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Module 1: Plant Cell Culture

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Bioreactors for Hairy Root Cultures

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So, last class I said we will be talking about Hairy Root Cultures.Apart from plant cell cultivations commercially for secondary metabolite production hairyroot cultures are also being used in industry.So, from where do the hairy root cultures come?They were actually a disease in the plant caused by agrobacterium rhizogenes.Now, we have already studied about agrobacterium transformations, if agrobacterium tumefaciensinfects the plant cells then we get crown gall disease, if agrobacterium rhizogenesinfect then we get adventitious roots which are called as hairy roots from the point ofinfection because of the raw root origin lucas present raul genes present in the T-DNA.Now, there are different commercial applications of hairy root cultures they are being testedfor phytoremediation studies, they are used for secondary metabolic production which Ihave already told you where same strategies can be applied for yield and productivityenhancement.But there are limitations in the scale of hairy root cultures by bioreactor designingplays an important role.Then production of medicinal secondary metabolites or use specially those which are endangeredthen it can be done for the medicinal plants for example, the production of high valuemetabolites.Then for metabolic engineering even for heterologousexpression of proteins then it becomes a more biosyntheticallyand biochemically stable system because there is a stable T-DNA gets integrated into theplant chromosome.But growth rate wise hairy route cultures being organized structures as well in comparisonto plant cells are slow because plant cells they are dispersed, more availability of nutrients,so the specific growth rates are higher, but then because biosynthetic capability and stabilityis more so people prefer hairy route cultures.Now, advantages as we said now they are fast growing when we say fast growing it meansrelative to the natural plant or natural root system, because in comparison to the naturalroot systems there are more number of mari stems present.There is extensive branching in hairy roots.So, therefore, you see that these specific growth rates are higher than the normal rootcultures.Then genetic stability because of the chromosomal integration.Then biochemical stability because there is organization organized structure, so thereis higher secondary metabolite yield.And moreover because they have new plastic in nature.So, again there are no hormones required because oximes synthesizing genes are already present,so there is no requirement of hormone in the medium, so cost effective in comparison toplant cell cultures.Now, the challenges in bioreactor cultivation or scale up.You can see the picture of hairy roots here.So, they grow like a mesh matrix.So, the the problem with hairy roots is that as the working volume would increase, evenfrom the day one depending on the size of the inoculum there can be gradients of nutrientlimitation specially oxygen and also nutrients if the size of the matrix is big as it grows.Now, hairy roots are self-immobilizing in nature.So, the growth rate begins the growth begins once they come together.So, as the growth will happen they will grow as single matrix and therefore, the innercore is generally found to be devoid of nutrients and gets necrotic.So, hairy roots now, there is shear sensitivity, it has been observed that if stirred tankreactor configurations are used forhairy root cultures then they break because these rootswill break and if the breakage happens the organism takes it as a wound created and therewill be callusing happening on the roots, thereby leading to inconsistent product formation.Then there is a requirement of support matrix because they are self-immobilizing in nature.It has been observed that the more dispersed they remain in the liquid media the growthis hampered.So, the growth does not begin.So, they are required, they are needed to come togetherfor it acts like a prerequisitefor the growth to begin.So, there is requirement for support matrix.They are highly sensitive to even the material of the support.Generally, stainless steelis the material which is used in the reactors, but it dependson the species sometimes it is found that it is very specific and sensitive to the materialof the inner parts of the reactors.So, inert matrix is needed.Restriction of nutrient or oxygen delivery to the central mass of the tissue, thenresistanceto flow due to the interlock matrix.Now, the size of the biomass itself like an immobilized system is quite big.So, there will be thicker boundary layers in comparison to your gas liquid boundarylayer.So, the mass transfer limitation in these systems it is much more severe than when youare working with plant cells or microbial cells because the cell size is small, so thesolid liquid boundary layer thickness is smaller, but here the solid liquid boundary layer limitationis higher in comparison to liquid gas.And even depending on the species on which you are working the root itself can be verythick.So, if the root is having a thicker morphology generally for tree species.If you compare with herbaceous species herbs will look like this [vocalized-noise.Generally, if you will work with herbs then you will have very fine roots, but if youare working with tree species then you may have very brittle morphology and thick morphology,like when we were working with azadirachta indica.Initially when hairy roots were induced, it was like a taproot system there was one singleroot and then branch and the branching was also less.So,it was very difficult to do liquid culture of those roots, growth rates were very slow,so we had to optimize the media for and then we applied gibberellic acid as one of thehormones and we could clearly see the morphology completely changed to the picture like this.So, where we had seen a complete difference in the morphology because by adding gibberellicacid what we observed the there were n number of mari stems which came out.So, more number of mari stems heavy branching gibberellic acid is known to increasebranchingin root systems.So, then there was heavy branching because of that the morphology became thin and itbecame easier to scale up the system and mass transfer limitations were also reduced.So,it has to be a balancing act between the biological needs of the tissue without inducingan additional biological response which means like for example, wounding I said which willinclude callusing in the roots.Now, the reactors for hairy root cultures they are divided into two types, liquid phasereactors and gas phase reactors.Liquid phase reactors are generally the kind of reactors which we work with the microbialfermentation or plant cell fermentations.They wherehow are the two different?In liquid phase reactors, the gas is the dispersed medium in the liquid.In gas phase reactors the liquid is the dispersed medium in the gaseous environment.So, we will talk about the liquid phase reactors.In liquid phase reactors the different kind of reactors which we already know, which havebeen used stirred tank bubble column where the kind of impeller which you use has beendeferred or submerged convective flow rotating drum and airlift reactors.I will show you the pictures.So, generally as I said because root cultures are self-immobilizing they need a support.So, even in these kind of liquid phase reactors a support is provided such that which canfacilitate self-immobilization of the hairy roots.So, you can see in picture a, which is a stirred tank reactor there is a mesh provided on towhich inoculation is done and the impeller is then and kept below the mesh such thatit cannot generate large shear forces on the inoculated roots.So, what are the limitations?But in all these, so you can see the picture of rotating drum bioreactors these are reactorswhich have already been used for hairy root cultures.So, there are reactors where there will be a support system, there will be aimpellerfor mixing, but generally you may also find that there can be two separate compartments,one can be culture compartment, the other can be your medium reservoir.So, if you want to avoid avoid your shear forces on to the biomass then the reservoiris aerated and mixed separately and then circulated through the culture chamber.Like you can see in the picture d.So, the configuration remains the same whatever we have studied be it bubble column, airliftreactors, but the geometry is deferred, so as to work around with the mass transfer limitationscan be for the nutrients can be for oxygen.And because support is needed they have self-immobilizing, so some structures inside the same reactorconfigurations are provided to work to balance out the mass transfer and the shear forces.So, the limitations which have been found with liquid phase reactors increase root headdensity increases the pressure drop across the reactor and limits mass transport.For example, if you are using this kind of reactor packed bed system.So, then, higher is the as the biomass will start growing there will be increased pressuredrop with the increased route density.So, then there will be mass transfer limitations.Similarly, even if the roots are suppose you people have used configurations where theyhave used small baskets polypropylene, mesh, and they inoculate the roots onto those meshand then they drop the entire mesh with the inoculated roots inside the liquid medium.Now, as the roots would grow around the mesh the branching would happen then you can imaginethat the inner core of the roots which is there the mesh will not have the same oxygenavailability what the outer surface would have.So, these are severe mass transfer challenges in hairy root cultures.So, then what can be a way to overcome this problem?You need support, the roots will grow like this, they are self-immobilizing you cannotdisperse them.So, how will you improve?What strategy would you use to improve mass transfer?What can you do?These are the kind of reactors which we know generally used.Now, we know what are the challenges.So, what are gas phase reactors?I said the reactors were at liquid is dispersed into the gaseous medium.Can you guess?Liquid being dispersed into the gaseous medium.Like for example, trickle bed reactors or missed bioreactors, where the media was providedin the form of cloud and missed on to the roots and the roots were kept on the supportsystem.And when we did this there was significant improvement in the growth of the roots.Now, why do you think it was it might be helping?There is a limitation of scale up, but this was working, hydrodynamic stress could beavoided, hyperhydricity.Roots when completely submerged in the liquid medium they they be also observed there wasa lot of mucilage which was getting collected on to the root surfaces which was inhibitingthe growth of the roots.So, when we had, so availability of the nutrients also does not mean that you keep it submergedin the liquid medium.Keeping it submerged all the time in the liquid medium was was also that we observed evenin the shake flask, that there was a lot of phenolics and mucilage which was getting collectedon to the root surface.So, then when we read literature and we found that they are sensitive to hyperhydricity,too much of water retention on to the roots.So, how to avoid that was using these mist.By changing, now you can even use trickle bed system, but there the droplet sites wouldbe greater or you use mist, so the droplet size has been reduced.Now, even in mist bioreactors the cycle of the mist, the size of the droplet can be optimizedfor best results.So, this is one kind of gas phase reactor system.So, reactors in which liquid is the dispersed phase and gas is the continuous phase.Roots are exposed to air and liquid nutrients are either sprayed on to the roots and deliveredas a mist.Now, coming back to the spray bioreactors.It is not that directly we, we were doing everything in parallel.So, one was we were trying to test the mist design.Now,before that we had also got custom made small setups where we were using a nozzleand this nozzle could create a spray of the liquid medium and this spray we first hadmade put the roots on to the support and beneath the support we had that spray.So, that directly it does not hit the roots, it used to go up to the head plate and fromthere it used to fall down the spray.But then it did not give us good results, but the we saw that there was lot of waterretention on to the roots the roots became necrotic by the end of 25 days.Similarly, so then we tried that let us put the nozzle on top and spray the roots withsmaller droplet size, but all these configurations did not work and the reason which we coulddecipher was probably the lot of water retention which was happening on to the roots.Although, we then can change the support also, we then initially the support did not haveany holes, then we created holes onto the support, so that the water could drain downand go back to the reservoir.But then that also did not help a lot because we could see lot of water retention on tothe roots and then the mist design came up which worked.But then scale up is an issue there.Things can work at small scale, but imagine and production scales so, quite costly.So, roots are exposed to air and liquid nutrients are either sprayed on to the roots or deliveredas a mist.So, these are mist bioreactor designs.Now, what can they do they can eliminate oxygen deficiency in the dense root beds and we couldsee that very clearly.Even when we opened up, once we harvested the reactors and we opened up the root meshand we could see that till the inner core we could see small branches coming out freshroots, so in total the biomass was one of the best in comparison to all the reactordesigns which we were choosing.Low shear environment.So, now, there is a lower shear environment because there are no moving parts.Complete control of gases in the gaseous environment.Now, this is one of the best.Now, one is that it can travel till the inner core, hyperhydricity is also removed.One of the bestor advantages was that there was good gas transfer happening.Why do you think?Better mass transfer of oxygen than in the liquid which could have been the reason forimprovement.So, oxygen is going to get to the roots through the mist.So, if I can stretch it then what she is trying to say is now the gas transfer is happeningdirectly from the air to the root, and in the all the problem which we have been discussingabout, about oxygen sparingly being soluble in the liquid which is not the case in air.So, there is better availability of oxygen for gas transfer to happen that could havebeen the reason to get significant change.Then nutrient mist reactors, then trickle bed reactors, radial flow reactors, gas spargedreactor.So, we can see some of the designs.So, this is what we also tried.So, in the center this was a mist chamber in which you can even use ultrasonic transducers,where the mist was created, it then goes to the culture chamber where you have inoculatedyour roots and the advantages are used.Then whatever condensate is obtained can then be recycled back to the reservoir.Then, trickle bed reactors, moreover I said thequality of support or the kind of supportyou use also matters.We tried glass beads as support, we tried polypropylene basket or polypropylene meshas support, we tried polyurethane foam as support and we tried stainless steel meshas support and all these different kind of supports gave us different results.So, the kind of material you use as a support can also become a critical factor for results.So, these are some of the other reactors which have been used whose in literature forotherorgan cultures of a shoot multiplication or somatic embryos.For somatic embryo scale up people have used balloon kind of bioreactors.Now, where generally you will observe either they are used as fill and draw kind of a systemto remove the phenomena of hyperhydricity or too much of water retention and there arecycles of water, like in rotating drum bioreactor the entire drum would rotate.