The mixotrophic growth of the diatom Phaeodactylum tricornutum UTEX-640 was studied using diverse substrates at different concentrations in discontinuous and fed-batch modes. The nutrients used were acetate (0.005-0.1 M), starch (0.5-5 g l(-1)), lactic acid (0.005-0.1 M), glycine (0.005-0.02 M), glucose (0.5-5 g l(-1)) and glycerol (0.005-0.1 M). Biomass concentration and biochemical profile were monitored. The capacity of the different nutrients to promote mixotrophic growth varied not only with its nature, but also with the concentration used for the experiment, showing how comparisons at the same concentration may be misleading. Subsequent fed-batch cultures using glycerol (0.1 M), and supplemented urea (0.01 M) and sodium nitrate (1 g l(-1)) as nitrogen sources, showed that repeated additions of organic substrate can sustain mixotrophic growth at very high density cultures. The best results were obtained using with urea (0.01 M), which resulted in maximum biomass and eicosapentaenoic acid productivities that were, respectively, 1.52 g l(-1) per day and 43.13 mg l(-1) per day, significantly higher than those obtained for the photoautotrophic control. Although the results reported here were obtained in flask cultures of only 11 working volume and under low irradiance (165 muEm(-2) s(-1)), similar data were reported for photoautotrophic growth on glycerol of this same strain in outdoor pilot-scale tubular photobioreactors (tube diameter 3 and 6 cm and to 50 and 2001 working volume, respectively), which suggest the possibility of using mixotrophy for the mass production of microalgae. (C) 2004 Elsevier Ltd. All rights reserved.