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Module 1: Plant Cell Culture

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. Today just to give you a wholesome pictureof whatever you have studied up till now; we will take up a Case Study which is a collationof all the data by one single research group. [gr]oup- How they began and how at every stageusing the strategies which you have studied in plant cell technology or plant cell bioprocessing.At every stage there could be an enhancement in the productivity up till the reactor level.So, the study was done for the secondary metabolite azadirachtin, which is a bio pesticide.So,I will not take up the advantages disadvantages of these.Bio pesticide everybody knows. So, basically the bio pesticide was azadirachtinazadirachtin is present in a ubiquitous plant, which isyour neem it is present everywhere.So,azadirachtin is found in all the parts of the plant; however, it is collected commerciallyfrom the seeds of this tree and this seedformation happens twice in a year. And moreover, butthere are disadvantages as I have mentioned before also; that generally ripe seeds arecollected which have maximum concentration of azadirachtin in ah.But this also has a lot of sugar content in it, because of which there are pathogeniccontaminations specially fungal pathogens. And it is known that and companies face problems;once they collect and there is a contamination with these fungal metabolites which some ofthese are carcinogenic like aflatoxin. Its a highly complex big molecules lot ofchiral centers. So, complete chemical synthesis , completechemical synthesis has not been reported yet. So, this is the molecule and it has a widemode of action in to number of pests known in different plant varieties. So, that isthe reason why it is a verylooked forward andhigh in demand bio pesticide by the inthe market. Now the reason being one is it has a very diverse mode of action then completelybiodegradable that is an big advantage and nontoxic to other organisms; it is it hasa specific mode of action towards pathogens. So, what they had proposed was a plant celland tissue culture based bioprocess for in vitro production of azadirachtin.Because the disadvantage associated with nature were, that you will find neem present everywhere.But the limitation is that if you collect it from different parts of the country youwill find that the yield of azadirachtin is variable. It is season dependent it is plantagedependent geographicallocation dependent and moreover the collection problems with thematerial. So, the two kinds of approaches were usedplant cell suspension based and the other was hairy root culture based. I will be talkingin detail about plant cell suspension later if we get time in subsequent classes onceyou are making the presentation. And if I find the need then I will also talk aboutthe hairy root culture process. So, for the plant cell suspension culturesthe objectives which were laid down were to establish the cell suspension of A indicafor production of azadirachtin under in vitro condition. So, one had to establish a highyielding fast growing plant cell suspension culture of azadirachta indica. Then to developdifferent strategies for enhancement of azadirachtin production in the cell suspension culture.So, which means that, you also develop strategies how to further improve the yield and productivityofazadirachtin. Now, yield was per unit biomass yield whichmeans, inherent capacity of the cell line to produce that product which was azadirachtin.And the second was the biomass also so, that the overall productivity or the titer levelscould improve because azadirachtin is an intracellular product. Then once you had a high yieldingcell lines and then on that cell line you develop the cell suspension culture. In thatdevelopment of cell suspension cultures you optimized all the strategies, which couldfurther improve the productivity. Now, all these optimized conditions were integratedin a bio reactor. So, first before taking it into the reactor level a suitable bio reactorwas selected. It based on the mass transfer and the mixing time characteristics. Ultimatelyit was based on substrate utilization rates product formation rates and biomass formationrates. So, once a suitable reactor was selected then in that reactor a batch kinetic studyhad to be established. Once the batch kinetic study was establish you get to know what whenwe say batch kinetic studies. They were able to see what is the kineticsof growth in that selected reactor, what is the kinetics of product formation. Once weknew that this is the kinetics of product formation substrate and growth; then all thesethree were made given a form of mathematical equation which was called as a model. Suchthat, this could be manipulated in silico to develop feeding strategies to further improveproductivity at the batch reactor level. Now, for further improving productivity frombatch what is generally done to remove the nutrient limitation which is done by feedingthe substrates. Now as you all know you have already studied that feeding can be done invarious different ways. So, what should be the feed concentration time of feed and theduration of feed and the way you feed? Whether exponential feeding strategy constant feedstrategy intermittent feeding so, all that you cannot afford to do experiments to designhit and trial. There will be n number of permutation andcombinations. So, that was the reason why modeling was important. Once model was inplace which was robust by fitting the batch data into those set of equations, then thismodel was extrapolated for fed batch and continuous cultivation. And in silico feeding strategieswere designed in fed batch and continuous cultivations and the best strategy which wasselected was experimentally verified. So, we will see how the complete steps led toenhancement in productivity and, which was more than what is present in nature.So, what was done I said what is important is, to begin with the high yielding cell line.So, first thing is to collect the different plant variety material from the country.So, almost I if if I am right there were some thirty seed varieties; because seeds are knownto be highest. So, 30 seed varieties across the country were selected and they were screenedfor the azadirachtin yield in them. Once the highest yielding seed variety was found tobe the trivendrum variety, which was then taken forward to develop cell lines.Now, assuming that as I have mentioned before a highest yielding plant would lead to a highyielding cell lines so, that was the logic behind. Once this was done callus was induced,now - each callus lines which will be induce will have a different genetic makeup and therefore,a different bio synthetic capacity. Again screening and selection of cell lines wasdone based on the growth index and azadirachtin yield. And then finally, the highest yieldingcell line was selected, now this cell line once callus line which was selected was takenforward to develop liquid culture which was suspension culture.Now, before going on to do any kind of optimization in suspension culture, what is important?To know the reference level, reference level means from what level I need to improve. So,for that I need to know once I have developed a suspension cultures again a batch kineticsin the shape flask at a small level was plotted batch kinetics was plotted.And it was found that which day should be the harvest day, in that batch kinetics. So,ultimately what is the summary? Now the reference point for us to have better productivitiesfor any kind of optimization would be what 12 days, the biomass is 5.9 grams per litrewhich is maximum biomass and your azadirachtin titter which is a club of biomass and yield.So, actually that is your reference point the azadirachtin titter is 9.3 m g per litreok, which is on the 12th day end of the log phase.Now this becomes our maximum possible azadirachtin titter in the given conditions of shake flask.Now when the suspension culture was developed it was developed in MS media. So, a numberof basal media were selected and then the callus line wasthe suspension culture wasdeveloped in different basal media, this was this is what was done.Different basal media which means known compositions like b 5 gamborg's medium arixon MS nish whitesthese are different predefined media for plant cell and tissue cultures we have discussedthat. So, in these media the cell line was grown, the suspension was developed and basedon the azadirachtin titter yield and biomass MS was selected for suspension culture forfurther optimization. Now in this because MS is generally what carbon source is usedin plant cell and tissue cultures sucrose. Now, it was important because sucrose is easilyavailable economical than the other carbon sources. Fructose can beonly the preferredbecause fructose is expensive, if it is really making a remarkable difference. So, therefore,what the group did was they picked up glucose because ultimately glucose is easily metabolisable.So, they did a kinetic study to see how in comparison to sucrose if glucose was givingmuch higher yields. So, they did this initial study and they found that glucose was givingbetter yields than thesucrose. So, for further optimization in MS, now MScontains major and minor salts carbon sources separate carbon is then glucose which wasutilized. So, this kinetics on theright hand side. So, then you can see that glucose wasfound to be a better carbon source than thesucrose. It will be nice to know what range shouldbe selected for studying the interactive effect. So, in order to select the range for the interactiveeffect study using statistical tools your design of experiments, single factor experimentswere carried out. In which each of these major components were varied in a range and theeffect on the biomass and azadirachtin was observed. Based on this they could get a roughidea that where it is highest. So, that the plus minus could be further increased or decreasedto get to the maximum, the optimum value using statistical design of experiments.So, they did for phosphate, nitrate, ammonia, glucose and they also did nitrate ammoniumratio study; because it may happen that the culture may not prefer ammonia over nitrateor may prefer nitrate over ammonia. So, then in that case it was important to study nitrateto ammonium ratio. Interestingly what they found in MS it is 30 is to 30 if I am right.Absence of ammonium is giving the highest azadirachtin yield. So, which means that youcan while studying the interactive effect, you can even discard ammonium or keep theammonium at very low concentrations for plus minus range.Then they also studied rest of the major factors, then based on their single factor resultsthen they created plus minus levels for the screening of the most significant factorsin these selected major components. There are 6 major components 1, 2, 3, 4, 5, 6 nowout of these 6 if you design and start optimizing using statistical design of experiments itwill go up to some more than hundred experiments. So, what will be more intelligent step wouldbe that first to see how significant each of them is and then select only the most significanttop 2 or top 3 top 4. So, this is what they did.They did a plackett burman design which is a screening design in which they could ableto find out which and how these 6 are impacting individually the growth and the azadirachtinyield. They could then rank these based on the nova results, then the statistical analysisis done and based on that analysis you can even rank the factors. Based on the model,which comes out and the coefficients associated with each of those main factors.So, then based on that what could they find? That the inoculum level calcium chloride,nitrate, glucose were critically affecting growth positively. And glucose, nitrate, phosphateand inoculum level were affecting azadirachtin. Now our idea is what to get maximum titterof azadirachtin so, but it is always recommended to do these optimizations for independentfactors it should not be aliased or it should not be clubbed.Because if you will take titter you will never be able to find how is one affecting thesetwo independent factors, the biomass and the azadirachtin yield. If you will take titteras your single response. So, because if you will do it individually you can even designa two stage cultivation, which can give you higher productivity than a single batch; ifyou will do it for only titter then you will this will work only for batch.And then you will have to apply other strategies for further yield or productivity enhancement.But if you will do individually who knows if it is a really completely non growth associatedproduct then it is better to run it as a two stage. So, for that, in the first stage youwould like to feed a nutrients which are best for growth. And in the second stage you wouldlike to feed a nutrients which are best for the secondary product, but it depends. Ifit is mixed growth associate or growth associated then you can take the titters.So, this was theanalysis done based on the response surfaces they could optimize. So,it effectively tells you that the x axis and the y axis component, how the pairs wouldbe the data would be affecting the growth or the azadirachtin. If you see clearly onthe azadirachtin plot there is no convergence point in the design space. And the convergencepoint was maximum azadirachtin is not it that was the goal.So, if you see that there is no e[ven]- even if in the range selected there is no datapoint which is converging. They could have added more contour plots that would give usmuch clear idea it is dependent on the user you can keep adding and see the convergencepoint. So, I am not very sure at the convergence point will lie within the design space, whichmeans that there is a need to go back and see the range selected.But, because for us it is dependent on both the biomass as well as the secondary metaboliteyield; so, then it can be even with whatever you have you can get to further improved resultfrom the un optimized. So, this is what happened here. Even with that, whatevermaximum theycould reach through that contour optimization they could define a media composition basedon this contour analysis. See there is no the model it is always a model. So, you cannotclaim that this is the maximum, that can always be scope. So, it is acceptable, but till thetime that it is giving you better results than the reference point.So, now the glucose nitrate phosphate and inoculum level were optimized. And if youcan see here the biomass the predicted was 15 and the azadirachtin was 3 m g and hereit is 2.9 m g per gram. Now, then what did they do? They didauxincytokinin growth regulators single factor studies in different ratios. And they couldfind that IBA and BA were found to be in this ratio could give maximum azadirachtin titters.Now, see this is also improved from your reference point.Then they did agitation speed study. Now what is important although mass transfer I saidwith agitation would improve, but the effect from the viability is also crucial, to seethe overall effect from the productivity or titters. So, they did a kinetic study in whichthey saw how the biomass is changing with time with increased r p m. So, if you cansee the plot you can see beyond 125 r p m, the viability of the cell started going down.So, 125 r p m was found to be theoptimum speed for maintaining the viability.Then p H temperature optimization was done being an interactive factors, again statisticaldesign was used. And then if you see here clearly the rangeseems to be fine; because they are effectively converging. And this will give you the idealresults what you expect for optimum values of p H and temperature.So, now if you see the comparison un optimized conditions the growth and the azadirachtinoverall it was 7.8 titter. But if you see the optimized conditions it had improved toforty 7.1 m g per litre. So, this is just through simple media optimization.Now, let us see there are other strategies, which can further be used to enhan[ce]- improvethe yields and productivities they did elicitation. These are the different kinds of signalingmolecules, a biotic biotic elicitors they tried these elicitors in different range.Now, in different range these were all singe factor experiments, they could select salicylicacid which was giving as high as 138 around m g per litre at that concentration.And also they could see that chitosan was also giving approximately the same enhancementup to 138, 140. So, then what did they do? They for multiplefold enhancement, they chose the three best elicitors at the range which they could seethrough the single factors. And they did an integrated study to have a synergistic effectthey did again CCD the statistical optimization. This statistical optimization again gave thembetter results. Now, for this the response would be what?It is an yield enhancement strategy. So, generally elicitor addition it is well known that itwould lead to reduction in biomass. So, time of exposure to the elicitor is crucial andthe response while selecting the elicitor can be yield rather than having titter. Becauseyou would never add the elicitor on the 0 day, you would add the elicitor for an optimumexposure time such that growth of the elicitor. So, while selecting the elicitor what didthey do? In this CCDCCD designed if you will see your response was azadirachtin yield.Based on this, they could find the optimum concentrations of these three selected elicitorswhich could lead to maximum enhancement in azadirachtin yield. So, now it has gone upto nearly 16 milligram per gram. Now, as I said they again did time of additionfor this selected elicitor pack, what should be the right time of addition? So, that thereis minimum loss in biomass. So, then they did and they found that with 48 hours of exposurewhich means adding the elicitor just before forty eight hours of the harvest. So, harvesttime for the batch was 12 days if you remember. So, they added 48 hours, 72 hours, 96 hours,24 hours before that the harvest day. And they found thatgiving a exposure period of48 hours is good enough to get the maximum yield enhancement. Later it has dipped maybe because of the viability of the cells getting affected.Now, this is a good way, but what else can be done then they also did precursor addition.Now, for precursor addition what is important to know the bio synthetic pathway? Now inthat bio synthetic pathway, the more you add exogenously one is the availability of theexogenously added precursor is crucial and the concentration of the precursor is crucial.So, they chose the upstream intermediate. So, that the probability is high of it beingavailable for the secondary metabolism. So, then they chose some of these precursors inthe bio synthetic pathway for ex exogenous addition in a different range.So, this was again a single factor study. And they could chose GPP, then GPP geranylpyrophosphate then squalene that is not going back mevalonic acid lactone. And what couldthey find they could find that GPP had certain concentration level was giving more than hundredazadirachtin. Then they did. So, these are all independentexperiments. So, there can be two ways of optimizing; one is you can run a relay systemthe other is you run a parallel system and then finally, integrate. So, because everythingif you wait for a relay system, it may take long time. So, generally you will observepeople do independent studies and then finally, integrate as a combined study to see synergisticeffects. So, then permeability enhancement was also checked.They used certain permeabilizing agents which are known in literature in concentration rangerange, which are well below the well below to affect the viability, but still becauseit is species dependent. So, viability was checked. So, at different concentration rangethese are all surfactance solvents. So, percentage volume by volume they were concentration rangewas added. And they could find that n hexadecane at 5percent volume could give you azadirachtin release up to 13 percent. Now, why do youthink there are otherthese permeability enhancers which are giving very high release? I seeit is going up to 70 percent decanol, but why they have chosen n hexadecane with 5 percent?. Cell viability, which will impact your biomassand these were added on 0 day. So, then once all these strategies and wecame to know that these are the strategies which are leading to enhancement you haveselected; then what is important to choose the right reactor for mass cultivation.For that what was done? Batch cultivations were carried out. First even in the stirredtank reactor I mentioned that the shear forces can be varied by changing the impeller design.So, then they did a study in which in the same stirred tank reactor, there were differentimpellers which were used based on their mass transfer coefficients and mixing time characteristics.So, they tried with steric impeller which is a low shear impeller known for plant cellcultivations. They tried with centrifugal impeller which is also a new designed wellknown for very low shear forces and very good mass transfer efficiency at that concentration.So, they tried these two configurations and they also did pneumatic reactors in whichthey used bubble column reactors. And for this then they drew the kinetics ineach of these reactors. So, first is the simple reactor, which is easy toscale up which isyour stirred tank configuration well known in industry. So, stirred tank configurationsetric impeller was used and the kinetics was drawn. So, what could you see as a changeonce it was brought from the shake flask to the reactor any advantage what was there inthe shake flask? .Very good. So, the productivity could be enhanced, because the kinetics was faster now and theproper reason could be the maintenance of controlled conditions.Then centrifugal impeller was used. Now, again the kinetics was drawn. So, youcan see that the biomass has been improved and the azadirachtin content also has improvedin 10 days. What could be the reason? They drewbiomassformation rate and product formation rate. And they could see that you can see from thefigures that centrifugal impellers leads to better biomass formation rates during thecultivation period and also better product formation rates. So, they wanted to justifywhat could be leading to this. So, they calculated K L a under the givenoperating conditions in which the reactor was run, they calculated K L a and they alsocalculated the mixing time. After calculating can you see which one is better, which oneis better ? .Why because K L a is better and . .Is less. So, then pneumatically agitator reactor was used if no moving parts are there in thereactors. So, they could see that the dry cell weight was equivalent, but the growthrates have gone low you are achieving it in 12 days. So, which means that,may be becauseof better mixing because of the moving part inside the reactor. So, one should prefera stirred tank configuration rather than pneumatic reactors. So, they did a comparison with thesetric, this was with the the improvement was with in comparison to the centrifugalimpeller. Where it had improve to 18.7 and 72.So, if when they did a comparison in terms of steric impeller, they could find that thebiomass formation rates and the product formation rates were better. But if you compare it withthe centrifugal impeller the bubble column was lower. So, finally, a stirred tank reactorconfiguration with centrifugal impeller was chosen for further studies .