Many organic carbon sources have been investigated for biodiesel production. These include various sugars such as glucose, galactose, fructose, and even some disaccharides. Polyhydric alcohols, such as glycerol, and some organic acids, such as acetate and propionate, have also been investigated. For example, Chlorella protothecoides can grow on a variety of carbon sources such as glucose (Shen et al. 2010; Xiong et al. 2008; Xu et al. 2006), fructose (Gao et al. 2009), sucrose (Gao et al. 2009), glycerol (Heredia-Arroyo et al. 2010), acetate (Heredia-Arroyo et al.

2010) , and reducing sugars from Jerusalem artichoke and sugarcane (Cheng et al.

2009) . Many species have also been reported to grow heterotrophically on ethanol (Ogbonna et al. 1998; Yokochi et al. 1998), lactose, galactose and mannose (Liang et al. 2009), and molasses (Andrade and Costa 2007). Schizochytrium limacinum produced palmitic acid (16:0) as 45-60 % of their dry weight when supplied with glucose, fructose, or glycerol (Yokochi et al. 1998; Chi et al. 2009). The effec­tiveness of these carbon sources varies with the species as well as on the culture conditions such as the light intensity, the pH, dissolved oxygen concentration, and on the presence of other carbon sources. Some carbon sources are good for mixotrophic culture, but not in heterotrophic cultures. For example, according to

Ceron Garcia et al. (2006), Phaeodactylum tricornutum UTEX-640 did not grow heterotrophically in media containing 0.005-0.2 M of fructose, glucose, mannose, lactose, or glycerol. However, addition of any of these organic carbons in mixo — trophic culture increased the biomass concentration and productivity relative to photoautotrophic controls. The biomass, lipids, eicosapentanoic acid (EPA), and pigment contents were considerably enhanced with glycerol and fructose in relation to photoautotrophic controls. The EPA content was barely affected by the sugars, but was more than twofold higher in glycerol-fed cultures than in photoautotrophic controls (Ceron Garcia et al. 2006).

Liu et al. (1999) compared several carbon sources and concluded that glucose was the best in terms of cell growth rate. This contradicts the work of Chen and Walker (2011) who reported that crude glycerol gave the highest growth of Chlorella protothecoides, followed by pure glycerol, while the least biomass concentration was obtained with glucose. In the case of Chlorella vulgaris, Kong et al. (2011) reported that glucose was the best carbon source for mixotrophic cultivation, followed by sucrose and then glycerin, while sodium acetate did not support good growth. Effectiveness of carbon source in supporting cell growth may depend on their energy content (Chojnacka and Marquez-Rocha 2004; Wang et al. 2012). For instance, glucose produces about 2.8 kJ/mol of energy compared to

0. 8 kJ/mol for acetate (Boyle and Morgan 2009), and glucose was more effective as a substrate for mixotrophic cultivation of Phaeodactylum tricornutum than acetate (Wang et al. 2012). The carbon source that gives high biomass productivity may not be the one that gives high oil production. Although for many strains, glucose has been reported to be the best in terms of cell growth, Das et al. (2011) ranked the effectiveness of different organic substrates in terms of intracellular lipid contents in mixotrophic culture in the following order: glycerol > sucrose > glucose.

The cost is another major factor determining the choice of carbon source for mixotrophic/heterotrophic cultures. The present cost of microalgae oil at US$2.4/L (Li et al. 2007; Xu et al. 2006) is 3-4 times higher than that of plant oils. However, Liu et al. (2010) estimated oil production cost of US$0.9/L for heterotrophic cul­tures of Chlorella zofingiensis using sugar as substrate. In heterotrophic/mixo — trophic cultures, the cost of organic carbon represents a very high percentage of the total production cost. Economic analysis shows that organic carbon source con­tributes 45.4 %; inorganic chemicals, 3.2 %; electricity, 30.6 %; steam, 14.2 %; and aseptic air, 6.6 % of the total production cost (Li et al. 2007; Xu et al. 2006). The cost of glucose has also been estimated to be about 80 % of the total medium cost (Li et al. 2007). Thus, there is a need to drastically reduce the cost of the organic carbon source. Many cheap carbon sources such as non-sugar carbon sources (Heredia-Arroyo et al. 2011), corn powder hydrolysate, impure glycerol, and molasses have been investigated.

Currently glycerol is an inexpensive and abundant carbon source generated as a by-product of biodiesel fuel production. About 0.45 kg of glycerol is produced per

4.5 kg of biodiesel, and the price of crude glycerol is now about 0.025USD/0.45 kg (Chen and Walker 2011). It has been reported that crude glycerol is better than pure glycerol and glucose (Chen and Walker 2011; Liang et al. 2010) because of the residual nitrogen in crude glycerol. The use of corn powder hydrolysate has also been widely investigated, and it has been reported that it is superior to glucose solution since it contains some other components that are beneficial for cell growth. For example, C. protothecoides produced 55.2 % crude lipids in a medium con­taining corn hydrolysate, with a cell dry weight concentration of 15.5 g/L (Xu et al.

2006) , which is higher than the values reported for glucose. Li et al. (2007) noted that if hydrolyzed starch is used as a carbon source for Chlorella, the cost of medium can be reduced to about 60-70 %. Cheng et al. (2009) used hydrolysate of Jerusalem artichoke tuber as a carbon source for heterotrophic cultivation of C. protothecoides, and the resulting biomass contained 44 % lipid. The lipid content of microalgae cultivated in the presence of the enzymatic hydrolyzates of sweet sor­ghum (which contains 10 g/L of reducing sugars) was 52.5 %. This is 35.7 % higher than the value obtained by cultivation using glucose (Gao et al. 2009). Anaerobi­cally digested dairy manure (Wang et al. 2010) and wastewater containing 85-90 % carpet mill effluents (Chinnasamy et al. 2010) were used as carbon sources for production of lipids for biofuel. Many agro-industrial wastes such as dry-grind ethanol thin stillage (TS) and soy whey (SW) have been used as nutrient feedstock for mixotrophic/heterotrophic cultivation of Chlorella vulgaris (Mitra et al. 2012). Both the cell concentration (9.8 g/L) and oil content (43 %) obtained with TS were higher than those obtained with modified basal medium containing glucose as the carbon source (8 g/L and 27 %, respectively) under mixotrophic conditions.

The optimal concentrations of these carbon sources vary with both strain and other culture conditions. Concentrations of glycerol used ranged from 3 to 12 % (Yokochi et al. 1998), while Liang et al. (2009) reported that 1 or 2 % glycerol resulted in a higher lipid content of microalgae compared to the value obtained with 5 % glycerol. The tolerable concentration of glycerol is within the range of

0. 7-10 % (Chi et al. 2009). The optimum cassava starch hydrolysate concentration for cell growth and lipid accumulation was 5 g/L, but the values were not signif­icantly different from those obtained with 10 g/L. A higher concentration of 15 g/L of hydrolysate resulted in lower biomass and lipid contents (Salim 2013). The optimum concentration of glucose for growth was 1 g/L, but there were no significant effects of varying acetate or starch concentrations between 0.5 and 5 g/L on cell growth. The highest values of the lipid content and lipid productivity with glucose in media were approximately 2.8 times (at 2.0 g/L glucose) and 4.6 times (at 1.0 g/L glucose) compared to control (photoautotrophic culture). As the content of glucose increased to 5.0 g/L, the total lipid content and lipid productivity decreased, but were still higher than the values obtained in photoautotrophic culture (Wang et al. 2012). On the other hand, starch in the medium did not influence the specific growth rate with concentrations below 1.0 g/L, but was inhibited signifi­cantly above 2.0 g/L (p < 0.05) (Wang et al. 2012).