## 3 Results
### Feeding-Rate-Controlled Fed-Batch Fermentation  
In the present study, the development and optimization of MEL production with the periodic addition of substrates were assessed to increase the titer. Similar to batch fermentation, the periodic addition of acid oil also supported cell growth and MEL pro duction. The effects of different feeding strategies on cell growth, substrate consumption, and MEL production are presented in Figure 3. Periodic oil feeding in four steps showed improvements in MEL production for all concentrations of acid oil. Figure 3a shows MEL production using an 80 g L\({}^{-1}\) substrate with variable feeding strategies. Here, MEL production increased from 1.42 to 2.72 g L\({}^{-1}\) with different feeding concentrations. With the presence of 120 g L\({}^{-1}\) of acid oil, the MEL concentration increased with increases in the biomass up to 10.14 g L\({}^{-1}\), corresponding to a titer of 3.51 g L\({}^{-1}\) (Figure 3b), by the periodic addition of substrates.  
Similarly, oil consumption, as well as cell growth, was improved and the MEL titer was found to be increased up to 4.96 g L\({}^{-1}\) and 5.56 g L\({}^{-1}\) with 160 g L\({}^{-1}\) and 200 g L\({}^{-1}\) periodic feedings of acid oil, respectively. Under different feeding rates, surprisingly, MEL formation was increased by 2.04 fold with fed-batch cultivation compared to batch cultivation. The controlled feeding strategy with a 200 g L\({}^{-1}\) substrate concentration improved the MEL production up to 0.07 g L\({}^{-1}\) titer after 7 days of fermentation (Figure 3d).  
From these results, we find that the feeding-rate-controlled fed-batch strategy had a distinct influence on the MEL titer. As the fermentation medium contained two immiscible phases, mass transfer played a vital role in the efficient production of MEL. Similar to other glycolipid surfactants, MEL was mainly produced during the stationary phase of cell growth [26]. As _P. antarctica_ is an aerobic microbe, the basic requirement for microbial growth is oxygen. During the periodic addition of acid oil, air-to-broth barrier formation was the least. So, the cell concentration, along with MEL production, was increased as a result of better aeration with the periodic addition of substrate.  
Further, the fermentation medium contained acid oil, a hydrophobic substrate; it is difficult for this substance to form a homogeneous phase. Hence, the mass transfer for the  
Figure 3: Production of MEL using _Pseudozyma antarctica_ by periodic addition of acid oil at different total substrate concentrations: (**a**) 80 g acid oil L\({}^{-1}\), (**b**) 120 g acid oil L\({}^{-1}\), (**c**) 160 g acid oil L\({}^{-1}\), (**d**) 200 g acid oil L\({}^{-1}\). Data are represented in the following formats: MEL titer g L\({}^{-1}\) (\(\blacktriangle\)), cell mass g L\({}^{-1}\) (\(\Leftarrow\)), and oil consumed g L\({}^{-1}\) (\(\Leftarrow\)). Data points are the average results of three independent cultivations.  
substrate in an aqueous nutrient medium may have acted as a limiting factor. Here, the mass transfer was achieved through emulsification, which requires some energy to disperse the hydrophobic phase in water. This amount of energy can be considered a measure of the thermodynamic instability of an emulsion. Changes in the interfacial area difference constitute the only way to minimize the energy required for emulsification [27]. In the case of periodic substrate addition, the thermodynamic energy requirement for stable emulsion formation was lower compared to the direct addition of the substrate. So, the minimum addition of oil at the time produced better emulsion, and hence, the utilization of oil became easy for microbes. Thus, the production rate of MEL can be improved by the addition of 20-50 g L\({}^{-1}\) of a hydrophobic substrate on each day after the stationary phase.