## 4 Discussion
Further research should focus on exploring the enzymatic mechanisms of hydrocarbon degradation in depth, especially for the strain _T. harzianum_ F26, which was the most successful. The experiments carried out in this work showed that the _Trichoderma_ strains tested tolerate the presence of used engine oil and that they can use it for their growth. The degradation efficiency, however, varies greatly depending on the single strains and species, as demonstrated in GC/MS tests. The causes may be different environmental growing conditions and substrate types, but especially adaptation to the presence of toxic agents such as pesticides, PAHs, and chlorinated agents [62; 63; 64; 65]. _T. harzianum_ F26 was the strain that best responded to all the tests, and it was hypothesized that this strain might have developed adaptive mechanisms to be able to degrade this type of hydrocarbon pollutant. Further studies are certainly needed to obtain more information on the chemical processes involved in bioremediation by _Trichoderma_. For this reason, an approach that integrates genetic, molecular, biochemical, and ecological studies may be a useful tool in understanding the behavior of this fungus and its metabolic processes. Besides this, genetic engineering could improve the degradative pathways and substrate specificity in _Trichoderma_ and allow the prospective development of modified strains capable of degrading specific families of pollutants. In conclusion, we can summarize the results of this work in three main messages:  
1. The efficiency of degradative activities seems to vary greatly depending on different species and single strains. 2. The use of strains isolated from contaminated environments provides an advantage in bioremediation activities. This may depend on the adaptation of fungi to stress conditions that limit their growth and on improved enzymatic response to the degradation of recalcitrant substances. 3. _T. harzianum_ F26 should be subject to further studies, including field studies, to deepen and enhance its potential as a bioremediation agent. **Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2076-3417/10/9/3152/s1](http://www.mdpi.com/2076-3417/10/9/3152/s1)]([http://www.mdpi.com/2076-3417/10/9/3152/s1](http://www.mdpi.com/2076-3417/10/9/3152/s1)), Table S1: Used engine oil fingerprint based on characteristic ions (m/z). Division based on [PERSON] at al. [46], Figure S1. Chromatograms of the used engine oil at T0 and after 45 gg of _Trichoderma_ treatment. The compounds were grouped in the macroclasses of aliphatics (black line), aromatics (red line) and other compounds (blue line). Treatment with _T. harzianum_ F12 at T0 (A), and at T45 (B); treatment with _T. harzianum_ F26 at T0 (C) and at T45 (D); treatment with _T. harzianum_ F58 at T0 (E) and at T45 (F); treatment with _T. aspergillus_ F1020 at T0 (G) and at T45 (H), Figure S2. Chromatograms of the used engine oil treated with _T. harzianum_ F26. The compounds were grouped in the microclasses of aliphatics C\({}_{1}\)-C\({}_{50}\) (black line), BTEX (red line), Alkyl Benzenes (green line), Alkyl Indones and Alkyl Tetralines (blue line), Alkyl Biphenyls (yellow line), Polycyclic Aromatics (purple blue) and other compounds (light blue line). **Author Contributions:** Conceptualization: C.D., M.E.E.T., L.N., S.T., B.M. ; methodology: C.D., M.E.E.T., L.N., B.M., F.C. ; software: L.N., M.E.E.T., C.D. ; data curation: M.E.E.T., L.N., C.D. ; writing--original draft preparation: C.D., L.N. ; writing--review and editing: L.N., C.D., S.T., G.C. ; visualization: M.E.E.T., C.D., L.N. ; supervision: S.T. ; project administration: S.T. ; funding acquisition: S.T. All authors have read and agreed to the published version of the manuscript.