# On-Line Tendency Control of Dissolved Oxygen Concentration during Aerobic Fed-Batch Fermentations
## 3 Results and Discussion
### Performance during P. pastoris Fermentation  
The time profile of cell concentrations and DO, as well as stirrer speed were depicted in Figure 7. Heterologous protein production by fed-batch culture with _P. pastoris_ is mainly divided into two phases: a growth phase to accumulate large amount of cells with glycerol as the carbon source, and an induction phase by feeding methanol for heterologous protein expression [12; 27].  
Figure 6: Performance in production phase during _C. glutamicum_ fermentation. (**A**) DO control using the tendency control strategy and (**B**) comparison of the adjustment of stirrer speed.  
As _P. pastoris_ is a slow-growing species, it requires a longer fermentation period during experimental set up. It has a comparatively longer DO time control period; therefore, the window size was set to 30 min. The results are shown in Figure 8 and Table 3.  
For the purpose of comparison, the results of the DO manual control experiments, which were carried out by step-wise manual increase or decrease in agitation, are also shown in these figures. Figure 8A depicts the pattern of changes in DO using DO-Stat ('traditional' or improved) during a 300 min period in the glycerol fed-batch phase.  
DO-Stat control relies on the presence of continuous oscillations in DO levels to function; when glycerol levels are depleted, the cells are forced to use nitrogen sources for the maintenance of metabolic and respiratory activities, which causes the DO level to begin to rise. The sharp increase in the DO level functions as a feedback indicator and leads to subsequent glycerol feeding; after the glycerol is supplied, cells will prioritize the use of glycerol, which will lead to the rapid decrease of DO levels. If the same  
\begin{table}
\begin{tabular}{c c} \hline
**Different Methods** & **From 1220 to 1509 min** \\ \hline Manual control & 99 times \\ Tendency control & 2 times \\ \hline \end{tabular}
\end{table}
Table 3: Action times of actuator for different methods.  
Figure 7: The time-course changes in _P. pastoris_ fermentation. (**A**) Changes in OD\({}_{600}\) (cell concentration) with time, (**B**) changes in DO concentration with time, and (**C**) changes in stirrer speed with time.  
actions are performed repeatedly, then DO will oscillate continuously. The DO-Stat ('traditional' or improved) glycerol feeding strategies are initialized with the aid of control programmes.  
As indicated in Figure 8B, the stirrer speed was increased from 600 rpm at 1220 min to approximately 800 rpm at 1509 min. As shown in Figure 8A, the range in the DO level was greater (10-95%). The actuator was also frequently under action during manual control of DO; in contrast, the actuator was much less active when the tendency control method was used. From 1220 to 1509 min, the actuator only acted two times. Although the actual values of DO were 9.9% at 1341 min and 9.8% at 1469 min, both of which were below the DO low limit of 10%, the average DO value was greater than 10%. Moreover, at 1469 min, the manual control failed to detect when the DO level fell below 10%, which produced a time delay of 6 min; during the DO tendency control experiments, all processes were initialized in a timely fashion. More importantly, the stirrer speed remained at an almost constant level after experiencing a sharp peak at approximately 1341-1348 min. For the aforementioned reasons, the automatic control of DO is necessary to conduct a fermentation process efficiently, and can lead to improved process reliability as well as reduced manpower input.