## 4 Discussion
Among the bioremediation strategies, biostimulation is an _in situ_ technology that involves the introduction of indigenous (obtained from the contaminated site) or exogenous hydrocarbonelastic microorganisms to a polluted site to enhance microbial degradation at the site. Many degradation studies are focused on water column degradation, and no attention has been given to sediment oil degradation. Oil biodegradation in sediments is significant for the heavy fractions of oil, which tend to settle after the oil spill. In addition, the sediment environmental conditions are different such as oxygen and nutrient concentrations. There are nevertheless some challenges associated with bioremediation of spilled petroleum [74], including: (i) the resistance of heavy fractions, especially asphaltenes and high molar mass polycyclic aromatic hydrocarbons, to degradation. Thus, it seems crucial to have microbial strains able to perform the biodegradation of heavy and extra-heavy oils such as _Alternaria_ sp. isolated and characterized in this work; (ii) eutrophication caused by biostimulation. Seawater is characterized by the low nitrogen and phosphorous concentrations that are limiting factors for oil degradation. The use of oleophilic fertilizers seems to be an effective nutrient type to be applied in marine environments to prevent eutrophication; (iii) unsustainability of bioaugmentation in the field. This can be reduced using well-adapted microorganisms, like, for instance, those adapted to sediment conditions. In addition, the encapsulation of the microorganism can protect it in the new environmental conditions; (iv) poor bioavailability of spilled petroleum. Oil biodegradation takes place at the oil-water interphase. Therefore, the widely used oil dispersion or solubilization by adding chemical or biological surfactants enhances biodegradation. However, the chemical agents could be potentially toxic to the environment; and (v) oil biodegradation inefficiency in semi-anoxic environments. As mentioned before, in deep sediments, there is a low oxygen concentration. It is also important to have microorganisms isolated from similar sediment conditions, such as the _Alternaria_ sp. isolated and characterized in this work. Considering all the above, microorganisms that, like _Alternaria_ sp., are capable of degrading some of the heavier and more recalcitrant components of these crude oils can be good candidates for further studies testing different environmental conditions in order to assess their use for bioremediation in the event of an oil spill. The following are available online at [[https://www.mdpi.com/article/10.3390/app11136090/s1](https://www.mdpi.com/article/10.3390/app11136090/s1)]([https://www.mdpi.com/article/10.3390/app11136090/s1](https://www.mdpi.com/article/10.3390/app11136090/s1)): Table S1: Geolocalization and data of stations sampled in the Gulf of Mexico. Table S2: Sampling stations and locations from where the fungal strains were isolated. Table S3: Reference DNA sequences of the ITS rDNA, 185 and \(\beta\)-tubulin markers used in the phylogenetic analyses. Figure S1: Protein content (average of three replicates) of _Penicillium_ sp. 1 (A) and _Penicillium_ sp. 2 (B) grown under different light crude oil concentrations. Figure S2: Chromatograms of whole extra-heavy crude oil and its saturates, aromatics and polars (SAP) fractions. Figure S3: _Alternaria_ sp. grown with (B, D) or without (A, C) 0.5% w/v of extra-heavy crude oil as sole carbon source after a month of incubation. Conceptualization, M.R. ; methodology, L.R.-H., M.D.C.-L., P.V. and I.B.-G.; investigation, L.R.-H.; writing--original draft preparation, L.R.-H.; writing--review and editing, M.R., P.V. and R.V.-D.; formal analysis, L.R.-H.; supervision, M.R. and R.V.-D.; project administration, M.R. ; and funding acquisition, M.R. All authors have read and agreed to the published version of the manuscript. This research was funded by the National Council of Science and Technology of Mexico-Mexican Ministry of Energy-Hydrocarbon Trust, project 201441.