## 4 Discussion  
Three factors with two levels were chosen for the optimization of GDL production by the Taguchi method as a fractional factorial experiment design. The conducted bioreactor cultures, supported by the Taguchi method, confirmed that the selection of appropriate cultivation conditions has a significant impact on the level of GDL biosynthesis. One of the factors determining the concentration of lactone in the medium is the carbon source. Biotransformations leading to the production of GDL described in the literature use castor oil, ricinoleic acid, or methyl ricinoleate [18]. These compounds are sources of carbon in the culture medium, necessary for the growth of microorganisms and maintenance of vital functions, as well as activators for enzymes in the pathway of GDL synthesis. The most widely used is castor oil (as in our research), a cheap, non-toxic, and readily available substrate. [PERSON] et al. [22] optimized the synthesis of GDL by _Y. lipolytica_ cells using castor oil at the concentration of 10-40 mL/L (\(v/v\)). The results of their research indicated that the highest concentration of aroma, at the level of approx. 0.053 g/L, occurred at the dose of castor oil in the range of 10-25 mL/L. The yeast _Rhodotorula aurantiaca_ A19 synthesized within 10 days of the biotransformation of 5.5 g/L, at the oil dose of 20 g/L. The concentration of lactone in the culture of these microorganisms did not increase in proportion to the concentration of the substrate. Higher concentrations of castor oil, in the range of 30-60 g/L, reduced the level of aroma synthesis [20]. Different conclusions result from the studies by [PERSON] and [PERSON] [23]. According to the authors, the use of higher concentrations of hydrophobic substrate, on the order of 60 g/L, in a 25 h cultivation of _Yarrowia_ yeast, enables the synthesis of approx. 5.4 g/L. At lower oil concentrations, a decrease in lactone concentration was observed after some time, until it was completely depleted. A similar relationship was observed in the culture of _Y. lipolytica_ Y-VTP5, where the concentration of 100 g/L made it possible to obtain 1.3 g/L of GDL [43]. In the case of ricinoleic acid, it has been shown that its high concentration may inhibit cell growth and, indirectly, lactone synthesis [22]. As shown by the Taguchi method, the factor that mainly influenced the GDL biosynthesis was the intensity of mixing. Appropriate mixing speed supports the proper oxygenation of the substrate and ensures the dispersion of the hydrophobic substrate (castor oil) in the water phase. The contact surface of fat droplets with the cells of multiplied organisms and the contact surface between the two liquid phases is considered to be a factor determining the degradation of the hydrophobic substrate, and thus the growth of cells and their aroma production [44]. Indications of the Taguchi method, confirmed by bioreactor culture (Figure 4, Table 5), showed that in the analyzed range of three parameters, the highest concentration of lactone was obtained at a variable mixing speed in the range of 200-500 rpm. This is also supported by literature data. Variable stirring speed, ranging from 300 to 600 rpm, used to maintain the proper level of dissolved oxygen in the medium, allowed the fed-batch culture of _Y. lipolytica_ to obtain the highest concentration of 70 mg/L [45]. Mixing at the level of 600 rpm was optimal for productivity and obtaining the maximum concentration of GDL in the study by [PERSON] et al. [46]. Under these conditions, more than 1 g of lactone per liter of medium was obtained, and the yield was at the level of