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Aims: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol.

Methods and Results: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied. The production of γ-linolenic acid reached 2·44 mg ml−1 (271 mg g−1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23°C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5°C.

Conclusions: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production.

Significance and Impact of the Study: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.