Dissolved Oxygen Control in Bioreactors

A DO cascade describes a specific sequence of actions that are employed to increase the DO concentration within bioreactors. As you move through the cascade, different actions of increasing intensity are initiated to ensure DO control is sufficient to optimize yield.

• Increased agitation - This is typically the first step of DO cascades and is achieved using an impeller, which is moved by a motor. The maximum agitation speed is not necessarily the theoretical maximum that can be achieved by the setup of choice. The user may decide to limit the maximum to a certain value, for example to avoid extensive shear stress to the cells.
• Air sparging - Once the maximum agitation speed has been reached, air sparging is increased in the system up to its maximum rate. Again, the maximum is user-defined.
• Oxygen enrichment - The air that is sparged into the system is gradually replaced by oxygen, until pure oxygen is sparged into the bioreactor.

Spargers are tools for introducing gasses into liquids through tiny pores. Different types of spargers exist with different functions in DO control, including macro-spargers and micro-spargers. Macro-spargers contain drilled holes that create larger gas bubbles. These are best suited for CO₂ removal, as they are capable of capturing more gas into the bubbles that rise to the top. However, the large bubbles released by macro-spargers require agitation to release oxygen and so macro-spargers are unsuitable for more sensitive cultures such as mammalian cells. Even bigger gas bubbles are created by open pipe spargers. Contrastingly, micro-spargers have a much smaller pore diameter of a few micrometers wide. They therefore introduce smaller bubbles that are more effective at transferring oxygen while minimizing cell stress in bioreactors.

In bioreactors, gasses introduced into the bioreactor can either be released into the headspace, or they can be released into the culture using spargers in a technique referred to as submerged gassing. Headspace gassing is often used to increase oxygen transfer into the culture, when agitation is too challenging or too detrimental to the cells. It is, however, less effective than submerged gassing. Submerged gassing is the most effective method of mass transfer of gasses in bioreactors. Spargers are typically located beneath the impeller to maximize the distribution of gas throughout the culture. However, sparging requires agitation for effective gas distribution, which can damage some types of cells.

Impellers - also known as agitators - are key components of bioreactors, providing agitation to increase oxygen transfer into the culture. A balance is needed to ensure that there is enough agitation for effective DO control, but not too much that it becomes damaging for more sensitive cell cultures, such as mammalian cells and stem cells. To achieve this, various impeller types are available that are suited towards different types of cultures and DO requirements. Blade orientation largely impacts the agitation of the impeller, with axial blades providing more gentle mixing than radial. Impellers suitable for more sensitive cell cultures include marine impellers, which have axial blades with convex back sides to provide gentle mixing. Another example is the pitched-blade impeller, which has blades oriented at a certain angle (often a 45° angle is used) to provide effective, yet gentle mixing for viscous or sensitive cell cultures. Impellers suitable for more robust cultures includes Rushton impellers, with flat, radial blades. Rushton impellers are commonly used for microbial fermentation in bioreactors. However, other factors related to the impeller design can influence the DO transfer rate, including the number of blades, the impeller speed and diameter.

The % DO is a measure of how much oxygen is dissolved in a solution. This is different to the total oxygen concentration, as DO depends on the solubility of oxygen in a solution. For example, 100 % DO in one solution could have a different total oxygen concentration than another solution with 100 % DO. Solubility can be impacted by several environmental factors such as the temperature and the pressure. Therefore, DO sensor recalibration is always required under different process conditions.

There are two main types of DO sensors, polarographic and optical. Polarographic DO sensors measure the electrical current within the solution to infer the oxygen concentration. Optical DO sensors measure the luminescence of a solution using blue light, with the presence of dissolved oxygen decreasing the luminescence. Find out more about the measurement principle of these sensor types and get guidance how to calibrate them:

k_LA and OTR measure the efficiency of DO transfer into the bioreactor culture, hence are important parameters for DO control. The k_LA is the volumetric mass transfer coefficient. This describes the rate of oxygen or gas transfer into the culture, per unit of volume, per hour. k_LA is strongly associated with features of bioreactor design, influenced by bubble size, agitation speed, impeller type and sparger type. OTR is the Oxygen Transfer Rate. It is equal the k_LA multiplied by the O₂ concentration gradient and therefore proportional to the k_LA.Therefore, k_LA and OTR values are paramount to the design of DO control systems. These are important values to consider when implementing new bioreactor equipment or when changing the operating conditions. When scaling-up for example, it is important to choose differently sized bioreactors with similar oxygen transfer capabilities to be able to reproduce the conditions optimized at small scale at larger scales.