Media Steaming Characterization
The fermenter must have an SIP cycle that controls temperature to ensure efficient sterilization for bioburden control, but
also avoids exposing the media components to excessive temperatures that could cause nutrient degradation. Evaporation or
condensation can occur during the SIP cycle. It is desirable to characterize (or eliminate) the change in fermenter weight
during SIP operations to ensure the target starting medium concentration is achieved. If significant evaporation or condensation
occurs, this should be compensated for in the amount of water that is used to batch the medium. Scale effects of the heat-up
and cool-down times of the medium as part of the SIP cycle also should be characterized because these durations can significantly
change with scale and may have an unexpected effect on media performance.
In this study, the fermenters were filled with water and three SIP cycles were performed in series with full cooling between
experiments, to understand variability related to evaporation or condensation.
Evaporative Losses During Media Hold Characterization
To allow for schedule flexibility during production and to ensure a robust process is implemented, the sterilized media must
be held for a period of time before inoculation. If the medium is agitated and air is applied to the fermenter, evaporation
may occur during this hold period even if a condenser is installed on the gas outlet line of the fermenter. Determining the
evaporation rate is essential for achieving the correct medium concentration at the time of inoculation. To compensate for
the evaporation rate, additional water can be added during medium preparation; however, this necessitates a fixed hold time
for every batch. Alternately, adjustments to the air flow rate can be made to minimize evaporative losses.
At the 300-L pilot scale, water was held under process conditions (temperature, air flow rate, volume, agitation, pressure).
Holding the medium at process conditions is advantageous because it eliminates the need for any equilibration time before
inoculation. Medium hold data from historical processes in the large scale fermenter (10,000-L) indicated significant weight
loss during hold periods. Therefore, for large-scale testing, the airflow rate was decreased from the process set point to
a pre-inoculation hold-specific set point. Fermenter weight was continually monitored and used to calculate the evaporation
rate at both scales. Weight was plotted against hold time and the slope was used as an estimate for evaporative loss occurring
during the media hold.
Results and Discussion
Heat Removal Characterization
The oxygen uptake rate (OUR) for the culture in question is approximately 300 mmol/L/h. The heat generated by the cells can
be estimated using the common method shown in Equation 3:
in which H M is metabolic heat (kJ/kg/h), OUR is the oxygen uptake rate (mmol/kg/h), and 5.2 x 10
is a constant.
Per Equation 3, the heat generated by the cells is approximately 150 kJ/kg/h. Typically, the next largest contributor of heat
to the fermenter is the agitator. This was measured at large scale using the agitator current draw and estimated to be approximately
18 kJ/kg/h. As such, it is considered a small contributor to the overall heat load, and the HTR required for the fermenters
is estimated as 150 kJ/kg/h.
Figure 1 shows the HTR calculated using Equation 1 for each fermenter weight tested at the pilot and large scale, respectively.
The calculated HTR at pilot scale varied between 555 and 750 kJ/kg/h with the fermenter weights from 150 to 300 kg, and at
large scale the HTR varied between 200 and 235 kJ/kg/h with the fermenter weights between 5,000 and 10,000 kg; therefore both
fermenters had sufficient HTR capacity to support the process need of 150 kJ/kg/h.
Figure 1. Heat transfer rate at pilot and large scales.
For both fermenters, only the bottom impeller was submerged at the lowest test weight. The maximum heat transfer rate was
observed at 200 and 7,000 kg, the pilot-scale and large-scale, respectively. The upper impeller is partially or just barely
submerged at these weights. The lowest HTR was observed at the maximum working volume of 300 kg for the pilot scale and 10,000
kg for the large-scale fermenter. The lower HTR at the maximum working volume is expected and likely caused by the decreased
surface area to volume ratio at the higher volume. Another factor that may have contributed to the heat removal rates was
the air sparge rate, which was kept constant for all fermenter weights; therefore, the volume of sparge air per volume of
water (VVM) decreased as the fermenter weight increased (shown in Figure 1). Introducing gas into the liquid likely reduces
the apparent density of the fluid and because of gas hold-up, the liquid comes in contact with additional cooling surface.