## 3 Results and Discussion
### Stability of the Emulsifying Properties of the Biosurfactant  
Biosurfactant production was evaluated using the emulsification index (E\({}_{24}\)%), and the value obtained against toluene was 47% (Figure S1). The biosurfactant showed high thermal stability (50% at 4 \({}^{\circ}\)C and 41% at 70 \({}^{\circ}\)C), with the E\({}_{24}\)% values being also stable at NaCl concentrations between 2 and 6% (53% for NaCl 2% and 4%, 47% for NaCl 6%), decreasing for higher 8% and 10% concentrations (31% and 28%, respectively). In our previous study, it  
Figure 1: _Y. lipolytica_ CMGB32 cell interactions with \(n\)-hexadecane droplets after three days of incubation (40\(\times\)) (plain arrow—yeast cells; dashed arrow—pseudohyphae; double arrow—\(n\)-hexadecane droplets).  
was shown that the biosurfactant produced by the _Y. lipolytica_ CMGB 32 strain is essentially a lipoprotein composed of a high amount of aromatic amino acids [31]. The aromatic amino acid residues, such as tyrosine and tryptophan, are involved in establishing energetically favorable interactions with molecules from the surrounding environment (interaction strongly influenced by temperature and ionic strength) [42; 43]. Thus, the stability of the biosurfactant at the tested temperature and the NaCl concentrations might indicate that the sequences of aromatic amino acids interfere in a limited way in the stabilization of the lipoprotein biosurfactant's functional structure. Moreover, the applicability of microbial biosurfactants in several fields is strongly dependent on their stability at different temperatures and salinity values. The high thermostability suggests that the biosurfactant produced by _Y. lipolytica_ CMGB32 can be used in food, pharmaceutical, and cosmetic industries, where heating is an important step in the manufacture process [44]. Additionally, it can be used in the bioremediation of habitats within a wide range of temperatures. Also, stability at high salinity concentrations is an important parameter when determining the applicability of biosurfactants in the bioremediation of spills in marine water [45]. As recorded previously [39], the biosurfactant produced by _Y. lipolytica_ CMGB32 in the presence of _n_-hexadecane had an E\({}_{24}\)% of 52% against petroleum. In addition, we showed that the strain _Y. lipolytica_ CMGB32 was able to synthesize stable biosurfactants also on a YP medium supplemented with 1% petroleum, with an E\({}_{24}\)% value of 52% against toluene [32]. Meanwhile, ref. [46] described the strain _Y. lipolytica_ IMUFRJ 50682 as producing biosurfactants with an emulsification index against hexadecane that reached a maximum of only 17.5% after 100 h of incubation on crude oil as a sole carbon substrate. Other studies reported an E\({}_{24}\)% of 62.4% for toluene emulsification using the bacterial strain _Streptomyces_ sp. MOE6 [47], and reported values ranging between 33.3 \(\pm\) 0.0 and 75.0 \(\pm\) 1.7 for various strains belonging to _Marinobacter_ genus [48].  
Toluene is one of the hydrocarbons found in crude oil's composition and represents one of the main industrial solvents used for paints, glues, cleaning products, etc. Long exposure to accidental oil spills and work conditions in the presence of toluene leads to renal and hepatocellular injuries, rhabdomy, and altered nervous system functions [49]. Moreover, toluene contamination can occur also in the presence of heavy metals, influencing the efficiency of bioremediation technologies and the composition of microbial communities with biodegradation abilities [50]. Therefore, the good ability of the _Y. lipolytica_ CMGB32 biosurfactant to emulsify toluene, and its high stability, might be considered as a solid base for its use as an additive for toluene removal from contaminated areas.