The overall goal of this procedure is to set up a bioreactor that will provide a controlled environment for large scale growth of bioengineered cells. This is accomplished by first assembling the vessel, calibrating the pH probe, and autoclaving the assembled vessel. The second step is to attach the autoclave vessel to the fermentor base and to set the parameters for impeller speed, temperature, pH, and oxygen saturation.
Next, the bioreactor run is initiated so that the desired impeller speed, temperature set point, and oxygen saturation are achieved after manual calibration of the oxygen probe. The final step is to inoculate the bioreactor with the bacterial culture and monitor the conditions of the bioreactor by the computer software. Ultimately, the growth characteristics of the culture can be analyzed and the culture can be processed for extraction and purification of the desired product.
In this video, we demonstrate the setup and operation of a bioreactor for bacterial fermentation. The visual demonstration of this method can be useful as the assembly calibration and monitoring of the bioreactor can be a challenging task for individuals not familiar with this technology. Okay, let's get started.
To begin, add bacterial medium that is not heat labile to the clean vessel Only add until maximum working volume is reached, which is 3.3 liters for a five liter total volume vessel. Mount the vessel head plate ensuring that the O-ring seal is properly seated. Install the exhaust gas condenser.
Install the harvest pipe into its 10 millimeter port. Place a piece of tubing on top of it. Connect to the sampling bottle and clamp the tubing off.
Install tubing and a 0.2 micron filter on the sparge inlet and then clamp this off. To calibrate the pH probe, gather two reference buffers in small beakers, a wash bottle and a wash beaker. Hook the pH probe up to the fermentor and turn it on.
Scroll to the pH parameter and then using the menu button, scroll to the cow option and press enter. Select two for two point calibration. Wash the pH probe tip and submerge it in the low reference buffer.
The value on the fermentor will stabilize using the plus and minus keys. Adjust the displayed value to match the value of the pH reference buffer. Once it stops blinking, press enter Next.
Wash the low reference buffer off the probe and insert the probe into the second reference buffer. Allow the value to stabilize and then use the keypad to make the displayed value match the high reference buffer value. Press enter to confirm.
Wash the probe tip and disconnect it from the fermentor. Put the protective cap on the connection and install it in the head plate. Open the oxygen probe tip and check to ensure that there is enough electrolyte to cover the tip.
Install the oxygen probe in the head plate. Make sure the autoclave cap is put on to protect its electrical connections as it will be sterilized prior to calibration. Next, obtain a bottle for reagent feed with a dip tube and air filter.
Add pump tubing to the dip tube port and attach the other end to the inlet port on the fermentor Place tubing on all unused ports and then clamp off the tubing. Install a 0.45 micron filter on the exhaust gas condenser. This filter ensures that the vessel does not pressurize in the autoclave.
Check that all tubes that go beneath the level of the media are clamped off to prevent media from leaking. Place the vessel in the autoclave and autoclave for 25 to 30 minutes at 121 degrees Celsius using a liquid cycle. Once the autoclave vessel has cooled, the next step is to install it on the fermentor base.
Then hook up the pH probe cable. Attach the oxygen probe cable and hook the sparger up to the gas addition. Rotter, turn the fermentor on while maintaining sterility.
Add one molar sodium hydroxide reagent to the bottle with the dip tube and install the pump head on the base pump head spindle. Put the temperature sensor in the thermo well on the head plate. Ensure that it goes all the way to the bottom of the thermo.
Well lower the agitation arm onto the vessels impeller coupling. Make sure that the fermentor is on. Check that water is available to the fermentor.
Turn on the air supply to the fermentor. Scroll to the temperature parameter and set the temperature to 37 degrees Celsius. Press the enter button to start the temperature control.
The vessel will heat up in 15 minutes or less. Turn the gas flow to one vessel volume of media per minute or less. For this setup, gas flow is three liters per minute.
Bubbles should appear at the bottom. Scroll to the agitation parameter and set to 300 RPM. Press enter to turn on the agitation.
Once the temperature has reached 37 degrees Celsius. Scroll to the pH parameter and set it to 6.8. Turn the pH control on by pressing the enter button.
Set agitation to the maximum speed of 1000 RPM for the run. Ensure that the oxygen probe is polarized properly to display proper values after two hours. Scroll to the oxygen parameter.
Use the menu button to select the calibrate function and select the one point calibration. After 15 minutes, use the cursors to set the value to 100 and press enter. This will calibrate the oxygen saturation.
Scroll to agitation and set to 300 RPM. Using the menu button, turn the cascade to on in the agitation menu. This will increase the speed of agitation in an effort to fragment air bubbles and force more oxygen into solution as demand increases due to growth.
Scroll to the oxygen parameter and set the value to 30. Press the enter button. Start the control software package on the pc.
Once the probes are calibrated, agitation is stable at 300 RPM. Temperature is at 37 degrees Celsius and pH is close to 6.8. It is time to inoculate using alcohol.
Sterilize the port that will be used for inoculation. Draw the inoculum into a syringe and add it into the sterilized port. Close the port mark, the time of inoculation in the computer log.
It is also important to take a sample at inoculation time. After opening the clamp, covering the harvest port, push a syringe plunger to clear the line. Then pull back on the syringe.
To draw a sample, unscrew the bottle and discard the first sample because it has been sitting in the pipe. Reattach the bottle and use the same syringe plunger to pull another sample. Remove the bottle and empty the contents into a vete.
For testing, push air back through the pipe using the syringe and close the clamp on the sampling port tubing. Determine the cell density and pH and log the values. With microbial cultures, it is useful to log values every hour, but the sampling interval is culture dependent.
The harvest methodology will depend on the intent for the culture after growth is completed. In this case, sample the culture a final time to receive endpoint values for cell density and possible pH or measurement of wet cell weight. Lastly, open the head plate, harvest the culture, and then dispose of the vessel content in a designated kill tank with bleach or other antimicrobial agents.
In this video, the setup and operation of a typical benchtop bioreactor system was demonstrated. This figure shows the bioreactor at the beginning of a run with all parts labeled and connected. The data output for a representative bioreactor run is presented in this figure.
At the start of a stirred tank run. The temperature was set at 30 degrees Celsius, impeller speed at 200 RPM pH at 6.5 and oxygen at 57.2%The parameters may be different for each experiment, but before addition of bacteria, the system should be at steady state. In this next figure, the change in oxygen concentration with the addition of the bacterial culture is shown by the blue line.
The feed pump delivers the bacterial seed culture at an OD of 0.1, causing an immediate drop. In the O2 level, the bioreactor responds to the changing O2 level with an increase in the impeller speed. This final screen image of the bioreactor software shows pH change over time represented by the dark blue line.
The pH of the culture was monitored continuously and over time it decreased. As lactic acid was produced and then increased. The entire run can be analyzed and parameters adjusted for subsequent experiments.
After watching this video, you should have a good understanding of how to set up and monitor a bioreactor for expression of a recombinant protein. Fermentation systems are used to provide an optimal growth environment for many different types of scale up cultures. The ability afforded by fermentors to carefully control temperature pH and dissolved oxygen concentrations in particular, make them essential to efficient, large scale growth and expression fermentation products.
Thank you for watching this video and best of luck with your experiments.
