(368f) A Modified Dynamic Method for Measuring kLa

The oxygen transfer rate (OTR) is a crucial parameter in aerobic and microaerobic processes, since it is often the rate-limiting step due to the low solubility in the broth. Therefore, it is essential parameter to accurately measure the mass transfer coefficient for process control, design, and scale-up of bioreactors. Both physical and chemical methods have been proposed for the measurement of the mass transfer coefficient,. Among them, a physical method named dynamic method¹has been widely used to measure the  with the presence of cells.

In general, the dynamic method consists of two steps: consumption and absorption. During consumption, aeration is stopped and dissolved oxygen (DO) decreases due to cell respiration, therefore the graphical slope of DO can be used to estimate the oxygen uptake rate; during absorption, aeration is resumed, and the DO increases until a steady-state is reached.  With determination of the oxygen uptake rate from the consumption process, the  can now be calculated from the absorption process.

Despite the many successful applications of the dynamic method, it has difficulties to obtain accurate  measurement in at least two common cases. First, when cell density is low while agitation is high, a linear decreases of DO during consumption process cannot be obtained, which makes the accurate estimate of oxygen pick up rates by the cells impossible. The departure from the linearly decreased DO is caused by the mass transfer between head-space and liquid phase, which is not considered in the dynamic methods. Second, when cell density is high while agitation is low, DO barely increases during the absorption process, which makes the estimate of  impossible. This is caused by the limited mass transfer between the bubbles and the liquid phase which is lower than the oxygen pick up rate.

In this work, a modified dynamic method is developed and tested to circumvent these difficulties. In the modified dynamic method, both mass transfer between the headspace and liquid phase, and mass transfer between bubbles and liquid phase are considered; in addition, during the consumption process, nitrogen gas is bubbled through the broth which not only expedites the desorption process, but also enables the  measurement this step alone.

We used Scheffersomyces stipitis as the model system to test our method.  Since S. stipitis is highly sensitive to oxygen supply, the determination of an optimal OTR is very important for improving product yield². We have conducted a series of experiments with different cell density and agitation speeds to validate the modified dynamic method.  Our results show that the parameters estimated from the consumption and absorption processes agree very well with each other; in addition, parameters estimated from liquid phase and headspace measurements also agree very well with each other.

Our experimental results show that modified dynamic method allows researchers in the biotechnology field to measure  more easily and accurately.  In addition, we found that S. stipites achieved a maximum  value at OD₆₀₀= 3.0 and then proceeded to steadily decay with cell density increases, which might be caused by the changing interfacial properties of the fermentation broth. The headspace mass transfer coefficient increased with agitation speed, while oxygen uptake rate increased with cell density and decreased with agitation speed.

References

1. Biotechnology Advances  (2009) 27: 153–176
2. Biotechnology and Bioengineering (1989) 34:398-402