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Aims:  To isolate a biosurfactant‐producing bacterial strain and to identify and characterize the chemical structure and properties of its biosurfactants. Methods and Results:  The bacterium Rhodococcus wratislaviensis BN38, isolated from soil, was found to produce glycolipid biosurfactants when grown on 2%n‐hexadecane. The glycolipids were isolated by chromatography on silica gel columns and their structures elucidated using a combination of multidimensional NMR and ESI‐MS/MS techniques. The main product was identified as 2,3,4,2′‐trehalose tetraester with molecular mass of 876 g mol−1. It was also noted that the biosurfactant was produced under nitrogen‐limiting conditions and could not be synthesized from water‐soluble substrates. The purified product showed extremely high surface‐active properties. Conclusions:  The glycolipid biosurfactant produced by the alkanothrophic strain R. wratislaviensis BN38 was characterized to be 2,3,4,2′‐trehalose tetraester which exhibited high surfactant activities. Significance and Impact of the Study:  Strain BN38 of R. wratislaviensis is a potential candidate for use in bioremediation applications or in biosurfactant exploration.

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