## 3 Results and Discussion
### Production of CMCase with Shifts in Aeration Rate and Agitation Speed of Pilot-Scale Bioreactor  
The optimal agitation speed of a 100 L pilot-scale bioreactor was calculated based on the optimal agitation speed of the 7 L bioreactor and the difference between diameters of impellers in 7 and 100 L bioreactors. The diameters of impellers with the same type in 7 and 100 L bioreactor were 6.0 and 16.6 cm. The impeller tip speed was an essential parameter when the agitating liquid contained solid particles [27]. The impeller tip speed at the agitation speeds of 416 and 500 rpm of a 7 L bioreactor were calculated to be 130.7 and 150.1 cm/s, respectively, which were almost the same as those at 150 and 180 rpm of a 100 L bioreactor. The optimal conditions of the aeration rates and agitation speeds of a 100 L bioreactor for the cell growth of _E. coli_ KACC 91335P were decided to be 0.50 vvm and 180 rpm, respectively, whereas those for its production of CMCase were 0.95 vvm and 150 rpm. As shown in Figure 5a,b, the cell growth of _E. coli_ KACC 91335P and its production of CMCase for 96 h with a fixed optimal conditions of the aeration rate and agitation speed were 5.58 g/L and 784.2 U/mL, respectively, whereas those with a fixed optimal conditions of the aeration rate and agitation speed for the production of CMCase were 3.45 g/L and 893.1 U/mL. However, the cell growth of _E. coli_ KACC 91335P and its production of CMCase with shifts in the optimal condition of the aeration rate and agitation speed from the cell growth to its production of CMCase at 36 h were 5.27 g/L and 1216.5 U/mL, respectively, as shown in Figure 5c. Figure 4: Relationship between cell growth and production of CMCase by _E. coli_ KACC 91335P (**a**) without shifts in agitation and/or aeration, (**b**) with shifts in agitation and/or aeration at 24 h, (**c**) with shifts in agitation and/or aeration at 26 h and (**d**) with shifts in agitation and/or aeration at 48 h.  
and agitation speed from the cell growth to its production of CMCase at 36 h, the mid-log phase of cell growth, was 0.052/h. However, \(\upmu_{\text{max}}\) with shifts in the optimal conditions of the aeration rate and agitation speed during a specific time was higher than those without shifts. As shown in Figure 6, the shifts in the optimal conditions of the aeration rate and agitation speed of the 100 L pilot-scale bioreactor from the cell growth to its production of CMCase at the mid-log phase resulted in a relatively more cells to participate in their production of CMCase, which in turn enhanced the production of CMCase. The production of CMCase by _E. coli_ KACC 91335P in a 100 L bioreactor with shifts in the optimal conditions of the aeration rate and agitation speed from the cell growth to its production of CMCase at the mid-log phase was 1.36 times higher than that with a fixed optimal condition of the aeration rate and agitation speed for the production of CMCase and even 1.54 times higher than that with those for the cell growth. During fermentation with the multistage bioreactors, the first bioreactor containing microorganisms in the growth phase was for the cell growth and the second bioreactor containing non-proliferating cells was for the production of metabolites [28]. The production of the arachidonic acid by _M. alpha_ was increased by using continuous multistage fermentation with precise control of the agitation speed and aeration rate at proper times [29]. The multistage chemostat system may be beneficial for improving the genetic stability and production of metabolites [20; 30]. The advantages of the continuous fermentation with optimized process conditions are higher volumetric and long-term