Stages in Batch Cultivation
Microorganism –
R. eutropha NRRL B14690. The culture is to be maintained on nutrient agar slants at 5ºC, and sub cultured monthly.
Media for shake flash cultivation –
A mineral salt medium which consisting of: 2.0 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.6 g/L Na2HPO4, 0.2 g/L MgSO4.7H2O, 20 mg/L CaCl2, 10 mL/L trace metal solution, 0.1 g/L yeast extract is to be used. The trace metal solution consists of: 1.3 mg/L ZnSO4.7H2O, 0.2 mg/L FeSO4.7H2O, 0.6 mg/L (NH4)6Mo7O24.4H2O and 0.6 mg/L H3BO3. Fructose is to be used as carbon source in concentration of 40 and 10 g/L for production media and inoculum development, respectively. Fructose, yeast extract and salt solutions are to be sterilized separately at 121ºC and then aseptically reconstituted at room temperature prior to inoculation. The pH of the resulting broth has to be adjusted to 7.0 with 2N NaOH/2N HCl.
Bioreactor cultivation –
Seed culture is to be prepared in a 1 L Erlenmeyer flask containing 200 mL media. Batch cultivation is to be carried out at 30ºC in a 7 L LF-1523 Bioengineering (Bioengineering AG, Switzerland) bioreactor containing 4 L of media. The reactor has to be sterilized in-situ at 121ºC for 20 min, cooled and then inoculated with 5% inoculum (v/v). Culture pH is to be maintained at 7.0 by automatic addition of acid or base by pH–mV controller M 7832 N. Dissolved oxygen concentration is to be maintained at 30% saturation value by manually adjusting the speed of the agitator and/or airflow rate. Samples are to be withdrawn at regular intervals and analysed for biomass, PHB and residual nutrients.
Protocol for operation of 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.