Continuous cultivation with cell recycling, denoted as perfusion culture, is a culture technique combining the advantages of both fed-batch and continuous culture systems, namely, avoiding the substrate inhibition and the inhibition caused by toxic metabolites produced by accumulated algal cells while maintaining high cell density and productivity

(Chen and Johns, 1995; Wen and Chen, 2002a). As illustrated by Figure 6.5b, in a perfusion culture system the algal cells are retained by a retention device, whereas the spent medium (cell-free) was removed from the bioreactor; at the same time, fresh medium was fed into the bioreactor to maintain sufficient nutrient supply. Wen and Chen (2002a) used the perfusion culture system to investigate the heterotrophic production of N. laevis. By employing an ex­ponential feeding of glucose and manipulating the rates of glucose feeding and spent me­dium perfusion, the optimal glucose concentration in the feed was determined to be 50 g L-1 (Figures 6.7a and 6.7b). With the feeding of optimized glucose concentration (S0 = 50 g L-1), a high cell density of 40 g L-1 was achieved in the perfusion culture of N. laevis (Figure 6.7c). Together with the relatively simple setup and operation as well as high biomass

yield coefficient based on glucose, the perfusion culture system potentially may be used to grow algae for heterotrophic production of bio-oils.

A modified perfusion culture system that introduces cell bleeding during perfusion oper­ation was also developed for heterotrophic production of algae (Figure 6.5c; Wen and Chen, 2001b). This system could potentially improve the biomass productivity but at the same time lower the cell density, e. g., from 40 g L-1 to less than 20 g L-1 (Wen and Chen, 2001b; Wen and Chen, 2002a).

It is worth mentioning that different algal species/strains may favor different culture systems to achieve maximized cell density, biomass productivity, and oil productivity. An experimental optimization is required for a selected algal strain to demonstrate which culture system is best for the heterotrophic production of oils. Regardless of the algal strain selected and culture system used, the key to optimizing a production system rests with the cost balance of output and input from a cost-effectiveness point of view.