Lipid accumulation

A given alga exhibits different lipid metabolism under different growth conditions. For example, the level of nutrients in the growth medium, the irradiance, the temperature, CO2 supplementation and salinity influence lipid accumulation and the types of lipids

accumulated (Shifrin and Chisholm, 1981; Roessler, 1990). These culture conditions determine the quality and amount of lipid produced (Hu et al., 2008; Rodolfi et al., 2009; Pruvost et al., 2011). Different algal species may respond differently to stressors (Shifrin and Chisholm, 1981). From the perspective of biofuels, modifications in the culture conditions have been used to enhance the lipid contents of the biomass (Illman et al., 2000; Liu et al., 2008; Mazzuca Sobczuk and Chisti, 2010). Lipid content and composition can also vary significantly in different phases of growth (Hu et al., 2008); under favorable growth conditions, more polar lipids may be synthesized (Cagliari et al., 2011) and less TAGs (Hu, 2004). Under stationary phase of growth or adverse growth conditions, production of neutral lipids in the form of TAGs is enhanced (Berge et al., 1995; Tonon et al., 2002; Hu et al., 2008).

The various factors that trigger lipid accumulation and affect its composition have been reviewed for certain algae (Hu et al., 2008; Leonardi et al., 2011; Sharma et al., 2012). Some of these factors are discussed briefly in the following sections.

23 2.4.1.1 Effect of nutrient starvation

Nutrient starvation can trigger lipid accumulation. Several nutrients including nitrogen, phosphorus, silicon and sulfur are known for inducing lipid accumulation (Sharma et al., 2012). Nitrogen deprivation has often been found to greatly increase the lipid content of many microalgae (Hsieh and Wu, 2009; Yeh and Chang, 2011; Praveenkumar et al., 2012). In addition to the concentration of nitrogen, the source of nitrogen also impacts the accumulation of lipids (Hsieh and Wu, 2009; Yeh and Chang, 2011). The first reported study on lipid accumulation under variable nitrate concentration was by Spoehr and Milner (1949) for Chlorella pyrenoidosa. An increase in lipid content of up to 85% was recorded. Extensive work has been reported on the effect of nitrogen deficient conditions on lipid accumulation in green microalgae (Largeau et al., 1981; Piorreck et al., 1984; Sawayama et al., 1992; Illman et al., 2000; Scragg et al., 2002; Khozin-Goldberg and Cohen, 2006; Griffiths and Harrison, 2009; Hsieh and Wu, 2009; Widjaja et al., 2009; Gardner et al., 2009; Chen et al., 2011; Praveenkumar et al., 2012) . Diverse responses are sometimes seen within different species of a given genus. For example,most Chlorella species accumulate neutral lipids under N starvation, but some species accumulate starch (Hu, 2004). In Tetraselmis suecica and some Dunaliella species, no change in lipids levels has been seen under N starvation (Borowitzka, 1988). Nannochloropsis species have been extensively studied for lipid production (Boussiba et al., 1987; Suen et al., 1987; Sukenik et al., 1989; Chini Zittelli et al., 1999; Hu and Gao, 2006). Except for Nannochloropsis salina (Boussiba et al., 1987), members of this genus have often been reported to accumulate lipids under a combination of nitrate depletion condition and high irradiance.

Diatoms (golden or brown algae) usually show multiple responses with regards to nitrate deprivation (Shifrin and Chisholm, 1981; Benemann and Oswald, 1996). Some

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diatoms show an increased lipid content, specifically the TAGs, under N deprivation. Examples of such diatoms are Cyclotella cryptica, Nitzschia palea, Navicula pelliculosa (Shifrin and Chisholm, 1981), Phaeodactylum tricornutum (Parrish and Wangersky, 1987), and Chaetoceros muelleri (McGinnis et al., 1997). Other diatoms may increase the lipid level only a little on N starvation. This happens in Skeletonema costatum, for example. Yet other diatoms, e.g. Synedra ulna and Biddulphia aurita, may show no increase on N starvation while in species such as Thalassiosira weissflogii nitrate starvation may actually decrease the lipid content (Shifrin and Chisholm, 1981). The silicate starvation has resulted in lipid content increase in diatoms (Shifrin and Chisholm, 1981). This happens, for example, in the diatoms Cyclotella cryptica (Werner, 1966; Shifrin and Chisholm, 1981; Roessler, 1990; Griffiths and Harrison, 2009), Amphiprora hyalina, Chaetoceros muelleri, Nitzschia dissipata and several species of Navicula (Griffiths and Harrison, 2009). In the case of Cyclotella cryptica, different silicon stress levels enhanced lipid accumulation significantly. Production of TAGs as well as saturated and monounsaturated fatty acids was also enhanced (Roessler, 1990).

A deficiency of other nutrients may also trigger lipid accumulation. For example, deficiency of phosphorus and sulfur may do this in some but not all algae. Phosphate deficiency induced lipid accumulation in Scenedesmus obliquus (Mandal and Mallick, 2009) and fatty acid production in Dunaliella tertiolecta (Siron et al., 1989), but decreased the lipid level in Nannochloris atomus and Tetraselmis sp, (Reitan et al., 1994). Deficiency of sulfur caused an increase in lipid content of Chlorella sp. and Chlamydomonas reinhardtii (Matthew et al., 2009). A deficiency or excess of micronutrients also influence lipid accumulation. For example, Fe3+ supplementation in certain stages of growth has increased the lipids content of Chlorella vulgaris (Liu et al., 2008).

25 In diatoms, phosphorus limitation increased the lipid content in Phaeodactylum tricornutum (Siron et al., 1989; Valenzuela et al., 2013), Chateoceros sp. (Reitan et al., 1994) and Chaetoceros gracilis (Lombardi and Wangersky, 1995).

2.4.1.2 Light stress

The level of light (both the extent of the photoperiod and the intensity of light) (Brenckmann et al., 1985) also influence lipid production. A high irradiance induces neutral lipid production (Spoehr and Milner, 1949; Orcutt and Patterson, 1974; Sukenik et al., 1989; Roessler, 1990; Napolitano, 1994; Brown et al., 1996) as they tend to protect the cell from photooxidative stress. The latter is usually associated with the production of secondary carotenoids (Rabbani et al., 1998; Zhekisheva et al., 2002). A low irradiance level increases the synthesis of membrane lipids such as glycolipids and phospholipids (Hu et al., 2008). In sunlight driven production processes the light level is not readily manipulated, unfortunately.

2.4.1.3 Temperature stress

Temperature is known to influence the lipid content and composition (Roessler, 1990; Hu, 2004; Guschina and Harwood, 2006; Hu et al., 2008), but no generalized patterns have been identified with respect to the effects of temperature (Hu et al., 2008). This is because of a relative lack of studies in this area possibly because temperature is not easily controlled in many outdoor large-scale commercial culture operations. Some algae have been reported to increase lipid content with increasing temperature. This occurs in the range of 15-30 °C in Ochromonas danica (Aaronson, 1973); in the range of 17-35 °C in Nannochloropsis salina (Boussiba et al., 1987); in the range of 20-25 °C in Nannochloropsis oculata; and in the range of 25-30 °C in Chlorella vulgaris

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(Converti et al., 2009). Lipid content of Chlorella sorokiniana has not shown any obvious effect under variable temperatures of 14 °C, 22 °C and 38 °C.

Interactive effects of temperature and light intensity have been reported (Sorokin and Krauss, 1962; Collins and Boylen, 1982).

2.4.1.4 Salinity stress

The effect of salinity on lipid accumulation has been reported for some green algae (Sonnekus, 2010). In some cases, salinity changes had no significant effect on lipid accumulation but affected the lipid composition (Vazquez-Duhalt and Arredondo-Vega, 1990; 1991). Dunaliella tertiolecta has been found to increase its lipid content with increasing salinity (Takagi et al., 2006). Lipid contents of Isochrysis sp. and Nannochloropsis oculata (Prymnesiophyceae and Eustigmatophyceae, respectively) were also elevated by increasing salinity (Renaud and Parry, 1994). Lipid contents of some diatoms declined at salinity of more than 35 ppt (Renaud and Parry, 1994; Sonnekus, 2010).

As outlined above, many factors can be used to influence lipid accumulation in microalgae. Some of these factors (e.g. temperature) are not easily manipulated in large scale outdoor operations, or are expensive to manipulate. Factors such as N starvation are broadly applicable, easy to control and may actually reduce the cost of producing the algal oils. What specific factors are used to influence oil productivity would depend very much on the specific alga (Pruvost et al., 2011).