregation happening.So, therefore, variable oxygen transfer rates are required during the entire cultivationperiod.More shear sensitive than microbial culture.So, this means what?That cultivation has to be done under low shear conditions.So, what can be done?People you will generally observe that rusting turbine is used for microbial fermentations,6 plate, it is the most commonly found configuration of the impellers.But, in plants and fermentations marine impellers or centric impellers or centrifugal impellerswhich can give better mass transport rates, because better KLA you will observe of ourpreferred and with less energy dissipated or with less shear forces generated are preferredover these high energy dissipating impellers like in microbial fermentations.Higherdiameter to width ratio is preferred, large diameter impellers are preferred.Why?Rather than small impellers with axial movement sorry radial impellers?In microbial fermenters radial impellers, rust in turbine is the radial impellerNow, in plant cell fermentations you will observe that axial impellers are preferredor separate which can provide both axial and radial.I said aggregation happens, if there is not much suspension created the cells will settledown which will eventually lead to loss in productivity in the critical phase when ithad to happen which was end of the log phase.So, then you need an good suspension ability and that higher suspension is created, thatI spoke about that the axial movement helps in suspending, suspension.So, and large diameter would create?mixing.Mixing, and higher suspensions axial impellers, and less shear generation.Now, cell to cell,cells often grow as aggregates or clumps unlike microbial fermenters cultures.So, mass transfer limitations, can limit the availability of nutrients to cells withinthe aggregates, then cultivation has to be done under low shear conditions.Cell to cell contact now we also have been now hearing that, even though what one wouldlike in a reactor is free suspension or well dispersed homogeneous suspension, but plantcells do not.If you try to create this is what they are also observing.Now, while preparing the inoculum even are optimizing the shake flask the what initiallypeople who have worked that micros, but they do not know the requirements of plant cells,you would try to keep your suspension as uniform as possible.So, but then eventually you will observe that there is a loss in yield of the product, becausewe now know that cell to cell contact leads to a differentiation signal which helps insecondary metabolite yield and productions.So, there has to be a balance between homogeneity and even cell to cell contacts.Plant cells are self-immobilizing, sometimes when you are working with tissues you willfind that the growth would not begin until unless all the tissues come together.What?I used to observe during my research when I was working with harry roots.When we used to inoculate roots, the roots used to be very small in size, we used tocut sections, 2 centimeter long, and we used to then inoculate into the shake flask.Initial 1 week, it used to take although we used to put the required amount of inoculum,but the culture or the growth you was not happening.Why?What we used to observe is until unless all the roots fragments which we had dispersedin the medium, the growth used to begin only when everything used to come together andthey used to form a clump, and then we used to see that log phase used to begin.So, which is an indication that is it depends on the species also, but self-immobilizationis a prerequisite for the growth to begin.So, that is why sometimes you will observe that support structures are needed, immobilizationof your cell mass is needed.Maybe you need anyimmobilizationlike polyurethane foam inside, this is what we used.We used polyurethane foam then while designing reactors where we could inoculate the roots.So, that they could stick together and then the lag phase could be reduced.So, then all that has to be optimized.Then, cell to cell contact can influence secondary metabolism in plant cells.So, optimal degree of aggregation self-immobilization is required for product synthesis, then volatilecompounds.Now,because you are trying to with high cell density in the reactor what you would do?You would try to increase the airflow rate, so that the oxygen transport rate can improve,but then you may observe that your secondary metabolite yield has gone down because thevolatile components like carbon dioxide or ethylene which is crucial for your secondarymetabolism it plays a role in say as a signalling compound, then which may affect the secondarymetabolite yield this is again what we observed with harry roots.We then eventually optimize the inlet gas composition with carbon dioxide also.So, plant cells, they uh are more sensitive to other gaseous components like carbon dioxideor ethylene, so which is needed to be optimized unlike the microbial fermenters or fermentations.So, what all things have to be kept in mind when you aredesigning reactors or reactoroperating strategies for plant cell fermentations?The sheer environment, in the reactor configuration, the oxygen transfer capacity of the reactor,mixing mechanism in the reactor.Foaming an aggregation, it is observed that in plants and fermentations you will see muchmore frequent foaming than the others because of theah large amount of sucrose which isused along with the proteinase is the growth hormones which you use which might alter thesurface tension, and because of which the air bubbles are not allowed to escape, sothey stabilize the foam.So, there is much more foaming in the plant cell fermenters.Then aggregation which can eventually lead to mass transfer limitation has to be takeninto account.Maintenance of a septic conditions for relatively longer durations.So, as I said earlier because the doubling time is in days.So, your batch time generally varies from 2 to 3 weeks and if you are planning to docontainers it can go even beyond one month or one and half months, so that is the timeperiod which we are looking for maintaining stability.Then capital investment; obviously, that is common, be it any fermentation that has tobe looked into.You cannot end up in making such a complicated design which is not able to get scaled upor which might be incurring a lot of cost.So, modulation of factors like mixing and aeration is to balance out the mass transferrequirements and the shear sensitivity.So, what are the factors which will affect the reactor design?Let us talk about the rheology now rheological characteristics of plant cell cultures, whatdoes it say?Plant cell cultures they are viscous.So, as the cell density increases they tend to become non-Newtonian in nature.Non-Newtonian means?. So, which means your viscosity will also,now it is not no more proportional to viscous force or your shear forces and shear rate,but it becomes a function of, it is not constant.It becomes a function of?Shear.Shear, shear rate or even density.Act as non-Newtonian fluid, so shear stress and shear rate correlation it depends on aggregatesize and cell concentration and thereby fluid properties.Now, culture illogical properties will get affected by medium viscosity and osmolarity.What is osmolarity?Now, viscosity would keep on changing so we can understand.What about osmolarity?What is osmolarity?. It is also used as one of the indirect estimationsways to indirectly estimate your biomass.So, what is it related to?Now, that you have guessed from osmo. solute concentration.Inside the cell.. So, salute concentration in the reactor, osmolalityper unit gram or moles of salute per unit, amount of the solvent, osmolarity is the amountof solute per unit.Solute.Volume of the.Volume of the.Solution.Now, cell aggregation foaming and wall growth in plant cell permutations.Now, plant cell suspension cultures they form aggregates, so diffusion limitation can happenwithin the aggregate.So, there can be external mass transfer limitation.So, when we are talking about oxygen transport rate, we are only taking into account what?The external mass transfer limitation which is your kl orkg.But and we neglect the ks part the solid liquid mass transfer.We were neglecting it till the point.Why we were neglecting it?Your kl is what?It is the overall mass transfer coefficient which involves only the gas liquid boundary,but we are neglecting the solid liquid boundary.There will be a solid liquid limitation, boundary limitation on, is not it.What solid?Because of the cell.Cell.So, why we were neglecting?Earlier the cells were very small.Very nice because it was made for?microbes.So, you could assume that, but if you are actually working with plant cells or tissuecultivations it depends on the size and the size of the aggregates.If your cell suspension is uniform you are much closer to that assumption you can neglect,but if you areworking with tissues much larger than the bubble size then can you neglectthat boundary, no.So, internal, and moreover when you are working with aggregates or when you are working withimmobilized cultures, can you neglect the internal mass transfer limitation.Internal means from the surface solid surface till the inner core.People have even found that when they are working with root cultures there are intracellulargradients of oxygen diffusion limitation.So, oxygengradients are observed till the inner because the size is appreciable.Now, foaming is frequently encountered in plant cell bioreactors with aeration, initial.So, I have already told you because of the proteinases medium sometimes or the proteinswhich come out once the cells are lysed because of the shear forces or the high sugar concentration.Now, wall growth due to cell flotation creates thick layer of necrotic cells.So, plant cells as I said earlier during my discussions that they have a tendency to adhereto the glass walls.So, you will observe in lab scales, special lab scale reactors or non-movable parts thoughbecause of the large size.So, once they get stuck there or once they adhere to those walls then subsequent growthwill cause a film or an aggregate size to increase, as it increases then the innermostlayer will be devoid of the nutrients, availability of nutrients, so then you will observe necrosis.So, mixing to keep the cells in suspension and to provide homogeneous environment.Now, at high cell densities culture viscosity he makes it makes the mixing difficult becausethey tend to aggregate.Now, there has to be a balance between homogeneity and suspension.So, now, both the things if you try to make it very homogeneous what kind of reactorswould you or impellers would you prefer?Large shear forces.To and even for good oxygen transfer.So, you would choose impellers which can create more amount of shear force or more energydissipated onto the medium, so that the bubbles can be well dispersed inside the medium andthere are more circulation velocities radial velocity is generated, so that it is homogeneousin nature.But then this will eventually lead to larger shear forces.So, you will observe that impeller design is a crucial thing in plant cell fermentation.Shear there, what is to be considered?The amount of shear which is generated power which is required because that will involvecost, so power dissipated, power required to drive the impeller, fluid circulation abilityof the impeller then under high viscosity.Then large; so, what is preferred is you will observe in plant cell fermentation large diameterimpellers are preferred because of their high homogenization, but less bubble dispersion.So, again although there is lot amount of you can improve mixing because you have largerdiameter impeller now, but what you are giving away is because then this will create lessershear force on the sparger for the air bubbles to be dispersed.So, what will happen?Eventually, you will be observe lesser dispersion of gas bubbles, lesser oxygen transport rate.So, things have to be manipulated during the fermentation as the plant cells would growand baffles.Now, baffles we know enhances mixing.But you will observe baffles is uh in literature are found to be detrimental for plant cellcultivation shear forces could be reduced around the baffles.So, people say that it is better to run plant cell fermentation without baffles becausemixing requirements or air dispersion or oxygen transfer requirements are much lesser thanthe microbial fermentations.So, you can afford to remove baffles.Now, in pneumatically agitated vessels superficial gas velocity affects the suspension efficiency.Now, there is no moving part.So, in your airlift reactors and bubble column reactors what is causing the circulation currents?The air bubbles.So, particle suspension and that is what is going to keep the cells in suspension suspendedstate, not allow them to settle.Now, particle suspension, improves with high reactor aspect ratio.This is what has been observed.Higher is the aspect ratio which is aspect ratio means high to diameter ratio higheris the aspect ratio better is the suspension ability.For the same power input per unit volume, so that is one of the power per unit volumeis?Scalar.Scalar criteria.So, for the same for power per unit volume keeping same, mixing time in airlift reactorshas been found to be much longer than the stirred tank, is not that obvious.Now, mixing is a limiting factor for pneumatically agitated reactors at high plant cell concentrations.So, even if you are choosing an airlift reactor for plant cell fermentation because thereare no moving parts and the air can keep it suspended, but then mixing is a limitation.So, that has to be taken care as the cell density would increase.So, which with, everything do not you think I am giving you a pro and a con, a merit anda demerit.What does that mean?So, how will you choose?Why am I giving you a merit and a demerit?Everything has a merit and a demerit that is what I can understand from this.So, what does this mean?What is needed?. Right.So, which means that depending on the species do not think that at not knowing your culturerequirement you can just begin with, ok, now I have optimized everything in shake flasklet me do a batch now in stirred tank reactor.No, it never works like that.Your batch once you have even optimize everything in shake flask, you need to take it to thereactor, choosing the right kind of a reactor configuration would be very crucial for reproducingthe results which you have obtained in shake flask at the reactive level.And then once you have chosen the right reactor configuration then the second stage of scaleup begins that what should be the right scale up criteria now to increase the volume ofthis reactor configuration.So, oxygen, demand and supply dissolved oxygen levels to be maintained above the criticalconcentration.So, that is same for any kind of fermentation.So, we need to ensure that the dissolved oxygen, the bulk oxygen concentration in the reactoris always maintained above the critical oxygen levels.High culture viscosity, cell shear sensitivity and cell aggregation would make your oxygentransfer in plant cell fermentation a challenging problem.Now, give me a way just think logically, I know that cells would be shear sensitive,my cells is our aggregating, assume they will be aggregating and I cannot increase the uhtoo much air and rpm because that will create shear.Shear.What would you do then?We have now learned about so many things in optimization strategies, what would you doto improve your biomass or youryield with this limitation?Cell shear sensitivity, culture viscosity, cell aggregation is making oxygen transferlimitation.So, we can as pure oxygen which will decrease.So, we we can optimize the inlet gas space concentration.What else?But that is an expensive strategy, you cannot afford to have pure oxygen in it.So, it is first the oxygen, so which which means what that oxygen transfer in plant cellfermentations are much more critical or the key limiting nutrient than the carbon.Generally, for microbial fermentations oxygen is not the limiting nutrient even in yourMonod's model, that s is never o oxygen, that s is carbon or nitrogen.At the mass when you work with products you will find some other critical nutrient whichmight be affecting the product.But in plants and fermentations are uh with harry root cultures I could find that becausecarbon was still available it was not going which means that we need a model where nowo 2 becomes the limiting factor and not c.So, that is how it shows that the plant cell fermentation, you cannot directly mimic whatyou know about microbial fermentations.So, you need to first understand the culture itself its requirement and then work and usethe, right configuration.So, impeller design or sparger design affects aeration and intern oxygen transfer.So, this is also critically needed.I will not go in to this.I will just show you.So, but in like for example, when we say.So, when we say that impeller design high homogenization or power requirements sheargenerations needed, just saying does not help.Shear generation means what how will you design an impeller taking into account shear generation,power requirement or circulation ability.How will you design?So, generally what is done is you look in literature and get to the empirical correlationswhich from where you first we need to define how would we define shear generation, howcan shear be quantified in terms of these reactor operating parameters.So, you can see literature you will find there are many correlations, empirical correlationswhere people have have correlated the shear forces generated with power input with impellerspeed, with impeller width.So, one of the the one which I showed you is one such empirical correlation which dependsthe amount of energy which would be dissipated on to the medium um depends on the power input,depends on the time duration and the volume of the reactor or the volume of the liquid.So, eventually this power generation is also connected like in your power number.Given a power number this power generation would eventually be correlated with the impellerspeed, the diameter of the impeller.So, now, you know how these operating parameters will be impacting the shear force.Now, first thing is you need to see the threshold of your culture.How would you see the threshold of your culture?You see do a simple experiment.I am just giving you an example.Do a simple experiment, keep on increasing the rpm, check the viability of the cells.The rpm once you know that this is the maximum rpm at which we can work, work backwards atthis rpm what will be the shear generated; so, which means this becomes your thresholdshear force.So, then you can fix up your rpm and similarly your power input or whatever the volume ofthe reactor if you need to know.Suppose you want to quantify how much at this volume will be the shear force generated orthis may even help you to choose the right kind of impeller, once you have fixed on thatthis can be the maximum shear which can be tolerated.Then you choose that kind of an impelleror the power input such that you go do not gobeyond this shear force generated.