Heterotrophic cultures use organic carbons as both sources of energy and carbon. There are many advantages of heterotrophic cultures over photoautotrophic cul­tures. These include the following: (i) the use conventional heterotrophic bioreac­tors that are simpler and easier to scale up, since the elimination of light requirements means that smaller reactor surface-to-volume ratios can be used; (ii) greater control of the cultivation process, since the cultures can be done indoors; and (iii) higher cell densities, which reduces the cost of harvesting the cells. The basic components of media for heterotrophic cultures are similar to those of the photoautotrophic media, with the addition of organic carbon sources. In addition, the growth rate and oil accumulation of heterotrophic cultures are affected by the C: N ratio in the medium.

Generally, the biomass concentrations obtained in heterotrophic cultures are much higher than those in photoautotrophic cultures (Ogbonna et al. 1998). Although the biomass concentration in most photoautotrophic cultures is less than 5 g/L, much higher concentrations of 15.5 g/L for Chlorella protothecoides (Xu et al. 2006), 28.8 g/L for Traselmis suecica (Azma et al. 2011), and even 53 g/L for

Chlorella zofingiensis (Sun et al. 2008) have been reported. Fed-batch cultures can be used to obtain even higher biomass concentrations. Furthermore, heterotrophi — cally grown microalgae usually accumulate more lipids than those cultivated photoautotrophically, as demonstrated for Chlorella species (Miao and Wu 2006; Xu et al. 2006; Agwa et al. 2013; Liu et al. 2008; Hsieh and Wu 2009). In the case of Chlorella vulgaris, for example, Wu et al. (2012) reported an increase from 15 % under photoautotrophic condition to more than 50 % under heterotrophic condition. Compared with photoautotrophic cultures, Jimenez et al. (2009) reported an 8 times increase in oil content of C. protothecoides under heterotrophic condition, and a 9 times increase in lipid yield was achieved in heterotrophic cultures fed with 30 g/ L of glucose (Liu et al. 2011).

The high biomass concentration and high lipid contents obtained in heterotro­phic cultures result in very high lipid productivities. A lipid productivity of 179 mg/ L/d in photoautotrophic culture is regarded as high (Chiu et al. 2008), but much higher productivities of 528.5 mg/L/d (Morales-Sanchez et al. 2013), 932 mg/L/d (Xu et al. 2006), 1.38 g/L/d (Liu et al. 2011), 2.43 g/L/d (Chen and Walker 2011),

3.0 g/L/d (Chen and Walker 2011), and 3.7 g/L/d (Xiong et al. 2008) have been reported for heterotrophic cultures. It is usually technically difficult to construct large-scale photoautotrophic photobioreactors; however, for heterotrophic cultures, conventional bioreactors can be used for large-scale processes. For example, a heterotrophic culture was scaled up from 5 to 750 L, and then 11,000 L, and the oil contents remained fairly stable at 46.1, 48.7, and 44.3 % of cell dry weight, respectively (Li et al. 2007).

It has also been reported that the quality of oil produced under heterotrophic cultures is more suitable for biodiesel production than those produced under pho­toautotrophic cultures with the same strains of microorganisms. Liu et al. (2011) reported that heterotrophic cells accumulated predominantly neutral lipids that accounted for 79.5 % of the total lipids, with 88.7 % triacylglycerol, while oleic acid accounted for 35.2 % of the total fatty acid. In contrast, photoautotrophic cells contained mainly the membrane lipids, glycolipids, and phospholipids. Further­more, C. saccharophila, C. vulgaris, N. laevis, Cylindrotheca fusiformis, Navicula incerta, and Tetraselmis suecica accumulate more lipids under heterotrophic than under photoautotrophic conditions, mainly in the form of triglycerides (Day et al. 1991; Tan and Johns 1991, 1996; Gladue and Maxey 1994). Conversely, photo­autotrophic cultures form more highly unsaturated fatty acids (polar lipids) (Tan and Johns 1991, 1996; Wen and Chen 2000a, b). Miao and Wu (2004) further noted that the oil obtained from heterotrophically grown cells possesses properties similar to those of fossil diesel in terms of oxygen content, heating value, density, and viscosity.

However, heterotrophic cultures have some limitations. Only a limited number of microalgae are capable of growing under heterotrophic conditions, and addition of an organic carbon source can significantly increase the cost of production. Furthermore, the presence of an organic carbon source further increases the risk of contamination, and depending on the species, the cell growth rate and lipid pro­ductivity in heterotrophic culture may be lower than the values obtained in mixotrophic culture. For example, Day and Tsavalos (1996) found that heterotro­phic culture of Tetraselmis with glucose yielded only about one-sixth of cellular lipid compared with the value obtained in mixotrophic culture.