## 4 Discussion
Although the efficiency of the performed GDL extraction is high, it is necessary to point out a disadvantage of using organic solvents in the media with the addition of lipids. In the target biotransformation media, the precursor of GDL synthesized by microorganisms is castor oil or ricinoleic acid. These are introduced into the media in excess, as they are also sources of carbon for the growth and maintenance of the vital functions of microorganisms. Not fully hydrolyzed castor oil and the ricinoleic acid not used in the \(\beta\)-oxidation cycle are extracted together with GDL, contaminating it significantly (unpublished data). Hence, the extraction technique itself is not sufficient and should be supported, e.g., with chromatography or fractional distillation. However, this technique is not always effective due to the small differences in the boiling points of individual compounds. Considering the significant contamination of GDL with the lipid fraction, which is extracted regardless of the solvent used, attempts were made in further studies to separate the lactone by hydrodistillation. In the extensive literature on the subject of GDL biosynthesis, only one publication was found in which [PERSON] et al. [7] used the hydrodistillation method to isolate GDL. Hydrodistillation is a traditional technique to recover essential oils and bioactive compounds from plants [41]. Despite its disadvantages, including long process time and low efficiency as well as partial degradation of unsaturated compounds due to thermal effects, it is widely used [41; 42; 43]. The use of hydrodistillation in this research to separate GDL from biotransformation media allowed for its recovery, and the efficiency of this technique was approximately 12-22% lower than in liquid-liquid extraction. It is assumed that the lower concentration of lactone in distillation may be partly due to its loss as a result of long-term heating of samples and possible hydrolysis of this compound [44]. Based on the data from Figure 1, it can be concluded that at the concentration of GDL in the biotransformation medium below 0.5 g/L, distillation exhibits low effectiveness. However, it should be emphasized that the purity of the distillates was higher than that of the extracts. In the distillates, apart from GDL, 3-hydroxy-\(\gamma\)-decalactone, dec-2-en-olide, and dec-3-en-olide were identified. These compounds had been previously detected during the fermentation process that was conducted by the yeast _Yarrowia lipolytica_ W29 on solid media, as mentioned by [PERSON] et al. [25]. Since the microbial synthesis of GDL is based on the transformation in the \(\beta\)-oxidation cycle of ricinoleic acid to 4-hydroxydecanoic acid, which subsequently cyclizes to GDL, it was justified to analyze the influence of the reaction environment on the level of acid cyclization, and hence, the final concentration of GDL in the distillate. Acidity turned out to be an important factor that partially determined the concentration of GDL in the distillate. Increasing the acidity of the reaction mixture from the natural pH of about 5.68 to the pH of 3 or 1 resulted in an increase in the concentration of the lactone in the biotechnological medium, ranging from 0.25 g to 0.75 g/L (percentage range 8-33%; Table 3). The results of [PERSON] et al. [45] confirm this research. The team carried out the bioconversion of methyl ricinoleate to gamma-decalactone using the yeast _Sporidiobolus ruineni_ and observed that acidification of the reaction medium before the extraction process increases the concentration of lactone in the aqueous phase. By changing the acidity of the post-reaction mixture from pH 6 to pH 2, an over 2.5-fold increase in the GDL concentration was observed. [PERSON] et al. [46] also emphasizes that lactonization of 4-hydroxydecanoic acid occurred particularly easily in an acidic environment. In the research on effective separation of GDL from biotransformation media, adsorption on porous adsorbents (Amberlite XAD-4 resin, zeolite, and vermiculite) was also tested.