Bacillus subtilis 3NA is a strain capable of reaching high cell densities. A surfactin producing sfp(+) variant of this strain, named JABs32, was utilized in fed-batch cultivation processes. Both a glucose and an ammonia solution were fed to set a steady growth rate mu of 0.1 h(-1). In this process, a cell dry weight of up to 88 g L-1 was reached after 38 h of cultivation, and surfactin titers of up to 26.5 g L-1 were detected in this high cell density fermentation process, achieving a Y-P/X value of 0.23 g g(-1) as well as a q(P/X) of 0.007 g g(-1) h(-1). In sum, a 21-fold increase in surfactin titer was obtained compared with cultivations in shake flasks. In contrast to fed-batch operations using Bacillus subtilis JABs24, an sfp(+) variant derived from B. subtilis 168, JABs32, reached an up to fourfold increase in surfactin titers using the same fed-batch protocol. Additionally, a two-stage feed process was established utilizing strain JABs32. Using an optimized mineral salt medium in this high cell density fermentation approach, after 31 h of cultivation, surfactin titers of 23.7 g L-1 were reached with a biomass concentration of 41.3 g L-1, thus achieving an enhanced Y-P/X value of 0.57 g g(-1) as well as a q(P/X) of 0.018 g g(-1) h(-1). The mutation of spo0A locus and an elongation of AbrB in the strain utilized in combination with a high cell density fed-batch process represents a promising new route for future enhancements on surfactin production.