A polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) with an inner arrangement consisting of eight equidistantly spaced rectangular strips mounted radially on a circular disk to provide additional surface area for microbial attachment was employed for protease production by two biofilm-forming bacteria, an intertidal gamma-Proteobacterium (DGII) and a chicken meat isolate, Virgibacillus pantothenticus. The flask design allowed comparison of protease production during cultivation with a hydrophilic (glass) or hydrophobic (PMMA) surface. Compared to the Erlenmeyer flask, the CCF allowed protease production that was 30% and 35% higher and growth that was 20% and 345% higher for DGII and V. pantothenticus, respectively. Protease production increased by 202% and 22% and growth by 19,275% and 940% for DGII and V. pantothenticus, respectively, in the presence of a hydrophobic as compared to a hydrophilic surface. This investigation pioneers the application of a vessel beyond the traditional shake-flask for enhancing protease production by biofilm-formers. (C) 2010 Elsevier Ltd. All rights reserved.