Agitation is performed by an electric motor that sits atop a drive shaft that goes through the head plate of the vessel,
Oxygen transfer rate and K L a determination Figure 3.5
connecting the motor’s power to the drive shaft in the vessel containing the impeller assembly. This agitation is a means of mixing cells, nutrients and oxygen bubbles throughout the liquid phase of the vessel, allowing uniform distribution and effi cient transfer of nutrients and oxygen to the growing cell population. Within the context of oxygen transfer, the agitation helps to increase residence time and breaks the bubbles into smaller sizes when air is fed through to the sparge unit below the impellor assembly.
This mixing also creates a uniform environment for the transfer of heat and the maintenance of a continuously stable pH.
Critical to the idea of effi cient mixing within a bioreactor, the vessel must be designed in such a way that gives the researcher the best chance to achieve this goal. There are three key structural components (all made of stainless steel) to the bioreactor vessel that contributes to this goal:
1. the impellers;
2. the baffl es; and
3. the aeration system or sparger.
All three of these components are present in stirred tank reactors. The differences in the design of these components lie with the actual size of the bioreactor vessels themselves.
As the volume of the reactor is scaled up, the component’s design can change in order to accommodate the mixing demands of the culture at the larger volume.
The impellers
The shapes and sizes of the impellers are probably the most varied in design of all the components for agitation. That is because the design of the impeller has the most impact on mixing, depending on what type of cell culture or fermentation
you are trying to achieve. There are two distinct designs of the impeller:
1. Radial fl ow impeller : such as a disc turbine or Rushton impeller; and
2. Axial fl ow impeller : such as a pitched blade or Marine propeller.
Since the 1940s, the Rushton impeller at one- third of the bioreactor’s diameter has been considered the most useful design for many fermentation processes. Radial fl ow impellers are used when high agitation and air fl ow rates are needed, especially for high cell density bacterial fermentations.
Compared to the disc turbine, the Marine impeller fl oods out much more readily and because of its axial fl ow, is less effi cient in breaking up the stream of air bubbles. Flooding of the impeller happens when the normal bulk fl ow of the media is lost and is replaced by a column of air coming up through the middle of the vessel. The fl ooding takes place due to the superfi cial velocity (Vs) of air that the impeller can handle. A radial fl ow impeller can handle six times the air fl ow than the Marine impeller, and thus is much less likely to fl ood out [76]. Typical spacing between the impellers is 1 to 1ẵ impeller diameters apart.
The baffl es
The baffl es are interconnected and independent from the vessel itself. They run vertically down the inside of the bioreactor vessel and are approximately one- tenth of the diameter of the vessel. There are usually four divided equally around the interior circumference of the vessel and are there to prevent a vortex from forming during agitation and to improve oxygen transfer within the liquid phase of the bioreactor vessel.
The sparge
The most common sparging unit in the fermentation industry is the ring sparger. It is usually stainless steel and forms a ring structure at the base of the reactor vessel, directly under the impeller shaft. This positioning gives the best chance at maximizing the aeration rate. The ring diameter is approximately three- quarters the diameter of the impeller and is perforated with tiny holes that are evenly spaced over the underside of the ring. The number and size of these holes are dependent on the size of the vessel itself.
The source of air should be clean and of low moisture.
Typically the air is fi ltered prior to entering the bioreactor control unit and then fi ltered again before it enters the bioreactor vessel itself. At times, depending on the level of moisture in the atmosphere, an air drying unit can be installed between the air outlet and the bioreactor control unit. The reasons for these precautions are obvious in that non- purifi ed air can be a source of contamination, especially given the large volume of air that fl ows through the vessel media.
Depending on the source of air, it may be recommended that the air is fi ltered between the air source and the bioreactor unit. A fi lter such as a BALSTON DFU Grade DQ, which is a microfi ber fi lter, can be used to remove any signifi cant moisture from the air prior to it entering the reactor.
If required, the sparge can be a mixture of both fi ltered air and pure oxygen. As the culture grows to higher optical densities, the amount of O 2 demand will increase. A bioreactor control unit should be able to call for a mixing of these two gases in order to compensate for this metabolic need.
In order to perform a fed- batch fermentation for the production of a recombinant protein, a number of high priced pieces of equipment must be acquired. These consist
of, but are not limited to, a centrifuge, a visible spectrophotometer, a bioreactor system with culture vessel and probes, an air/O 2 supply and an autoclave.
When setting up for a fermentation run, sterility is important and every manipulation of the sterilized vessel should be done with care. When opening up a feed line or an addition port, the line or port should be sprayed before and after with 70% IPA. This will dramatically reduce the possibility of contamination.
10-liter bioreactor for E. coli fermentation Figure 3.6