Lipase production in two-step fed-batch culture of organic solvent-tolerant Pseudomonas aeruginosa LST-03

Efficient lipase production by two-step fed-batch culture of an organic sol vent-tolerant bacterium, Pseudomonas aeruginosa LST-03, was investigated. W hen FB synthetic medium was used in flask culture, no lipase activity was d etected, whereas lipase was produced at 2.3 I.U./ml in C2 complex medium. H owever, lipase production was induced in FB medium when a fatty acid was ad ded to the culture broth in the stationary phase. Among fatty acids tested, long chain saturated fatty acids, such as C18 (stearic acid) and C20 (arac hidic acid), were found to function as effective inducers for the productio n of lipase, giving an activity level almost the same as that obtained in C 2 medium in flask culture. Two-step lipase production, comprised of a growt h phase in fed-batch mode and a production phase in which lipase was induce d by the addition of 5% (v/v) stearic acid, was carried out in a jar-fermen ter. In the growth phase, the maximum cell concentration at 16 h was only 2 0 in terms of the optical density at 660 nm (OD660), and a low level of lip ase production (8 I.U./ml) was obtained after 167 h. This was considered to be due to the exhaustion of several medium components brought about by the use of an unsuitable medium or feeding solution. After analyzing the conte nts of the compounds in the culture broth by inductively coupled plasma spe ctrometry for metal ions and HPLC for anions, a modified FB medium was desi gned. When this modified FB medium was used in two-step fed-batch culture, the maximum cell concentration reached an OD660 of 55 (30.2 g-dry cells/l) at 16.5 h, and lipase was produced at 96 I.U./ml after 35 h, which is appro ximately 40 times higher than the production level obtained in flask cultur e using C2 medium.