A newly isolated indigenous strain Pseudomonas aeruginosa J16 was able to produce a glycolipid biosurfactant (i.e., rhamnolipid). Analyses with (13)C NMR and mass spectrometry show that the biosurfactant consisted mainly of mono-rhamnolipid (RL1) and di-rhamnolipid (RL2). Since RL2 possesses higher surface activity than RL1 and is of better applicability and commercial benefits, the present work attempted to enhance the production of RL2 over RL1 in A aeruginosa J16 from the perspective of medium improvement. The results show that the abundance and composition of RL1 and RL2 varied with the type of carbon and nitrogen sources used. When glycerol and NH(4)Cl were used as the carbon and nitrogen sources, respectively, an RL2 production of 448.3 mg/L was obtained with a volumetric productivity of 4.67 mg/(L h) and a RL2/RL1 ratio of 4.22 (by mass). Replacing NH(4)Cl by (NH(4))(2)SO(4), the RL2 concentration and volumetric productivity were improved to 2121 mg/L and 22.1 mg/(L h), respectively, with the RL2/RL1 ratio of 3.0. To further enhance RL2 production, statistical experimental design methodology was applied to optimize the culture medium composition favoring RL2 synthesis. Three key parameters (glycerol, (NH(4))(2)SO(4), and MgSO(4).7H(2)O) were selected by two-level factorial design. Response surface methodology was then used to identify the optimal composition of the three key parameters, giving an optimal concentration of 0.38 M, 33.3 mM, and 577 mu M for glycerol, (NH(4))(2)SO(4), and MgSO(4).7H(2)O, respectively. With this optimal medium, the RL2 production could be markedly elevated to a maximum concentration of 3190 mg/L and a volumetric productivity of 44.3 mg/(L h), while the RL2/RL1 ratio maintained nearly constant at a value of 3.0. (C) 2008 Elsevier Ltd. All rights reserved.