Mixotrophic cultures are culture systems where light and organic carbons are used as the energy source, while inorganic and organic carbons are used as the carbon source. Although it is sometimes used interchangeably with photoheterotrophic culture, in a strict sense, photoheterotrophic culture involves the use of light as the energy source, while organic carbon is used as the carbon source. In other words, light is required to metabolize the organic carbon source in photoheterotrophic culture. Photoheterotrophic cultivation requires both organic carbons and light at the same time, whereas in mixotrophic culture, both are present, but either can be used without the other. From practical point of view, both mixotrophic and pho­toheterotrophic cultures can be regarded as culture systems where light, organic carbon, and inorganic carbon are present at the same time.

As already discussed, heterotrophic culture has many advantages over photo­autotrophic cultures. However, there are many metabolites whose syntheses are promoted by light, and thus are not efficiently produced in heterotrophic cultures (Chen and Zhang 1997; Lee and Zhang 1999; Cohen 1999; Sukenik et al. 1991). This disadvantage can be overcome by mixotrophic culture which involves simultaneous use of light and organic carbon sources. Mixotrophic cultures have many advantages over other culture systems. For example, inhibition of photo­synthesis by high dissolved oxygen concentration is a major problem in photoau­totrophic cultures, while oxygen limitation is a major problem in heterotrophic cultures. In mixotrophic culture, dissolved oxygen concentration does not increase to inhibitory levels since it is simultaneously used for heterotrophic metabolism of the organic carbon. On the other hand, organic carbon assimilation is hardly limited by dissolved oxygen concentration since oxygen is constantly produced by pho­tosynthetic activities. Furthermore, heterotrophic growth generates carbon dioxide which is used for photoautotrophic growth (photosynthesis).

In mixotrophic cultures, the presence of an organic substrate means that cell growth is not strictly dependent on photosynthesis, and hence, light is not an indispensable growth factor. Read et al. (1989) and Fernandez Sevilla et al. (2004) have reported that mixotrophic growth requires relatively low light intensities and, consequently, can reduce energy costs. In some strains, it has been found that mixotrophic cultures reduced photoinhibition and that the growth rates are higher than those observed in both photoautotrophic and heterotrophic cultures. Further­more, mixotrophic cultivation reduces biomass loss at night and decreases the amount of organic substances utilized during growth (Chojnacka and Noworyta 2004).

In mixotrophic cultures of many strains of microalgae, there are additive or synergistic effects of photoautotrophic and heterotrophic metabolic activities, leading to increases in productivity. Park et al. (2012) reported higher biomass and fatty acid productivities of 14 species of microalgae in mixotrophic culture over photoautotrophic culture. Bhatnagar et al. (2011) found that the mixotrophic growth of Chlamydomonas globosa, Chlorella minutissima, and Scenedesmus bijuga resulted in 3-10 times more biomass production compared to those obtained under photoautotrophic growth conditions. It has also been shown that the addition of glycerol as the carbon source resulted in increased biomass productivity of Phae — odactylum tricornutum (Ceron Garcia et al. 2005, 2006; Moraisa et al. 2009). One of the possible reasons for better growth in mixotrophic cultures is the stability of pH, since carbon dioxide is simultaneously assimilated and released during pho­tosynthesis and respiration. In photoautotrophic cultures, the pH increased to more than 10, but remained stable around 7 in mixotrophic culture (Kong et al. 2011). It is important to note, however, that biomass productivity in mixotrophic cultures depends on many factors such as the strain, type, and concentration of the carbon source, and other medium components, as well as the light intensity. In some strains, for example, the addition of some carbon sources to photoautotrophic cultures inhibits growth, while others stimulate growth (Heredia-Arroyo et al.

2011) . This is because photosynthesis and oxidative phosphorylation of organic carbon substrates seem to function independently in some algae, and growth rates in mixotrophic cultures are the sum of those in photoautotrophic and heterotrophic cultures. This has been reported for Chlorella sp., Spirulina sp., and Haemato — coccus (Ogawa and Aiba 1981; Marquez et al. 1993; Martinez and Orus 1991; Hata et al. 2001). Under certain culture conditions, the presence of organic carbon in some microalgae depresses photosynthetic O2 evolution and inhibits respiration and enzymes of Calvin cycle (Liu et al. 2009). In mixotrophic cultures, photosynthetic fixation of inorganic carbon is influenced by light intensity, while the heterotrophic assimilation of carbon is influenced by the availability of organic carbon. Thus, the ratio of photoautotrophic growth to heterotrophic growth depends on the light intensity, type, and concentration of organic carbon and carbon dioxide concen­tration (Ogbonna et al. 2002a, b). These factors must be controlled to ensure high rates of growth and lipid accumulation.

Aside from increased biomass concentration and productivities (Lodi et al. 2005), mixotrophic cultures can lead to increases in lipid accumulation over the values obtained in photoautotrophic cultures. This has been reported for several species of microalgae such as Chlorella sp. (glucose), P. tricornutum (glycerol) (Fernandez Sevilla et al. 2004), Nannochloropsis sp. (glycerol) (Wood et al. 1999; Liang et al. 2009), and C. vulgaris (Kong et al. 2011). However, the oil contents of the cells in mixotrophic cultures are dependent on the nature of the carbon source. In some cases, the lipid contents of the cells are even lower or the same as those in the photoautotrophic cultures (Park et al. 2012). Nevertheless, because of the higher growth rate, the lipid productivities are, generally, much higher than those in photoautotrophic cultures. Ratha et al. (2013) reported that lipid production by twenty different strains of cyanobacteria and green algae was highest under mixotrophic condition, compared to heterotrophic and photoautotrophic cultures. With either glucose, starch, or acetate, the maximum lipid productivities of Phaeodactylumtricornutum in mixotrophic cultures were several times higher than those obtained in the corresponding photoautotrophic control cultures (Wang et al.

2012) .

Lipid productivity in mixotrophic culture is also dependent on the strain used. For example, the lipid content and lipid productivity were higher under mixotrophic conditions as compared to both photoautotrophic and heterotrophic cultures in all the members of Chlorococcales tested. Yet, the filamentous alga Ulothrix and all the cyanobacterial strains had slightly higher lipid content and lipid productivity in photoautotrophic cultures (Ratha et al. 2013). The increases in fatty acid produc­tivity under mixotrophic conditions can result from the combined increases in biomass productivity and fatty acid content, or from increased biomass productivity at relatively constant fatty acid content. In some strains and under certain culture conditions, there is no positive effect of mixotrophic culture on cell lipid content; thus, the increase in lipid productivity is mainly due to increases in biomass pro­ductivity, shown for C. vulgaris with various carbon sources (Kong et al. 2011). In contrast to photoautotrophic cultures, where conditions that favor lipid accumula­tion often suppress cell growth (Chisti 2007; Hu et al. 2008), in mixotrophic cultures, there can be a linear relationship between biomass and fatty acid pro­ductivities (Griffiths and Harrison 2009; Park et al. 2012).

Mixotrophic cultivation affects the fatty acid profile of microalgae. In 10 out of 14 isolates grown under mixotrophic condition with acetate as the organic carbon source, the percentage of oleic acid content increased significantly (Park et al.

2012) . However, the fatty acid profile was not affected when glycerol was used (Fernandez Sevilla et al. 2004), indicating that high oleic acid content is not a general feature of fatty acids in mixotrophically grown cells and that the carbon source is likely to be an important determinant of the fatty acid profile.

Other advantages of mixotrophic cultures include the feasibility of using open ponds for large-scale cultivation (Perez-Garcia et al. 2011), and the use of waste­waters as sources of organic carbon and other nutrients for reduced production costs (Zhao et al. 2012). When open ponds or non-sterilized bioreactors are used, the addition of the organic carbon sources must be controlled to avoid contamination by fast-growing heterotrophs. In some cases, the organic carbon substrate is only introduced during daylight hours, or alternatively is added only once toward the end of the culture to avoid bacterial contaminants from accumulating to unacceptable levels (Abeliovich and Weisman 1978; Lee 2001).

The main disadvantages of mixotrophic culture, as with heterotrophic culture, are that the cost of carbon source can be high and an excess/uncontrolled addition of organic substrates in an open system is likely to stimulate growth of invasive heterotrophic bacteria, resulting in a low microalgae biomass yield. There is also the problem of photoinhibition of organic carbon metabolism, in some cases, while maintaining an optimum balance of photoautotrophic to heterotrophic metabolic activities can be challenging.