Steps in batch anaerobic microbial cultivation in a bioreactor
Culture –
Clostridium acetobutylicurn strain ATCC 824 is capable of producing Acetone – Butanol and Ethanol.
Culture tubes –
Culture maintenance storage spore activation has to be done in hungate tube (1.5 cm in diameter and 15 cm long) Seal the screw threaded top of the hungate tube with a silicon septum and open screw caps to facilitate the insertion of sterile hypodermic needles for aseptic culture transfers to and from the hungate tubes.
Fermentation flasks –
Growth of the inoculum and some preliminary studies on the performance of the culture can be done in custom made 500 mL flasks Replace the mouth of the flask by serum bottle mouth cover by a rubber stopper and seal by aluminium cap by means of crimper. Sparge sterile high purity nitrogen gas for one hour to flush out the air from the flasks and provide an anaerobic environment for inoculum growth. Use constant temperature gyratory shaker to agitate the flask and maintain 200 rpm.
Maintenance medium –
Reinforced clostridial medium (RCM) is to be used for the maintenance of the culture and activation of the spores.
Cultivation media –
A standard culture media with respect to ammonium sulfate and yeast extract for the specific glucose concentrations has to be used in this study while maintaining the same concentrations of minor nutrients. It typically consists of the following components in g/L: glucose, 50; ammonium sulfate, 9.0; yeast extract, 11.0; K2HP04, 0.8; KH2 P04 0.8; MgS04, 0.3; MnS04 .H20, 0.02; NaCI, 0.02; FeS04 . 7H20, 0.02; and L-cysteine, 0.5. Temperature 37ºC, pH 4.5 or 6.0
Flask and bioreactor cultivation –
Heat shock the spores maintained in RCM by placing the test tube in a boiling water bath (100ºC for one minutes). This is to be followed by cooling to 4ºC for 3 minutes. The temperature of the tube is normalized to room temperature for one hour. 3 % inoculum is to be transferred into 15 mL hungate cap test tubes containing 10 mL of the RCM. The test tube containing the spore solution were kept for spore activation at 37ºC in an anaerobic jar for 20-24 hours. Adequate growth for subsequent transfer of the inoculums was characterized by the start of gas production. 3 % inoculums was transferred into a liquid glucose medium (50 g/L) in a shake flask. After 10 hours of cultivation, samples were withdrawn from the culture flask every hour. The pH of the inoculum was not allowed to fall below 4.6 by manual addition of 0.1 N NaOH. The appropriate timing for inoculation (14-15 hours) was identified by the culture acid break point through the analysis of extracellular products by gas chromatography. A 3 % inoculum is transferred to the fermenter containing sterilized 50 g/L glucose medium at pH 4.6 Before inoculation the fermenter was sparged in the head space by Nitrogen. The nitrogen sparging was continued until the gases were being produced by the culture. The culture was allowed to grow in a batch mode.
Analytical protocols
Cell Dry Weight –
20 ml sample of the fresh fermentation broth is centrifuged at 10,000 g and 4ºC for 15 minutes. The supernatant is frozen in deep freezer for extracellular metabolite and glucose analysis. The biomass is washed three times with equal amount of distilled water. The pellet is re suspended in 10-15 mL of distilled water, transferred to preweighed aluminum dish and dried in an oven at 100ºC for 24 hours. After cooling the aluminum dish to room temperature in a desiccator the dry weight of the cells is measured.
Glucose –
Glucose concentration can be measured by DNS method or by hexokinase enzymatic analysis. A direct relationship between glucose concentration and OD340 is obtained in the range 0 – 4 g/L.
Solvent and acid concentrations –
These products are to be analyzed by Gas-Liquid Chromatograph equipped with 183 cm by 0.32 cm O.D. nickel column packed with Chromosorb 101 80/100 mesh using following conditions –
Carrier gas – Helium, Injection, detector, column port temperature 165ºC, Carrier gas flow 30 mL/min, Attenuation -2, Sample size 2µl, Detector- FID.
During fermentation appropriate amounts of samples are to be withdrawn and filtered by 0.45 µ filter One ml sample is acidified with 50µl of 6N HCl to ensure conversion of un-dissociated acids to their dissociated acidic forms. 2µl of this sample is injected in to the gas chromatograph equipped with the data integration software. The unknown samples are compared with the peaks of standard mixture to find the concentrations of acids & solvents.
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.