Journal of Applied Phycology 12: 239–248, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. 239 Mixotrophic growth of Phaeodactylum tricornutum on glycerol: growth rate and fatty acid profile M.C. Cer´ on Garc´ ıa, J.M. Fern´ andez Sevilla, F.G. Aci´ en Fern´ andez, E. Molina Grima & F. Garc´ ıa Camacho ∗ Departamento de Ingenier´ ıa Qu´ ımica, Universidad de Almer´ ıa, E-04071, Almer´ ıa, Spain ( ∗ Author for correspondence; fax +34-950215484; e-mail fgarcia@ualm.es) Received 5 October 1999; revised 26 January 2000; accepted 27 January 2000 Key words: eicosapentaenoic acid, microalga, mixotrophic growth, Phaeodactylum tricornutum Abstract Mixotrophic growth of the eicosapentaenoic acid (EPA) producing diatom Phaeodactylum tricornutum UTEX 640 was carried out in 1-L batch cultures under an external irradiance of 165 μmol photons m −2 s −1 by supplementing the inorganic culture medium with glycerol. The effect on the growth and the fatty acid profile was studied for different initial glycerol concentrations (0–0.1 M). The optimal glycerol concentration was 0.1 M. A lag phase was observed at high glycerol concentrations. The present study also shows that successive additions of glycerol at 0.1M concentration and using ammonium chloride as a nitrogen source remarkably increased the maximum biomass concentration (16.2 g L −1 ) and maximum biomass productivity (61.5 mg L −1 h −1 ). These values were respectively 9 and 8-fold higher than in the photoautotrophically grown control. The level of saponifiable lipids in mixotrophically cultured cells was significantly higher than in photoautotrophically cultured cells and increased with the glycerol concentration in the medium. The concentration of storage lipids, saturated and monounsaturated fatty acids, were enhanced but the EPA content did not change significantly. The EPA content was around 2.2% of biomass dry weight. The maximum EPA yield was 33.5 mg L −1 d −1 and was obtained in a culture containing 0.1 M glycerol, supplemented periodically by ammonium chloride. This productivity was 10-fold higher than the EPA productivity obtained under mixotrophic conditions. Introduction Microalgae are potential sources of high value chemic- als and pharmaceuticals such as polyunsaturated fatty acids (PUFAs) and bioactive compounds. Many mi- croalgae have been reported to have a high content of PUFAs (Dunstan et al., 1994; Yongmanitchai & Ward, 1989), especially eicosapentaenoic acid (Mo- lina Grima et al., 1994a). However, the EPA productiv- ity of microalgae is low compared with bacteria and fungi (Bajpai & Bajpai, 1993). The reason for the low EPA productivity is mainly a slow growth rate under photoautotrophicconditions. To promote the EPA pro- ductivity, culture conditions such as nitrate, ammonia and urea concentrations, pH (Yongmanitchai & Ward, 1991), aeration rate and irradiance (Sánchez Pérez, 1994), culture temperature (Seto et al., 1984) and cell concentration (Cohen et al., 1988; Chrismadha & Borowitzka, 1994) have been investigated. Nevertheless, under mixotrophic conditions some microalgae are known to grow rapidly and to have a higher growth rate than under photoautotrophic condi- tions (Samejima & Myers, 1958). For example, Lee et al. (1996) cultured Chlorella sorokiniana mixotroph- ically in an outdoor enclosed tubular photobioreactor reaching an optimum biomass productivity of 10.2 g L −1 d −1 during the day and 5.9 g L −1 d −1 during the night using an initial glucose concentration of 0.1 M. The daily volumetric productivity of photosynthetic Chlorella cultures in a similar tubular photobioreactor was about 3 times lower (Lee & Low, 1992). These