Background: Microbial biodegradation of oil-hydrocarbons is one of the sustainable and cost-effective methods to remove petroleum spills from contaminated environments. The current study aimed to investigate the biodegradation abilities of three Fusarium isolates from oil reservoirs in Saudi Arabia. The novelty of the current work is that the biodegradation ability of these isolates was never tested against some natural hydrocarbons of variable compositions, such as Crude oil, and those of known components such as kerosene and diesel oils. Methods: The isolates were treated with five selected hydrocarbons. The hydrocarbon tolerance test in solid and liquid media was performed. The scanning electron microscope (SEM) investigated the morphological changes of treated fungi. 2, 6-Dichlorophenol Indophenol (DCPIP), drop collapse, emulsification activity, and oil Spreading assays investigated the biodegradation ability. The amount of produced biosurfactants was measured, and their safety profile was estimated by the germination assay of tomato seeds. Results: The tolerance test showed enhanced fungal growth of all isolates, whereas the highest dose inhibition response (DIR) was 77% for Fusarium proliferatum treated with the used oil (p < 0.05). SEM showed morphological changes in all isolates. DCPIP results showed that used oil had the highest biodegradation by Fusarium verticillioides and Fusarium oxysporum. Mixed oil induced the highest effect in oil spreading, drop collapse, and emulsification assay caused by F. proliferatum. The highest recovery of biosurfactants was obtained by the solvent extraction method for F. verticillioides (4.6 g/L), F. proliferatum (4.22 g/L), and F. oxysporum (3.73 g/L). The biosurfactants produced by the three isolates stimulated tomato seeds’ germination more than in control experiments. Conclusion: The current study suggested the possible oil-biodegradation activities induced by three Fusarium isolates from Riyadh, Saudi Arabia. The produced biosurfactants are not toxic against tomato seed germination, emphasizing their environmental sustainability. Further studies are required to investigate the mechanism of biodegradation activities and the chemical composition of the biosurfactants produced by these species.

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