Abstract: The current study was aimed for isolation, identification and preservation of mycobiota associated with the olive oil processing wastes (Fatura) collected from different cities in Al-Gabal Al-Gharby, Libya, screening the cold-active lipolytic activity of the isolated fungi and selection of the highest cold-active lipase producers. 31 fungal species belong to 12 genera were isolated from these samples with total CFUs of 29560. Fusarium was the most common genus at total CFUs of 9020 and comprising 30.51% from all fungi, followed by Aspergillus, that recorded 25.44% from all fungi. Penicillium was ranked third, nine different species were present. A total of 100% of samples were found to have CFUs of 5140 and 17.4% of all fungi. On lipase production agar medium at two temperatures, 10 and 20°C, 102 fungal isolates from 31 species were tested for their lipolytic activity. The majority of fungi could produce lipase activity at 20°C, where 98 out of 102 isolates the highest lipase producers was higher at 10°C (25) than at 20°C (16). The most active isolates were Alternaria, Fusarium, and Penicillium. Molecular identification of the most active four isolates was carried out by sequencing their internal transcribed spacer region (ITS).

 Keyword: Olive oil, cold active enzymes, Lipase, fungi, lipolytic activity, Fatura.