A novel extractive fermentation procedure, tentatively named an extractive liquid-surface immobilization (Ext-LSI) system, for the production of water-insoluble secondary metabolites with fungi was developed. The system using a unique polymeric micro-material, a ballooned polyacrylonitrile microshpere (MS), was applied to the production of 6-pentyl-alpha-pyrone (6PP), a fungicidal secondary metabolite, with a newly isolated Trichoderma atroviride AG2755-5 and its nitrosoguanidine (NTG)mutant. The resulted mutant AG2755-5NM398 was immobilized on the surface of a liquid medium by using the MS. Following the formation of a fungus-MS mat, low toxic hydrophobic solvent, dimethyl silicone oil, was added onto the fungus-MS mat. The optimum carbon and nitrogen sources were fructose and malt extract, respectively. The higher the initial medium pH, the more the 6PP-accumulation was observed. The best MS and extractive organic solvent were MFL-80SDE (non-coated type) and KF-96L-1CS (dimethyl silicone oil). Thus, produced 6PP was spontaneously extracted from cells to the organic phase to reach 7.1 g L(-1) of the accumulation in the organic phase. (C) 2009 Elsevier Ltd. All rights reserved.