Background: Biodiesel is a long chain of fatty acid alkyl esters derived from renewable feedstock and it was used as a partial or full substitute for petrol diesel. Results: Among 36 Egyptian filamentous fungi that were screened for their abilities to produce lipid by using Nile red staining assay, Fusarium oxysporum NRC2017 was selected and identified based on the molecular method. Optimization of culture conditions for F. oxysporum NRC2017 maximum lipid production were investigated by using a basal liquid medium with initial pH 5.0, incubation temperature 30°C, and after 9 days of incubation at a static condition. Six types of agro-industrial by-products were used as components of semi-solid-state fermentation, namely breaking rice, rice straw, wheat bran, corn stalk, sweet sorghum, and bagasse. The highest lipid accumulation was obtained when baggase 98.3 mg/g was used. Gas chromatography (GC) analysis of F. oxysporum NRC 2017 biodiesel showed the presence of 2.2% palmitoleic acid, 11.1% oleic acid, 5.6% linoleic acid, 3.0% αlinoleic acid, 2.5% eicosatrienoic acid, 6.8% eicosapentaenoic acid, 16.1% erucic acid, 4.2% margaric acid, and 3.9% behenic acid. Biodiesel from F. oxysporum NRC2017 was blended with petroleum diesel in 5% "B5" and the physical properties results were density 0.84 g/ml, viscosity 2.55 mm 2 /s, cloud point 0°C, iodine value 92.71 gI2/100 g, acid value 1.6 mg/g, and pour point − 1.3°C. Conclusion: This work revealed the possibility of biodiesel production from the Egyptian promising fungal strain F. oxysporum NRC2017.
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