Steps in Plant Cell Cultivation in the bioreactor
Development of callus culture –
For callus culture development, seeds are to be depulped and surface sterilized in 70% v/v ethanol for 1 minute followed by 3-4 times rinsing with Sterile Distilled Water (SDW). Seeds are thereafter sterilized in 0.1% w/v HgCl2 for 8 minutes and rinsed with SDW 4 - 5 times. This has to be followed by dissection of seeds. Seed kernel (along with embryo) has to be, thereafter, excised and used as an explant for callus induction on Murashige & Skoog medium (MS). It may be noted that a high azadirachtin containing callus culture will be developed from higher azadirachtin containing seed kernels. MS medium supplemented with naphthalene acetic acid (10µM), benzyladenine (2.5µM) with 8 g/L agar (Hi-media) has been reported to suitable for callus development. Callus culture can be maintained fresh by regular sub-culturing and incubations at 25ºC for 16/8hr light/dark period on the same medium.
Experimental Protocol
Plant cell suspension culture of A. Indica –
The plant cell suspension culture can be initiated by transferring the friable callus in 50 mL liquid MS medium. Growing callus cultures from shake flasks are transferred to modified Murashige & Skoog. The medium ( statistically optimized) containing glucose: 25.0 g/L, nitrate: 5.7 g/L, phosphate: 0.094 g/L, inoculum: 5 g/L (dry cell weight basis), 8 mg/L indole butyric acid and 4 mg/L benzyladenine (with rest of the constituents same as that of MS media). The cultures are incubated in gyratory shaker rotating at 125 rpm and maintained at 25ºC under dark conditions. Periodic sampling is done to figure out the right time for the subsequent transfer to the bioreactor.
Batch cultivation of cells of A. indica plant in 3 L stirred tank bioreactor –
A. indica cells can be cultivated in 3 - 5 L stirred tank bioreactor (Applicon, Dependable Instruments, The Netherlands) with 1.5 L working volume. The statistically optimized medium recipe (as described above) can be used for the study of growth and product formation in batch cultivation. Mixing in the bioreactor has to be done by low shear setric impeller at an agitation rate of 125 rpm. Dissolved oxygen has to be maintained above 30% in the reactor by manually varying the air-flow rate. The temperature and pH in the bioreactor is to be maintained at pre-optimized values 27ºC and 5.8 respectively by bio-controller (ADI 1030, Applicon, Dependable Instruments, The Netherlands). Samples (25 ml) are to be collected every 2nd day and analyzed for dry cell weight, azadirachtin content, residual glucose, nitrate and phosphate concentrations to establish the growth and production kinetics in the bioreactor.
Protocol for operation of model simulator
Bioreactor –
The Bioreactor portion illustrates the different components which are adequately described in the theory link. It also describes the purging of the sterile air (please see the air filter in the air inlet) from the sparger and rotation of the impeller. The impeller is bottom driven. This kind of arrangement is possible with BioEngineering AG Switzerland Bioreactor. This kind of agitation gives more space on the lid so that there is no overcrowding of different inlet ports & sensors and it is particularly useful when flame is used to connect acid /alkali pipes and when transfer of inoculum is done to the reactor. The constant temperature water flows in the jacket of the reactor (marked green) to maintain the temperature of the reactor.
Select parameters –
Beneath Select parameters label there are spaces for Simulation Start time, Time increment step and Simulation end time wherein the User have the option to enter different relevant numbers to start the simulations using different time intervals till the end of the simulation. The maximum limits are also indicated User has to ensure that the values do not exceed the maximum values.
Beneath above table there is another table which allows the Users to input the values of different model parameters (the minimum and maximum range is indicated in the brackets and the users has to ensure the input with in the range).
Underneath above table there is yet another table which allows the Users to input the initial conditions for the model simulation at time t=0.
Run Simulation –
After entering the above entries the User can enter the RUN Simulation button and the graph and table for X, S, P vs time is printer on the screen.
More graphs are available for Batch Microbial Cultivation wherein the user also sees the rate specific rates and yields of the biomass substrate and product vs time. This data along with the kinetic profile can be used to understand the culture behavior.
Out put Graph –
This section describes the kinetic profile of different fermentations in graphs.
Save Results –
By pressing this button the User goes to the Excel Sheet where all the values of kinetic profile is tabulated which can used for the detailed analysis of the system and /or making more desired trends in the Excel Sheet.