## 4 Discussion  
DOT is a critical fermentation parameter for aerobic micro-organisms to produce their metabolites. When microbial metabolite production is correlated to OUR, DOT should be maintained above a certain threshold because sufficient DOT leads to a higher OUR [15; 16]. Reductions in microbial metabolite yields have been reported when DOT fell below a critical level [17; 18]. To maintain a suitable DOT level, it is important that oxygen transfer takes place faster than oxygen uptake, especially in the log phase, when the microbial population undergoes exponential growth. Both agitation and aeration have been applied to solve this problem, elevating \(K_{L}a\) in bioreactors [10; 11; 19]. In the case of surfactin production using _Bacillus_ spp., oxygen-transfer efficacy plays an important role in production kinetics. Increases in agitation speed and aeration rate reportedly resulted in maximum surfactin production [9; 10]. However, a reduction in surfactin production was observed at a higher agitation speed due to vigorous foam production [11]. Rapid foam formation caused the culture to overflow, resulting in decreased  
Figure 6: Profiles of volumetric surfactin productivity and spore ratio when an original and a mesh-type sparger were used. Cultivations were performed in 5 L jar bioreactors at 30 \({}^{\circ}\)C at 400 rpm.  
Figure 7: Surfactin production when a mesh-type sparger was used. Cultivations were performed in 5 L jar fermenters at 30 \({}^{\circ}\)C for 48 h.  
cell mass and reduced surfactin production. In our study, an increase in agitation speed led to an increase in \(K_{L}a\), resulting in an increment in surfactin production. Similar to findings by [PERSON] et al. [11], maximal surfactin concentration was not observed at the highest agitation speed of 500 rpm, but at 400 rpm. However, the reason for avoiding high agitation speed differed in our study. Since foam formation was perfectly controlled by an antifoam agent, there was no foam formation at 500 rpm. However, high agitation speed led to early spore formation, while no spore formation was observed at low agitation speeds. Considering that the volumetric productivity of surfactin was reduced when endospores were produced, sporulation of _B. subtilis_ Y9 seems to have a negative effect on surfactin production.  
Since sufficient oxygen supply is required for surfactin production, a mesh-type sparger was applied to improve the oxygen-transfer rate without increasing agitation speed. Use of the mesh-type sparger led to an increase in the gas-liquid interfacial area by generating microsized bubbles. \(K_{L}a\) with the mesh-type sparger was higher than that with the original sparger, showing the highest surfactin production. Surfactin production rate from 12 h to 24 h was substantially higher than that in the case of the original sparger under all agitation speeds. Under agitation at 500 rpm with the mesh sparger, however, endospore appearance was observed from 24 h, resulting in a reduction in surfactin concentration to 584.0 mg/L\(\cdot\)h (data not shown).  
There are numerous reports that a high DOT level has a positive effect on spore production of _Bacillus_ spp. [20; 21; 22]. The higher the oxygen supply is, especially \(K_{L}a\), the higher the spore productivity [21; 23]. In our study, high DOT resulted from increased agitation speed, leading to fast sporulation. Considering that sporulation might have an adverse effect on surfactin production in the submerged culture of _B. subtilis_ Y9, the increase in agitation speed to improve DOT level should be limited. Instead, we attempted to promote \(K_{L}a\) by installing the mesh-type sparger. As shown in Figure 5, both the highest volumetric productivity and prolonged production period were observed, although sporulation started in the stationary stage of culture (1.2% of spore ratio at 36 h).