Biosurfactants are currently not a feasible alternative to chemically synthesized surfactants as a result of their potentially high production costs. One of the most widely studied and surface active biosurfactants is Surfactin, produced by Bacillus subtilis ATCC 21332. In this study, we have demonstrated a link between the enhanced production of Surfactin and the onset of nitrate-limited growth, by using a defined medium with ammonium nitrate as a nitrogen source. The utilization of nitrate occurred following the depletion of ammonium in the medium, and in anaerobic conditions in which dissimilatory nitrate metabolism was induced. A far higher Yp/x was reached in nitrate-limited oxygen-depleted conditions, (Yp/x = 0.075) than in ammonium-limited oxygen-depleted (0.012), carbon-limited oxygen-depleted (0.0069), aerobic carbon-limited (0.0068), or aerobic nitrogen-limited (0.021) cultures.