The heterotrophic culture of transgenic Chlorella containing the rabbit defensin (NP-1) gene was studied in a 5 L stirred tank bioreactor. An initial batch culture experiment indicated substrate shortage in the later phase of the culture. A preliminary fed-batch culture with glucose and KNO3 feeding as well as pH, and DO control was conducted. The transgenic Chlorella cell density reached about 9.6 g/L, which was 92% higher than the batch culture. The NP-1 gene expression capability maintained basically the same level. To enhance cell growth, a second fed-batch culture with a more finely designed scheme of feeding and process control was conducted, in which both the timing and frequency of substrate feeding varied. As a result, the final cell accumulation achieved about 17.0 g/L, which was almost double the preliminary experiment while the NP-1 gene expression capability showed no significant changes. (c) 2005 Elsevier Ltd. All rights reserved.