Steps in Fed-Batch Microbial Cultivation in the bioreactor
Microorganism and Maintenance –
Sorbitol - commercial grade (70% w/w) is to be used. NRRL B-72 strain of Acetobacter suboxydans facilitates bioconversion of sorbitol to sorbose Cultures are to be maintained on agar slants having the composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; agar 20. The initial pH has to be kept as 6.0. Take samples after 48 h growth at 30º C and preserve the cultures at 4º C.
Inoculum Development –
A loop full of microorganism from the slant has to be transferred into 10 mL medium in test tubes having the composition (g/L): Sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH 6.0. The test tubes are to be incubated at 30º C for 72 h. The bacterial growth is characterized by the appearance of a thick pellicle on the surface of the medium and uniform turbidity. It is then ready for transfer to next stage. The growing inoculums from the test tube is then transferred into 1.0 liter capacity flasks containing 100 mL medium of composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH, 6.0. The flasks are then incubated in a rotary shaker (Adolf Kuhner, Switzerland) at 30º C and 250 rpm. Subsequent transfer of inoculums from the shake flask to the fermenter has to be done when the biomass concentration in shake flask is about 2.8 to 3.0 g/L.
Batch Fermentation –
The microbial batch cultivation can be carried out in a 7.0 litre capacity fermenter (Bioengineering A.G., Switzerland) / or equivalent equipped with two sets of flat blade turbine impellers. The working volume can be kept as 4.5 litres. The medium is prepared and filled in the bioreactor. The reactor is autoclaved in-situ and the medium is allowed to cool down to room temperature. The actively growing inoculum is transferred from shake flask to the bioreactor. The aeration rate and agitation speed have to be kept at 2.2 vvm and 700 rpm respectively. The temperature is to be maintained at 300 C and the pH at 6.0 by the automatic addition of 3 N NaOH and 3 N HCl. Intermittant samples are taken to assess the biomass, sorbitol and sorbose concentrations.
Fed-batch Cultivation –
A number of fed-batch cultivation can be designed using the mathematical model. As has been indicated, fed-batch cultivation features availability of disappearing nutrients particularly during exponentially growing phase of the culture. The batch cultivation is allowed to proceed till it reaches the exponential growth phase and then a number of feeding strategies (constant feed, linearly increasing / decreasing feed rate, exponential feed rates) can be simulated off line and selected few (which give high product concentration and productivity) can be shortlisted for experimental validation. The advantages/disadvantages of adoption of one nutrient feeding strategy over other has been described in detail in the theory section of the experiment.
To execute a fed-batch cultivation, start the cultivation as batch with an initial sorbitol concentration of 100 g/L. The concentration (g/L) of other nutrients have to be kept as; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0 and magnesium sulphate, 1.0. Sorbitol concentration in the feed bottle has to be kept as high as possible so that it gives minimum dilution to the fermentation broth in the bioreactor and the contents of the reactor are not excessively diluted by the incoming feed. (Usually 600 g/L sorbitol can be used in the feed bottle). The rest of the medium components have to be increased in same proportion so that they are not limiting during the cultivation. The fed-batch cultivation has to be stopped when all the sorbitol in the bioreactor is totally consumed as unconverted substrate (sorbitol) is a loss to the company and also it give rise to problems in recovery of product (sorbose). The completion of sorbitol (end of the fermentation) is reflected by increased DO signal in the bioreactor.
Biomass Concentration –
Optical density (OD) of the suitably diluted fermentation broth samples is to be measured at 600 nm in a UVIKON 930 spectrophotometer (Kontron Instruments, USA). Biomass can be measured from an OD vs concentration (g/L) correlation, which has to be determined a priori as follows:
Determine the Biomass concentration of a known volume of the fermentation broth samples gravimetrically after measuring the OD at 600 nm. Plot a standard curve between OD of the samples and their respective biomass concentrations to arrive at the following correlation.
Biomass (g/L) = 0.73 × OD600
Sorbitol and Sorbose Concentrations –
Sorbitol and sorbose concentrations are to be measured by HPLC (Waters Associate USA) using a supelcosil (LC-NH2) column (Supelco USA) 25 cm X 4.6 cm ID equipped with RI detector and using acetonitrile-water (75:25) as eluent with a flow rate of 1 ml/min at ambient temperatures.
Protocol for operation of model simulator
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.
The Simulator is designed in such a way that the USER has the option to change the different values of the start / stop time, time interval. The model parameters can also be changed. Even the Model Structure can be modified but putting relevant coefficients as zero. It can simulate different bioreactor operating strategies (fed-batch, Continuous) which can then be implemented experimentally.