Statistical screening experimental designs were applied to identify the significant culture variables for biomass production of Aurantiochytrium limacinum SR21 and their optimal levels were found using a combination of Artificial Neural Networks, genetic algorithms and graphical analysis. The biomass value obtained (40.3 g cell dry weight l(-1)) employing the selected culture conditions agreed with that predicted by the model. Subsequently, two significant culture conditions for docosahexaenoic acid (DHA) production were determined, finding that an inoculum of 10% (v/v), obtained from the previous (statistically optimized) stage, should be used in a DHA production medium having a molar C:N ratio of 55: 1, to reach a production of 7.8 g DHA l(-1) d(-1). The production step was thereafter scaled in a 3.5 1 bioreactor, and DHA productivity of 3.7 g l(-1) d(-1) was obtained. This two-stage strategy: statistically optimized inoculum production (fist step) and a DHA production step, is presented for the first time to optimize a bioprocess conducive to the obtention of microbial DHA. (C) 2009 Elsevier Ltd. All rights reserved.