Last class, we were discussing about characteristics of plant cells, then how are they differentfrom microbial fermentations, so what impact bioreactor different operating parameterswould have in plant on plant cell fermentations.So, something which is peculiar to plant cell fermentation we spoke about cell aggregation,which is more frequented plants and fermentations, then the foamingand also the wall growth.Then we spoke about mixing where we need radial as well as axial mixing and I also said thatin plant cell fermentationbecause they are heavy more dense as the cell density increases,so suspension efficiency has to be enough and is an important aspect which has to betaken care apart from dispersion of the gas bubbles or the mixing or homogeneous insidethe reactor.So, for which the impeller design is crucial.So, generally, if you remember I said for plant cell fermentations high width to diameterratios are preferred.Andmoreover the distance between the sparger and the impeller and the kind of spargers,like for example, points spargers if they will be used then they will have a more instabilityin terms of there is higher chance, lesser dispersion possible.Butwith more radial mixing dispersion improves, but then there is a demerit to suspensionaxial movement.So, therefore, helical ribbon orah setric impellers, centrifugal impellers, these arespecific impeller designs which are frequently used for plant cell fermentations becausethey can create both axial and radial movements and better k L a in comparison toused impellerslike radial impellers for microbial fermentations and less shear forces on the cells.So, pneumatically agitated reactors mixing is a limitation, but obviously, because thereare no moving parts so shear rates are less Ma'am, what is the pneumatically agitated? Pneumatically agitated means air driven, thereare no moving parts.Whatever mixing is being provided is provided by the air flow rates sparging.So, dissolved oxygen levels can should be maintained above the critical oxygen concentration.Now, high culture viscosity, cell shear sensitivity, cell aggregation all these phenomena's makeoxygen transfer in plant cell cultures a challenging problem.So, there impeller design and sparger design as we were talking about will help in overcomingthese problems.Now, factors which will affect the k L a which is mass transfer efficiency in the reactorswhich will include superficial gas velocity culture viscosity and reactor geometry.So, when we say on basis of what that I spoke about that these factors will be affectingk L a, there will be you will find a literature enough empirical co-relationships betweenmass transfer, characteristics, how they are related to various operating parameters ofthe reactors, for example, impeller width, impeller diameter, then gas superficial, gasflow velocity, power input to the impellers.So, these are some of the parameters in the reactors which have been found to be relatedwith the mass transfer coefficient of the that reactor vessel.So, whyplant cells in comparison to microbial fermentations are less shear tolerant?Because was see now there is an inverse relationship that when the plant cells are in the earlylog phase rapidly multiplying, then they are more flexible.But as the cell density would increase they become lesser flexible toshear forces.So, reactor configurations which you will see in literature these are all pneumaticallydriven reactors.The first one is your stirred tank reactor.Now, number of impellers also can be one of the factors for mixing, the amount of mixingwhich is needed, which is quantified in terms of mixing time.Then in the pneumatically driven reactors you will find, there are tapered bubble columnreactors, then there will be airlift reactors, external internal loop reactors all these.Now, knowing that these are pneumatically driven reactors, what is the limitation ofsuch kind of reactors?Mixing.Mixing is a limitation.So, any changes like making an external loop to it or making it tapered in the design isimproving the mixing time or trying to overcome the limitation of the pneumatically drivenreactors.The position of the sparger, where the sparger should be placed in an airlift of a bubblecolumn will also determine whether there can be dead zones at the bottom or not.If the like for example, I will show you a picture.It will come later.Where you will see in literature they did a study where the position of the spargerwas changed and they could find that in an inner loop reactor when the position of thesparger is well above the loop the draft tube then the circulation currents were betterbecause the bubbles could drive the currents from inside to outside.But because if the sparger was placed well below the draft tube, then there is a chancethat there would be dead zones created because the bubbles will get dispersed at the bottom,rather than all the bubbles flowing through that channel creating density gradient andcirculation currency.So, let us talk about the different convocations.Now, stirred tank reactor is a well-known configuration which is very well used formicrobial fermentations.Now, stirred tank reactor, the difference which you will find in plant cell fermentationis with respect to the sparger and the impeller design, absence and presence of baffles.Now, even people are working out with the aspect ratio.Now, the kind of reactors which I was talking about for example disposable bioreactors whereorwave bioreactors they will have more surface to volume ratio.Now, the kind of impellers, like anchor impeller, propeller 6,flat blade turbine this is wellused for microbial fermentations.Now, why these designs?Because these designs as I said will impact your flooding transitions or flooding andloading.Now, this impacts the dispersion of the gas bubbles in the reactors, more is the dispersionof the gas bubbles better is the mixing, but there is a limitation of creating suspensionof the cells.So,sparger whether you should use points sparger, whether you should use a rings sparger alsodepends on the reactor geometry.So, all these aspects have to be taken into account when you are designing reactors.Now, alternative designs which you will see, uh these are disc turbine curved blade reactors,then large diameter impellers.Airlift reactors, these are pneumatically driven reactors.So, the first picture is your inner loop reactor, the second is the outer loop reactor.Now, outer loop reactor which will improve mass transfer.Why?It is said that the outer loop improved mass transfer of oxygen, gas liquid mass transferwas improved.Becauseair is being sparged and that is why.Because?Air is being sparged and that is why.In both the cases air is being sparged.Ma'am,how it is that external loop works ? It is a simple enclosure which is in continuitywith the inner reactor.String also.So, what is increasing?You are having an extra path?Surface area.So, what?Time . So, you are giving more time for gas holdup.More the time, more is the time given for exchange to happen.So, more circulation time.Then, this is what I was talking about.The position of the sparger if you see, so when you want effective mass transfer to happenin pneumatically driven reactors you want the suspension also to happen mixing withsuspension.So, what is causing this suspension and mixing?It is only the circulation currents.And what is causing the circulation currents?The density gradient.What is the cost for the density gradient?The gas sparging.So, if you can see in the picture it is very well demonstrated that if you change the positionof the sparger, a little up in the draft tube then the gas bubbles would drive in the directionin which it is desired, thereby creating the right circulation currents and improving mixing.So, lesser chance for dead zones at the bottom of the reactor.The other thing which they have shown here is that if the lower edges can be filled ratherthan keeping it very erect edgy at the bottom and they have been made circular there byfilling it with glass then this will improve the circulation rates of the medium, avoidingthe dead zones in at the bottom of the reactor.The disposable bioreactors, we have already discussed about the way bioreactor.This is a well-known bioreactor used for animal cells, but people have also started usingit for plant cell fermentations.It works on the principle of wave and undertow.Wave everybody knows.Undertow?It works on the principle of wave and undertow mechanism.So, why this mechanism has been spoken about?There is no moving part.That is.So, what is causing mixing?This motion, rocking motion.Now, this rocking motion would create what?Ripples, currents.So, it will create wave that at the surface.Now, even; so, the same principle what works at the beach, when the wave comes to the beachthen there is a large mass flux of the liquid towards the beach, but there is an equivalentmass flux against the beach which is beneath the turf.So, that is undertow those currents which are back flow, beneath your wave.So, there are they work in the opposite directions.So, this is what will cause mixing to happen when this wave gets created, so that is whatis called as wave and undertow mechanism.Slug bubble reactors they are very high aspect ratio reactors.High aspect ratio means very high, high to diameter ratio is large.So, in this such that a single bubble is created which can span the entire column, entire reactorvolume as it move slowly and the mass transfer would take place, as it would move on theedges of the bubble, it is said that it will create a plug flow.So, on the edges of the bubbles there is a very thin film of liquid which is left adjacentto the glass column or the reactor wall.So, through that the liquid and the gas transferred between the liquid and the gas takes placethrough that thin film which is adjacent to the bubble and the wall at the top of thebubble, if you can see on the sides of that bubble which is shown in filled grey color.So, these are called slug bubbles because they will create slug flow.Slow motion, but and you can assume it closer to plug flow, no boundary layers.Now, bioreactor operating conditions.Apart from choosing the right kind of configuration if you want to improve the productivity.Now, we have spoken about different strategies which you can use to improve the yield ofthe product or the productivity of the product.Then, you under those conditions you choose the right kind of bioreactor, you apply thoseconditions, you get to some x productivity Now, what is the limitation?Now, I need to improve further, beyond this.Is it done?Is the bioprocess?Now, optimized no there is still ample scope for productivity enhancement.In what way, we have learned about different strategies?You can count now, there are n number of strategies which we have learned which can improve theproduct yield secondary metabolite yield and productivity.Now, the limitation in the reactor would be a time will come when I will have to stopthe reactor beat any reactor.What will be that time?That time would be invariably when the nutrients are done.Can I do anything to never allow nutrients to be done?. Till whatever point I want.So, that is what you can overcome with your mode of cultivation.Now, in that I will have to design which nutrient to feed and at what time to feed, at whatflow rate to feed, and should I be using suppose I optimize the nutrient composition; it cameas 30 grams per litercarbon and nitrogen.And I saw that oh as she said secondary metabolite, I did statistical optimization and I foundthat at carbon is the most critical nutrient for biomass, but phosphate is the highestimpacting nutrient for mysecondary metabolite.So, then if I want to improve the productivity which is a sum of the biomass and the productwhat should I do?I would say it is better to run in one the one reactor itself and have a two stage.Initially, we will do Cultivation.We will increase the biomass, then we will change the substrate.Ha.So, which means that what should be the composition of the field will also be governed by whatI have studied in the batch, whatever optimization I did in the shake flask.So, that is where what concentration of the feed and what composition of the feed shouldI be feeding in will be determined now at the reactor level which can then further improvein the same reactor the productivity.So, that is what is bioreactor operating strategies where you can even do it as a fed batch ora continuous cultivation in the chosen reactor configuration.Now, an effective bioreactor operating strategy provides a high productivity, a high productyield.So, we have been talking about high product yield with respect to biomass and high productyield with respect to substrate.Now, high product yield with respect to biomass will reduce my which cost?Upstream, downstream?. High product yield with respect to biomasswill reduce I said the cost which costs will it reduce?. Upstream, downstream?Downstream Downstream.High product yield with respect to substrate.Upstream.Will reduce my production cost.So, that is why these two factors should also be optimized can be optimized separately.The operating strategy is determined based on the pattern of product formation, to relatethe pattern of product synthesis, to cell growth appropriate index for cell growth isneeded.Now, modes of cultivations which you will come across will be multistage batch fed batchreactors single and multi-stage continuous chemostat then repeated batch which are alsocalled as fill and draw systems.See you will hear many different terminologies, but if you will read them, see the details,it will you will be very able, you will be able to see that there is a lot of overlapbetween one or more of these.Perfusion reactors, perfusion reactors are nothing, but cell recycle reactors; so chemostatwith cell retention.So, cell retention devices, so they are called as perfusion reactors also and immobilizedbioreactors.Now, immobilize reactors for plan sense what do you need?Good bulk mixing required to minimize external mass transfer limitations.So, that there are lesser boundary layers around the beads or the stagnated biofilm.Adequate exposure to air or the aerated medium.So, sometimes you will see that whenthe culture reactor is separate the from the medium.So, it is called as a reservoir and the biomass is kept in a separate reactor.If suppose it is being worked as a packed column reactor, packed bed reactor then thereis a reservoir the media is circulated from the reservoir continuously and the spent mediais again recirculated to the reservoir So, why do you think this is being done notin one reactor?How will that help?There is a separate reservoir because it will help you to aerate the medium.Once it comes out you separately in the reservoir the medium is aerated such that you have enoughdissolved oxygen and then it is circulated because mass transfer is a limitation in immobilizedreactor systems or the external mass transfers.Now, immobilization can also affect plant cell metabolism.So, cell viability has to be tested.So, different reactor designs they involve what?Surface attached cells are as biofilms or these will facilitate what cell to cell contactand generally you will see that as the cell to cell contacts improve this may also impactthe secondary metabolite biosynthesis.These are some of the examples in literature people have used immobilized reactors.I was also talking about hollow fiber reactors where either inside the tube the cells canbe immobilized or outside the tube the cells can be immobilized.But again with growth there would be mass transfer limitations, so the flow rates haveto be optimized.Now, packed bed reactors, the cells are.Now, what is the limitation of packed bed reactors?There will be higher pressure drops in packed bed reactors because there is more resistanceto flow andbut that limitation is taken away in well mixed immobilized reactors.You use the cells and you make them freely suspended in solution.So, they they become like reactors with cell retention.So, there the limitation of mass, external mass transfer can be avoided.Then fluidized bed reactors in which from the packed bed reactors there is fluid movementwith to improve the mass transfer, but then the only way they are the cells are kept suspendeddepends on the flow rate of the gas or the fluid flowing rather than gas because as thefluid flow rate would determine the suspension efficiency of the cell mass also.Now, if I improve the flow rate there will be better suspension, but then there is lessertime given for the mass transfer to happen, is not it.So, then what is done is you may see that there arecirculation developed through reticulatematrices such that you drive you the liquid currents or the fluid is forced to spend moretime with the cells or you will see that lot of gas sparging is done.Now, gas sparging is done in these reactors to improve the bulk mixing and to facilitatemass transfer between the gas bubbles and the liquid medium.So, that is one way of improving the mass transfer of oxygen in these reactors.But then what is the limitation?The limitation would be that you are having high gas velocities with liquid circulationcurrents, so the limitation can be high amount of shear which may get generated.Membrane reactors, we have already spoken about hollow fiber reactors.You can go through, whatever is written here.Now, despite all this there are known limitations and plan cell fermentations.One thing is the minute it is brought to in vitro cell lines the yields drop.So, and moreover plant cells we know that there iswhat is the limitation?. With plant cells.Callus; plan cells suspensions are created from callus, is not it, cell lines.So, there is somaclonal variation.So, because of which the biosynthetic capacity may vary.Now, in order to avoid this people came unexploited theagro bacteria mediated transformed culturesspecially the hairy root cultures.Why?Because they are organ cultures and heterologous expression can even be done it will becomemore stable because the there is genome integration of the T-DNA into the plant chromosome.So, it is more stable transformation event.And there is,the other limitation in plant cell fermentation you need hormones, but inhairy root cultures the cost will come down because they do not need hormones for growth.