Welcome back, soas we were discussing the azadirachtin case studywhere the authors havetheselected the high yielding cell line for azadirachtin production followed by optimization of thecultureconditions for maximum productivity of azadirachtin. And then they also implemented yield enhancementstrategies like, precursor addition and elicitor addition to improve the yield of azadirachtinin the cell line. Then they went on to doestablish batch kineticsin different types of reactors where it was stir tank reactor, then bubble column reactor.And even in stir tank reactor they tried different impellers there bychanging the mass transfercharacteristics and the mixing time time and they saw the effect on the batch kinetics.So, after a suitable reactor configuration waschosen for maximum productivity of azadirachtinin batch. They also then wanted to develop nutrient feeding strategies in the reactorto further enhance the e the yield and productivity of azadirachtin in the reactors. Now, fordoing thisone way is to do hit and trial hell with respect to time of addition of the feed,the concentration of the feed in the reactor and the manner in which the feed is done ah.So, either one can do a hit and trial or there can be a more rational way of of simulatingand coming and selectingfeed nutrient feeding strategy e based onkinetic model. They developeda mathematical model that gives an adequate description of growth and production kineticsof A indica cells in the suspension culture. Now, when they formulated the mathematicalmodel following points were kept in mind, one is that they did a separate inhibitionsubstrate inhibition study e with respect to the critical substrates in the medium.To see if beyond a certain concentration the substrates were inhibiting the growth rateof the culture. Thenthey incorporated this inhibition effectin to their batch growth kinetics and thenwith the model which was a description of substrateutilization kinetics, product formation kinetics, and biomass, or growth kinetics. They simulatedthese equations optimized evaluated the parameters of the model equation.Then then they did parameter sensitivity analysis in the model to drop out the insignificantparameters to reduce the number of parameters in the model there by making the model moresimple for simulation. So, this was modeled redefining and then finally, this redefinedmodel was used to simulate nutrient feeding strategies under fed batch or continuous cultivations.Now, before we we see the case study let us understand what is mathematical model. So,becauseit is a quite complex system um and plant cells they take longer cultivation timesthegeneral experimentation is more of a hit and trial approach. Hence mathematical model approachcan give you a better understanding of the system.Now, it is based on the earlier knowledge of the system behavior it is more therefore,logical. Easy offline simulation can be done instead of doing hit and trial experimentsand there by the number of experiments which one would take to reach to the optimum orenhanced productivities would be less. So, what they did while developing the model?They began with certain assumptions, they had the batch kinetic data. and the inhibitiondata in batch. Now, they formulated the model equations to describe the system behaviorthen they determined the model parameters based on initial guesses.Then further these model parameters were evaluated using an iterative process and parameter sensitivityanalysis was done to select the significant parameter there by defi[ning] redefining themodel. And finally, the redefined model was simulated to predict the nutrient concentrationsor the feeding strategies in the fed batch cultivation and also in a continuous cultivation.So, before beginning to develop the mathematical model they did inhibition studies in whichdifferent concentrations of the limiting nutrients were tested for possible inhibition in growthof the culture. So, this was done to account for inhibition of growth at higher concentrationsof substrate which will be fed during the fed batch cultivation.So,the different model equations which were incorporated to account for growth and forinhibition were as follows. If you can see on the slide for growth they picked up Monod'smodel and for defining inhibition they picked up luongs model and also the asymptotic inhibitionkinetics. They tried to fit the data for substrate inhibition for different rate limiting substrates.So, the substrate inhibition studies were carried out with respect to glucose, nitrate,and phosphate. So, the model assumptions were that the majornutrients were carbon nitrate, ammonium, and phosphate. Rest of the nutrients were availablein excess in the culture broth, and the culture environment like the parameters like temperatureand pH remained constant throughout the cultivation. This was the model equation used which hadthe limiting terms with respect to all the limiting nutrients taken up in the model.The glucose nitrate and phosphate concentration and then they also incorporated the inhibitionterms with respect to the same 3 substrate to account for substrate inhibition duringfed batch cultivation. Then luedeking piret model was used to defineproduct formation kinetics, the last three equations they demonstrate the specific substrateutilization rates. Where m S, m S 1, m s 2, and m S 3 stand for the maintenance coefficientswith respect to each of the three critical nutrients. And Y X by X stands for the yieldcoefficients with respect to substrates and mu is the specific growth rate of the culture.A non-linear regression linear regression technique was then applied which was assistedby a computer programmer to minimize the deviation between the model and the experimental valuesfor all the data points and process variables. So, an objective function which was calledas sum of squares of weighed residues was used to simulate the model. So, this was basedon original method of rosenbrock to find the minimum value of the objective function.These were the optimized parameter values of the model. Now, this picture shows thathow the fitting was done using the batch kinetic data with the model. Now, in the batch modelwas extra polluted for fed batch cultivation and continuous cultivation to develop offlinefeeding strategies. And then finally, the selected feeding strategy which was givingmaximum productivity was experimentally verified. In order to extrapolate the batch model forfed batch or continuous cultivation this is what they use they did the feeding of allthe three critical nutrients and this was a constant feed rate strategy. And then theydefined as done in the batch poodle the substrate utilization rates for all the three criticalsubstrates and the product formation rates. Now, being a fed batch cultivation the dilutionterms were added to the model and then the parameters this model was then simulated fordeveloping nutrient feeding strategies offline. Where parameters including initial workingvolume, start of feeding, feed rate, concentration of substrate in the feed were varied. Thestrategy which resulted in maximum overall volumetric productivity with minimum residualsubstrate concentration in the medium after a given time was chosen for experimental validation.So, the feeding strategy which was selected for fed batch cultivation was as follows,batch cultivation was carried out in the reactor till 8th day. Then from 8th till 12th dayglucose feeding of 500 grams per liter and phosphate of 1 gram per liter was done ata rate of 0.05 liters per day in the reactor. Then from 13th to 14th day the glucose feedingwas stopped and nitrate additional feed started at 35 grams per liter.And the phosphate feed was continued, but at a reduced concentration of 0.5 grams perliter with the flow rate of all the feeds at 0.5 liters per day. So, of if you noticethe glucose feeding was stopped, the phosphate feeding was reduced the feed concentrationand the nitrate feeding was started. This strategy as predicted by the model gave atotal of nearly 22 grams per liter of biomass, and 90 milligrams per liter of azadirachtinin 14 days. So, these were the conditions which were usedfor the fed batch cultivation and then the fed batch cultivation was experimentally validated.So, if you see the kinetics here the plot shows that as the feeding has started from8th day you can see the residual nitrate the residual phosphate and glucose concentrationsgetting high and then gradually the biomass increasing and the azadirachtin feed alsoincreasing. Now, continuously the dry cell weight is increasingup till 14 days and the azadirachtin is also increasing continuously till 14th day. Whileresidual nitrate was becoming limiting around 12th day, so possibly that is the reason wherethe feeding was again started for the nitrate on day 12.Then the batch model was further used to develop or strategies nutrient feeding strategiesin continuous cultivation. Where again the same model parameters were used to designand design equations for the continuous cultivation and then the model was simulated.And experimentally validated one of the strategies which could give high azadirachtin productivities;then in fed batch or in batch was chosen for experimental validation in continuous cultivation.The MS medium salts with glucose of 75 grams per liter the nitrate feed of 10 gram perliter and phosphate of 0.5 grams per liter at a flow rate of 0.5 liters per day was chosenfor the experimental study. It the this continuous reactor which was runwas a cell retention reactor set up in which the feed was sent in the annular space ofthe reactor outside the filter till the 26th day. And the medium was withdrawn at the samerate from inside the spin filter. The model in this case predicted nearly 140 grams perliter of biomass and 130 milligrams per liter of azadirachtin in 26 days.So, if you see the plots you can clearly see that as the feeding was done in the continuousreactor with cell retention it led to increase continuous increase in the biomass and alsoin the azadirachtin productivity. So, experimentally the biomass which could be achieved was around88 grams per liter, and the azadirachtin concentration or titters which could be achieved now wereat a level of 280 milligrams per liter in 26 days.After achieving and designing nutrient feeding strategies for continuous enhancement in theproductivity of azadirachtin. Then, the authors went on to do process integration in whichthey added the elicitor and precursor selected from the shake flask studies to further enhancethe yield of azadirachtin and there by the productivity So, the cells and bioreactorswere during the batch mode, but treated with optimum concentration of elicitors after 8days of cultivation. So, these elicitors were chosen from the previousstudy of optimization with elicitors. So, salicylic acid, jasmonic acid, and chitosanwere selected at their optimum concentrations based on the design of experiments statisticaloptimization was done. And then these elicitors were added on 8th day and growth and azadirachtinproduction in the reactor was studied. And after addition the biomass was nearly thesame which was 14 grams per liter, while the azadirachtin content increased to 11.5 milligramsper gram in 10 days which was much higher than that obtained in the batch reactor inthe absence of elicitor. Then continuous cultivation with nutrientaddition, elicitor addition, precursor addition, and permeabilization was all done togetherto see an integrated effect where the cells were grown in bioreactor in batch mode. Thenthe cultivation was converted to continuous mode with cell retention with nutrient additionof carbon nitrogen and phosphate source with elicitor feeding from 8th till 40 days.And the cultivation was continued thereafter in batch mode till 44 days and before theharvest of the biomass was done for azadirachtin analysis. So, if you see on the right handside sodium acetate was used as a precursor which was added on second day. Then feedingof the MS salts with glucose, nitrate, and phosphate in the feed which was optimizedoffline was done from 8th day till 40th day. Along with the addition of salicylic acid,jasmonic acid, and chitosan which was optimized previously using central composite designat a feed rate of 500 ML per day. Then permeability enhancers were added on 41st day when thereactor was continued in batch mode till 44th day to enable the release of the product fromthe cells where 5 percent n-hexadecane was added in the reactor.So, what did they achieve? The maximum dry weight which could be achieved was now 62grams per liter and the maximum azadirachtin concentration or titters achieved were morethan 7 30 milligrams per liter. Now, after addition of hexadecane 751 milligrams perliter of azadirachtin was obtained in the thus from the final harvest, while 14 percentrelease in the culture medium was observed. So, this is what was done a summary of whatthe authors did in this case study, they began with a high azadirachtin containing tree basedcell line. They developed callus culture and then suspension culture among five differentmedia tested Murashige and Skoog medium demonstrated highest growth and azadirachtin production.Then optimum concentration of the medium nutrients culture conditions was obtained using statisticaldesign of experiments which will minimize the number of hit. And trial and then a afterthis they were able to achieve 15 grams per liter of biomass and nearly 50 mg per literof azadirachtin under optimum conditions in shake flask. The cells were found to be sensitiveto shear to increase in the rotational speed where the viability were found to drop morethan 85 percent above 150 rpm in shake flask. So, addition of sodium acetate was done asa precursor where azadirachtin productivity was improved to 162 mg per liter in 12 days.Then further permeability enhancers were optimized and hexadecane 5 percent was chosen whichcould result in 13 percent release of azadirachtin in the medium while maintaining the viabilityup as 10 percent. Then elicitors were chosen and their concentrationswere optimized using statistical design of experiments where salicylic acid, jasmonicacid, and chitosan were chosen. Then cumulative addition of these elicitors on 8th day for48 hours before harvest led to the yield increase up to 15 nearly 16 milligrams per gram.So, if you see a snapshot of how theproductivity improved from in different modes of cultivationswhen they did batch with steric impeller the productivity was 5. When they did batch witha centrifugal impeller which had improved mass transfer characteristics and mixing timecharacteristics the productivity improved to 7.2.Then they did continuous cultivation with cell retention device where the productivitywas found to further improve and then batch cultivation with elicitor addition was seento increase the productivity to very high levels almost doubling or three folds rather.Then, continuous cultivation with an integrated study which included addition of elicitor,precursor, permeability, enhancers and nutrient feeds led to the maximum enhancement in productivityup to 17 milligrams per liter per day. So, if you compare literature what they foundwas that they could achieve maximum yields of azadirachtin in the biomass by using thisrational and a more systematic manner of optimization. So, I hope this gives you an overall pictureof how plant cell bioprocessing different strategies can be implemented to achieve maximumenhancements in productivities of plant secondary metabolites .