BACKGROUND: Lipopeptide production is strongly influenced by trace metals. The availability of free Fe2+ in the media throughout the process of fermentation was found to be very critical. Since free Fe2+ was reported to be sequestered by the lipopeptide as it was produced, intermittent feeding of Fe2+ was strategized and optimized for enhanced lipopeptide production by marine Bacillus megaterium in glucose mineral salts medium (GMSM). RESULTS: Studies with the single-dose Fe2+ (0.48 mmol L-1) supplementation after 8 h of fermentation resulted in lipopeptide concentration of 3.3 +/- 0.1 g L-1. Lipopeptide production was further enhanced to 4.2 +/- 0.15 g L-1 by adopting a multi-dose Fe2+ feeding strategy. The maximum product yield (YP/S) of 0.24 +/- 0.02 g g-1 with specific product formation rate (qp) of 0.124 +/- 0.01 g g-1 h-1 was achieved when 0.48 mmol L-1 Fe2+ was fed intermittently at different times as per the designed strategy. CONCLUSION: Lipopeptide concentration was improved 4.7-fold by single-dosing and 5.8-fold by multiple dosing of Fe2+, when compared with GMSM without Fe2+ supplementation. Copyright (c) 2012 Society of Chemical Industry