The sparge is a non- limiting supply of oxygen that can be in the form of air or a mixture of air and pure oxygen. Dissolved oxygen is one of the most important indicators in a fermentation or bioreactor process. It determines the potential for growth. Oxygen requirements for each mole of glucose metabolized is 6 moles of O 2 , which equals about 25 mmoles of oxygen per gram of dry cells. When considering the stoichiometry of respiration and the complete oxidation of glucose, the mass equation is
C 6 H 12 O 6 + 6O 2 = 6H 2 O + 6CO 2 [3.6]
This means that in order to oxidize 180 g of glucose, you need 192 g of oxygen. Given the fact that oxygen is 6000 times less soluble in water than glucose, the need for a consistent and plentiful oxygen supply is critical. To complicate matters further, it is shown that the demand for oxygen is dependent on the source of carbon in the media
and that the more reduced the carbon molecule the greater the oxygen demand will be [76]. Thus, pick your carbon substrate carefully, with oxygen demands/requirements in mind. If you want to reach high cell densities in your fermentation process, a more oxidized form of substrate may be better. To get around this constraint, the researcher could choose glucose, a highly reduced carbon source, and feed the culture at a lower rate where the oxygen demand would be better controlled.
Oxygen, by its nature, is an insoluble gas in aqueous media. At 20 °C, water holds about 9 ppm of oxygen. At 37 °C it is even less, at 7 ppm of oxygen. The solubility of oxygen in water is directly proportional to the partial pressure of oxygen in the gas phase. This is can be described mathematically by Henry’s Law [77]:
C* = 1/Hʹ × PO 2 [3.7]
Hʹ is a Henry’s Law constant based on molar concentration and the partial pressure (PO 2 ) is in atmospheres. Henry’s Law can be described as: “At a constant temperature, the amount of a given gas that dissolves in a given type and volume of liquid is directly proportional to the partial pressure of that gas in equilibrium with that liquid.”
Therefore, in order to achieve suffi cient oxygen transfer that will support a fermentative culture, the dissolved oxygen is generally kept between 20 and 40%. Oxygen is sparged into the bioreactor vessel using a sparge ring and a clean source of air. Measurement of the sparge rate is usually defi ned as the volume of air introduced into the vessel per volume of the liquid phase of the vessel per minute (VVM). Pure oxygen can also be supplied to the vessel as needed to maintain this percent dissolved oxygen. It was established in the 1950s [78] that the transfer of O 2 from the air to the cell happens in three stages (Figure 3.2):
1. transfer of oxygen from the air bubble into solution;
2. transfer of the dissolved oxygen through the fermentation medium to the microbial cell; and
3. uptake of the dissolved oxygen by the cell.
Bartholomew’s research team demonstrated that the limiting step was the transfer of the oxygen from the air into the medium [79]. This transfer can be described by the equation:
dC L /dt = K L a(C* − C L ) [3.8]
where CL is the concentration of dissolved oxygen in the fermentation broth (mmoles/dm 3 ); t is time (hours); dCL/dt is the change in oxygen concentration over a time period (mmoles O 2 /dm 3 h); KL is the mass transfer coeffi cient (cm/
hr); a is the gas/liquid interface area per liquid volume (cm 2 / cm); and C* is the saturated dissolved oxygen concentration (mmoles/dm 3 ).
Because it is diffi cult to measure both K L and ‘a’ in a fermentation, the two terms are used together (K L a) as the volumetric mass- transfer coeffi cient (h −1 ) and is used to measure the aeration capacity of the bioreactor. A large K L a means a higher aeration capacity of the fermentation system.
The design of the fermentor, along with fermentation conditions, will have a direct effect on the K L a value.
Variables such as aeration rate, agitation rate and impellor design are critical in maximizing oxygen transfer. The dissolved oxygen concentration is only the oxygen that is available to the cells in the media. It refl ects the balance between the supply side (air sparge) and the oxygen demand from the growing culture. Thus, if the K L a is low and the demand for oxygen by the organism is high, the dissolved oxygen concentration will decrease below a critical level for optimal growth. If the K L a is high, the dissolved oxygen concentration is likely to be close to saturation and will
support the continued growth of the recombinant culture throughout the fermentation. Therefore, we can conclude that the oxygen transfer rate is related to both the K L a value and the dissolved oxygen concentration in the liquid media.
Measurements of the dissolved oxygen in the liquid medium are made by an oxygen electrode (probe) that is immersed in the medium. There are generally three factors that affect the amount of dissolved oxygen in a given volume of water or media:
1. the atmospheric pressure of the air surrounding the fermentation vessel;
2. the temperature of the liquid medium; and
3. the amount of other dissolved substances such as sugars, salts or other gases in the liquid medium [80].
Oxygen transport within the cell Figure 3.2
This probe records dissolved oxygen activity or dissolved oxygen tension (DOT). The solubility of oxygen is affected by dissolved solutes so that pure water and a fermentation medium saturated with oxygen would have different dissolved oxygen concentrations yet have the same DOT. In order to translate DOT into concentration, the solubility of oxygen in the fermentation medium must be known [79,80].
When assessing the bioreactor vessel’s aeration effi ciency (soluble O 2 ), determination of the oxygen transfer coeffi cient K L a is essential in establishing an adequate supply of oxygen to the bioreactor. This becomes even more critical as the researcher scales up to larger bioreactor volumes. Changes in aeration rates, impellor design and micro- bubble dispersion have been studied in an attempt to maintain the K L a at the larger scale [79,80]. There are a few different techniques that have been used to determine K L a in the fermentation system:
■ the sulfi te oxidation technique (not recommended);
■ the gassing out techniques (static and dynamic methods);
and
■ the oxygen balance technique.
The oxygen balance technique is viable, but much more technical and will not be discussed here.
Sulfi te oxidation method
The sulfi te oxidation method relies on the conversion of a 0.5 M solution of sodium sulfi te to sodium sulfate in the presence of O 2 and a copper or cobalt catalyst. This method has the advantage of being simple, but due to the sulfate determination (can be up to 3 h) has been shown to be historically inaccurate [80].
Gassing out method
The gassing out method relies on purging the media in the bioreactor of O 2 to a low level and then initiating the aeration and agitation and measuring the dissolved oxygen over time using a dissolved oxygen probe.
Static method
In the static method, the O 2 is removed by purging the liquid phase, which can contain media or water but no live culture, with nitrogen until the dissolved oxygen level is very low.
Begin aeration and agitation and record the increase in % dissolved oxygen (%DO) every 10 s until the % DO reaches above 100% (C*), graph %DO change versus time ( Figure 3.3 ). To determine the oxygen transfer rate at a given time, draw a tangent to the curve at this time (50 s). The slope of the tangent is the oxygen transfer rate.
The K L a is then determined from the equation OTR/C* = K L a.
Another method of determining K L a is to plot the natural log (ln) of (C* − C L ) versus time ( Figure 3.4 ). The slope of this line is −K L a. This method is recommended for 10 L or less.
Dynamic method
The dynamic method requires that K L a determination takes place within the background of a live culture. In 1966, Taguchi and Humphrey [81] made use of a growing culture to lower the level of O 2 in the bioreactor to low levels prior to the aeration portion of the method. As can be seen in Figure 3.5 , this period gives the researcher a measure of the respiration rate of the culture (A-B). The aeration is resumed and the rate at which the DO reaches a maximum is directly related to the oxygen transfer rate and thus K L a (B-C). K L a is
%DO versus time Figure 3.3
ln (C* − C L ) versus Δ time (s) Figure 3.4
then determined by measuring the slope of the tangent of BC at differing DO concentrations. Obviously, the cell culture must not be too dense when performing this procedure, due to the high respiration rate.
Maintaining suffi cient oxygen transfer rates, and thus K L a values, for bioreactor systems is crucial to the successful development of a robust fermentation process. Factors that affect the K L a include air fl ow rate, agitation rate and rheological properties of the media, as well as temperature and the presence of antifoam agents. Another parameter that is helpful in maintaining dissolved oxygen is agitation. This will be addressed more fully in the next section.