Crude glycerol is the primary by-product in the biodiesel industry, which is too costly to be purified into to higher quality products used in the health and cosmetics industries. This work investigated the potential of using the crude glycerol to produce docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the microalga Schizochytrium limacinum. The results showed that crude glycerol supported alga growth and DHA production, with 75-100 g/L concentration being the optimal range. Among other medium and environmental factors influencing DHA production, temperature, trace metal (PI) solution concentration, ammonium acetate, and NH4Cl had significant effects (P < 0.1). Their optimal values were determined 30 mL/L of PI, 0.04 g/L of NH4Cl, 1.0 g/L of ammonium acetate, and 19.2 degrees C. A highest DHA yield of 4.91 g/L with 22.1 g/L cell dry weight was obtained. The results suggested that biodiesel-derived crude glycerol is a promising feedstock for production of DHA from heterotrophic algal culture. (C) 2007 Elsevier Ltd. All rights reserved.