Various species of microalgae can be used for biodiesel oil production using either photoautotrophic, heterotrophic, or mixotrophic culture system. However, the suitability of each depends on the strain, availability of facilities, nature of organic carbon source, and other culture conditions. In a comparison of the lipid produc­tivity in 20 species of photosynthetic microorganisms, three strains produced more lipid in heterotrophic cultures when compared to photoautotrophic cultures and 11 strains produced more in mixotrophic cultures than in photoautotrophic cultures (Ratha et al. 2013).

Lipid productivity is a product of lipid content and biomass concentration and gives the total amount of lipid formed per unit culture volume and time (g-lipid/L. d). Thus, strategies for improving productivity aims to increase lipid content without significant reduction in cell growth, increase cell growth without significant reduc­tion in oil content, or increase both cell growth and lipid accumulation per cell. Several species of microalgae can be induced to overproduce lipids by the choice of culture system as well as by manipulations of the culture medium such as the source and concentrations of carbon, nitrogen, phosphorous, and silicate as well as by manipulation of culture conditions such as temperature, pH, and oxygen tension.

Nitrogen starvation is one of the most studied methods of inducing oil accumu­lation in microalgae. As a result of nitrogen starvation, the lipid content as high as 70-85 % of dry weight has been reported (Becker 1994). The effectiveness of nitrogen limitation in increasing lipid contents of microalgae has been demonstrated with many strains such as Prophyridium cruentum (Becker 1994), Chlorella vulgaris (Widjaja et al. 2009; Converti et al. 2009), Chlorellaprotothecoides (Miao and Wu

2004) , as well as many strains of cyanobacteria and other green algae (Shifrin and Chisholm 1981; Illman et al. 2000; Takagi et al. 2000; Li et al. 2008a, b). However, using nitrogen limitation to increase intracellular lipid content may have negative effects on cell growth and thus lipid productivity, which has been reported for several strains (Hsieh and Wu 2009; Xiong et al. 2008). On the whole, the effectiveness of nitrogen limitation in increasing lipid productivity depends on the strain. In the case of diatoms, such as Achnanthes brevipes and Tetraselmis sp. for example, nitrogen limitation leads to accumulation of carbohydrates, rather than lipids (Guerrini et al. 2000; Gladue and Maxey 1994). Under nitrogen starvation, accu­mulation of lipids has been attributed to mobilization of lipids from chloroplast membranes as chloroplastic nitrogen is relocated by 1.5-biphosphate carboxylase/ oxygenase (E. C. 4.1.1.39, Rubisco) (Garcia-Ferris et al. 1996). Some other reports have shown that increased lipid content of cells at low nitrogen concentration may be due to the high C/N ratio, rather than the absolute nitrogen concentration. For example, in culture of Chlorella sorokiniana, a C/N ratio of 20 gave the lowest cell lipid content, but increased at both higher and lower C/N values (Chen and Johns 1991). Furthermore, in marine (Cryptheconidium conhii) and freshwater (Chlorella sorokiniana) algae, accumulation of lipids may not be dependent on nitrogen exhaustion but on an excess of carbon in the culture media. Hence, in heterotrophic cultures, lipid accumulation was attributed to consumption of sugars at a rate higher than the rate of cell generation, leading to conversion of excess sugar into lipids (Chen and Johns 1991; Ratledge and Wynn 2002; de Swaaf et al. 2003).

In the case of diatoms, lipid accumulation is related to depletion of silicates because of their dependence on silica for growth (Roessler 1988; Wen and Chen 2000a, b, 2003; Wilhelm et al. 2006). Roessler (1988) reported that silicon defi­ciency could induce lipid accumulation in Cyclotella cryptica by two distinct processes: (1) an increase in the proportion of newly assimilated carbons which are converted to lipids and (2) a slow conversion of previously assimilated carbon from non-lipid compounds to lipids.

Phosphorus starvation has also been used to induce lipid synthesis (Zhila et al. 2005; Weldy and Huesemann 2007). Khozin-Goldberg and Cohen (2006) found that phosphate limitation could cause significant changes in the fatty acid and lipid composition of Monodus subterraneus. However, in other species such as Nan — nochloris atomus and Tetraselmis sp., phosphorus deficiency led to reduced lipid content of the cells (Reitan et al. 1994).

Lipid synthesis may also be induced under other stress culture conditions such as high light intensity (Guedes et al. 2010; Khotimchenko and Yakovleva 2005; Qin 2005; Weldy and Huesemann 2007), low temperature (Renaud et al. 2002; Qin

2005) , high salinity (Kotlova and Shadrin 2003; Takagi et al. 2006; Qin 2005; Wu and Hsieh 2008), pH control (Guckert and Cooksey 1990), CO2 concentration (Chiu et al. 2009; de Morais and Costa 2007), and high iron concentration (Liu et al. 2008). The polyunsaturated fatty acid contents of microalgae tend to increase at low temperatures. The high PUFA content at low temperature might be explained by the need for the algae to produce more PUFAs to maintain cell membrane fluidity. Another reason might be that low temperature could lead to a high level of intracellular molecular oxygen and hence improve the activity of the desaturase and elongase involved in the biosynthesis of PUFAs (Jiang and Chen 2000).

Aside from lipid productivity, it is important to consider the quality of lipids produced by microalgae, since the quality of the oil influences the quality of the biodiesel. European Biodiesel standards (EN 14214 and 14213) limit the contents of fatty acid methyl esters with four and more double bonds to a maximum of 1 % (mol/mol). According to the EN 14214, for example, the linolenic acid (C18:3) should not exceed 12 % (mol/mol) (Knothe 2006). These oils require additional

treatment, such as partial catalytic hydrogenation (Dijkstra 2006). An advantage of microalgae oil is that the composition of the oils can be controlled by controlling the culture condition, as previously discussed. In some strains, it has been reported that nutrient limitation results in a change in lipid composition from free fatty acids to TAGs which are more suitable for biodiesel production (Widjaja et al. 2009). In some strains, nitrogen starvation might not result in an increase in total lipid content, but a change in lipid composition. Zhila et al. (2005) reported that nitrogen limitation increased oleic acid contents of Botryococcus braunii, yet the content of total lipids and triacylglycerols did not change. The saturation of fatty acids is directly dependent on the amount of excess sugar and the culture conditions (Tan and Johns 1991; Wood et al. 1999; Wen and Chen 2000a, b). Wood et al. (1999) noted that as the concentration of sugar increased, the fatty acid became more saturated. The type of nitrogen source also affects the quality of oil. In cultures of N. laevis, ammonia favored saturated and monounsaturated fatty acids (C14:0, C16:0, C16:1), and nitrate and urea promoted polyunsaturated fatty acids (C20:4 and C20:5) (Wen and Chen 2001a, b).

Genetic and metabolic engineering has high potentials for improving both the lipid quality and lipid productivity and therefore the economy of microalgae bio­diesel. Much work has been done on genetic engineering of cyanobacteria, and many functional genes have been successfully cloned in cyanobacteria (Qin et al. 1999). For example, Acc1 (a kind of restriction enzyme) has been cloned from Cylclotella cryptica (an oceanic diatom) for the production of biofuel (Roessler 1988). Genetic engineering of diatoms has increased the lipid contents from 5-20 % to over 60 % under laboratory conditions. The improvement of lipid content in genetically engineered microalgae is mainly due to the high expression of acetyl — coA carboxylase gene, which plays an important role in the control of the level of lipid accumulation (Huang et al. 2010). Due to the various advantages of hetero­trophic cultures, attempts have also been made to genetically modify obligate photoautotrophs for heterotrophic growth. For example, Glut 1 gene that encodes the glucose transporter protein was introduced into Phaeodactylum tricornutum (which is an obligate phototroph), thereby making it possible to grow it hetero — trophically (Zaslavskaia et al. 2001).