## 4 Discussion
In this case, the only strain that showed statistically significant results was _T. harzianum_ F26, which showed better degrading action on the BTEX and alkylbenzene family, increasing the content of C\({}_{1}\)-C\({}_{20}\) alkanes. An explanation for this may be that _T. harzianum_ F26 is more selective and prefers the degradation of BTEX and alkylbenzenes, while the other strains have more widespread and less marked degradation in all microclasses, making it more challenging to appreciate the results from a statistical point of view. Many authors have indicated _T. harzianum_ as one of the _Trichoderma_ species able to degrade unsaturated hydrocarbons, BTEX, and resins, as well as PAHs [21; 54]. This species, in fact, can degrade up to about 10% of anthracene at a concentration of 400 mg kg\({}^{-1}\) and 24.7% of pyrene at a concentration of 10 mg kg\({}^{-1}\)[55]. The results obtained by the action of _T. harzianum_ F26 partially agree with the literature as it acts more on BTEX and alkylbenzenes. Other works, however, have also demonstrated its ability to grow even on a mixture of complex hydrocarbons, such as crude oil, up to a concentration of 3% [56]. Finally, the colorimetric tests were aimed at identifying the ligninolytic enzymatic activities often associated with hydrocarbon degradation. Regarding the gallic acid test, only _T. asperellum_ F1020 and _T. harzianum_ F12 showed indications of ligninolytic enzyme production, and the guaiaocl test showed that _T. asperellum_ F1020 and _T. harzianum_ F12 were able to produce Lac and Per. However, the guaiaocl test also revealed that _T. harzianum_ F26 and _T. harzianum_ F58 showed slight activity. This result suggests that the guaiaocl test may be more sensitive to the production of Lac and Per than the gallic acid test, even detecting milder activity. The RBBR test was used to determine the strains' ability to degrade PAHs and indicate the presence of Lac [41]. _T. harzianum_ F58 and _T. harzianum_ F12 showed positive results, but _T. harzianum_ F26 was the most active. F1020 showed no activity, consistent with the results obtained by GC/MS. The production of Lac and Per enzymes by these _Trichoderma_ species has already been demonstrated by other authors [43; 55]. [PERSON] et al. [26] reported that fungal catechol 1,2 dioxygenase, lactose, and peroxidase are involved in the degradation of PAHs by _T. asperellum_. Considering the results of the colorimetric screening, _T. asperellum_ F1020 was an excellent producer of Lac and Per. However, this result was not demonstrated in the GC/MS test, suggesting the action of other enzymes in the degradation of used engine oil. Many authors have shown the metabolic pathways activated by fungi to degrade hydrocarbons, and what emerges is that microfungi, in particular, mainly use cytochrome P450 (CYP) monoxygenases (EC:1.14.13.12) to degrade recalcitrant substances [11; 56; 57; 58; 59; 60]. Moreover, as already mentioned, adaptation to the pollutant is fundamental for its efficient degradation. On the contrary, the strains that generated significant results under GC/MS did not provide equally evident signals in colorimetric tests. The reasons for this may be that _T. harzianum_ F12, _T. harzianum_ F26, and _T. harzianum_ F58 could take greater advantage of the CYP complex mentioned above or could increase their production of Lac and Per in the presence of used engine oil. In fact, the colorimetric tests were performed on MEA without the pollutant present, as required by the protocol, but this may not have stimulated the production of the enzymes of interest. It is known that the concentration of lignin products and organic pollutants can induce the regulation of Lac and Per, especially in white-rot fungi [61; 62], but a similar effect has been noted by [PERSON] et al. [57] in _T. harzianum_. They found that the concentrations of Lac and Per produced by a strain of _T. harzianum_ increased when the fungus grew in a polluted medium of crude oil compared with in oil-free medium.