Culture medium elements were analysed by a screening DoE to identify their influence in surfactin specific production by a surfactin constitutive overproducing Bacillus subtilis strain. Statistics pointed the major enhancement caused by high glutamic acid concentrations, as well as a minor positive influence of tryptophan and glucose. Successively, a central composite design was performed in microplate bioreactors using a BioLector((R)), in which variations of these impressive parameters, glucose, glutamic acid and tryptophan concentrations were selected for optimization of product-biomass yield (Y-P/X). Results were exploited in combination with a RSM. In absolute terms, experiments attained an Y-P/X 3.28-fold higher than those obtained in Landy medium, a usual culture medium used for lipopeptide production by B. subtilis. Therefore, two medium compositions for enhancing biomass and surfactin specific production were proposed and tested in continuous regime in a bubbleless membrane bioreactor. An Y-P/X increase of 2.26-fold was observed in bioreactor scale.