In the heterotrophic batch cultures, high initial concentration of substrates, e. g., sugars, is usually used to provide sufficient carbons for obtaining high cell density. Accompanying the high substrate concentration, however, is the occurrence of possible growth inhibition. For instance, the optimal sugar concentration for growing C. zofingiensis was reported below 20 g L-1, above which the inhibition of algal growth was observed (Ip and Chen, 2005; Liu et al., 2012a). The substrate-based inhibition caused not only the decreased specific growth rate but also the lowered biomass yield coefficient based on sugars (Sun et al., 2008; Liu et al., 2012a), contributing accordingly to the increased cost input. To overcome the inhibition issue associated with batch cultures, fed-batch cultivation is a commonly used strategy in which the substrate is fed into the algal cultures step by step to maintain it sufficiently for cell growth but below the level of inhibition threshold. There have been a number of reports employing fed-batch strategy to grow algae heterotrophically with the aim of avoiding the possible inhibition caused by the initial high substrate and improving the production poten­tial of biomass as well as of oils (Xu et al., 2006; Li et al., 2007a; Sun et al., 2008; Xiong et al., 2008; Liu et al., 2010; 2012a; De la Hoz Siegler et al., 2011; Yan et al., 2011; Chen and Walker,

2012) . Liu et al. (2010) investigated the heterotrophic oil production by C. zofingiensis using fed-batch cultures in a 3.7-L bioreactor. A two-stage feeding was adopted: three times of feed­ing with glucose-containing nutrients (to maintain linear growth) followed by four times of glucose feeding alone (to further increase biomass and induce oil accumulation; Figure 6.4). Glucose concentration of the cultures was maintained between 5 and 20 g L-1. The maximum lipid yield and lipid productivity achieved in the fed-batch cultures were 20.7 g L-1 and 1.38 g L-1 day-1, respectively, representing around a 2.9-fold increase of the those obtained in batch cultures.

Although the employment of fed-batch culture strategy proves able to eliminate the sub­strate inhibition, it cannot overcome the inhibition caused by the toxic metabolites that would be produced by the algal cultures and accumulate as the cells build up, preventing further enhancement of cell density.

FIGURE 6.4 (A) Growth and glucose consump­

tion and (B) lipid production in a two-stage fed-batch A» fermentation of C. zofingiensis in a 3.7-L fermentor. (O) cell biomass; (□) glucose concentration; (column) lipid content; (△) lipid yield; (#) glucose-containing medium feeding; (##) glucose feeding alone. Adapted E from Liu et al. (2010) and the permission for reprint requested.

6.5.1 Continuous Cultivation

The term continuous cultivation refers to the fresh medium being continuously added to a well-mixed culture while cells or products are simultaneously removed to keep the culture volume constant. It allows the steady state of kinetic parameters such as specific growth rate, cell density, and productivity and is thus considered an important system for studying the basic physiological behavior of heterotrophic algal cells. Figure 6.5a shows the schematic di­agram of the continuous cultivation system. This system is capable of effectively eliminating the metabolite-driven inhibition. There are several reports of continuous cultivation of algae in both photoautotrophic (Molina Grima et al., 1994; Otero et al., 1997) and heterotrophic (Wen and Chen, 2002b; Ethier et al., 2011) growth modes. Ethier et al (2011) investigated the continuous production of oils by the microalga Schizochytrium limacinum with various di­lution rates (D) and feed glycerol concentrations (S0). The yields and productivities of bio­mass, total fatty acids (TFA), and docosahexaenoic acid (DHA), shown in Figure 6.6, were over the range of D from 0.2 to 0.6 day-1 (S0 fixed at 90 g L-1) and the range of S0 from 15 to 120 g L-1 (D fixed at 0.3 day-1). The highest biomass productivity is 3.9 gL-1 day-1, obtained with the 0.3 day-1 of D and 60 g L-1 of S0 (Figure 6.6b). The maximum productivities of both TFA and DHA were also achieved at the same D but with a higher S0 of 90 g L-1 (Figures 6.6d and 6.6f). Liu et al (2012a) surveyed the feasibility of using a semicontinuous C. zofingiensis culture fed with waste molasses for oil production. The waste molasses contains relatively high levels of metal ions and salt that are inhibitory to algal growth, causing the

FIGURE 6.5 Schematic diagram of (A) continuous, (B) perfusion, and (C) perfusion­bleeding culture systems. X, cell concentration; V, culture volume; S, carbon concentration in medium; F, flow rate of feed; F1, flow rate of per­fusion; F2, flow rate of bleeding; S0, carbon con­centration in feed. The flow rates are controlled to keep the culture volume constant.

failure of molasses-based fed-batch cultivation when molasses was not pretreated; in con­trast, C. zofingiensis in the semicontinuous culture fed with diluted raw molasses showed comparable growth rate and sugar utilization to that with pretreated molasses (Liu et al., 2012a). Although continuous cultivation can promote the productivity, it is worth to mention that accompanying the increase of dilution rate is the drop of cell density as well as of sub­strate utilization efficiency (Wen and Chen, 2002b). From a cost-effectiveness point of view, this is undesirable in that the residual substrate is wasted with the effluent and more energy input is required to harvest the diluted cells.

FIGURE 6.6 Algal growth, TFA and DHA production of continuous Schizochytrium limacinum in a 7.5-L fermentor with various dilution rates (D) (A, C, E; S0 = 90 gL-1) and feed glycerol concentrations (S0) (B, D, F; D = 0.3 day-1). Adapted from Ethier et al. (2011) and the permission for reprint requested.