Heterotrophic growth of algae requires organic carbons, water, and inorganic salts. The growth, lipid content, and fatty acid composition are species/strain specific and can be greatly influenced by a variety of medium nutrients and environmental factors.

Carbon is the main component of algal biomass and accounts for ca 50% of dry weight. Sugars, particularly glucose, are the commonly used organic carbon sources for heterotro­phic growth of algae (Table 6.1). Different algae may prefer diverse sugars for heterotrophic growth. Liu et al (2010) studied the effect of various monosaccharides and disaccharides on growth of C. zofingiensis and found that glucose, fructose, mannose, and sucrose were effi­ciently consumed by the cells for rapid growth, whereas lactose and galactose were poorly assimilated and hardly supported the algal growth. In contrast, C. protothecoides may be un­able to directly assimilate sucrose, and pretreatment using invertase is required to release glu­cose and fructose (Yan et al., 2011). The growth, lipid content, and fatty acid profile of heterotrophically grown C. zofingiensis were slightly affected by the sugar species, namely, glucose, fructose, mannose, and sucrose (Liu et al., 2010) but were influenced to a large extent by the initial concentration of sugars (Liu et al., 2012a). Within the tested range of sugar con­centrations (5 to 50 g L-1), higher sugar concentrations gave C. zofingiensis higher cell density but at the same time lower specific growth rate (Figure 6.3a). The slow growth at high sugar concentrations is due likely to the substrate inhibition, a common issue confronted in batch cultures. High sugar concentrations also favored the intracellular lipid accumulation of C. zofingiensis, in which the lipid content at 30 g L-1 sugar was 0.5 g g-1, 79% greater than that at 5 g L-1 sugar (Figure 6.3b). In addition, the lipid distribution was found to be associated with sugar concentrations. Neutral lipid (NL) is the major lipid class, the proportion of which increased with increased sugar concentrations and could account for up to 85.5% of total lipids. Similar to NL, TAG levels were promoted by higher sugar concentrations (Figure 6.3c). In contrast, the membrane lipids phospholipid (PL) and glycolipid (GL) decreased in re­sponse to the increased sugar concentrations (Figure 6.3c). The fatty acid profiles of hetero­trophic C. zofingiensis were investigated in response to different sugar concentrations (Liu et al., 2012a). C16:0, C16:2, C18:1, C18:2, and C18:3 are the major fatty acids and represented more than 85% of total fatty acids. The levels of C16:0, C16:2, and C18:2 remained nearly unchanged under all tested sugar concentrations. In contrast, C18:1 and C18:3 levels were significantly affected: The former was promoted by higher sugar concentrations, whereas the latter by lower sugar concentrations. In addition, the content of total fatty acids based on dry weight ascended as the sugar concentration increased and could reach as high as 42.2%. Although the mechanism underlying sugar-induced lipid accumulation remains largely unknown, preliminary data suggested the involvement of glucose in triggering the great up-regulation of fatty acid biosynthetic genes, e. g., acetyl-CoA carboxylase and stearoyl-ACP desaturase (Liu et al., 2010; Liu et al., 2012b). Glucose catabolism provides not only energy for lipid/fatty acid synthesis but also acetyl-CoA, the direct precursor of fatty acids. The high sugar levels cause the formation of excess carbon for cell generation, and the carbon flux can be directed to lipid synthesis.

It is worth noting that some algal species prefer other carbon sources over glucose in het­erotrophic mode. For example, feeding pure acetic acid enabled Crypthecodinium cohnii to yield much higher productivity of docosahexaenoic acid (DHA) of 1,152 mg L-1 d-1; the su­periority of acetic acid to glucose might be because in this alga, the conversion of glucose to acetyl-CoA needs several steps, whereas acetate only needs a single-step action to be activated to acetyl-CoA directly by acetyl-CoA synthetase (de Swaaf et al., 2003). Another alternative carbon source, glycerol, has been commonly used for those algal species naturally occurring in habitats with high osmolarity, such as seawater or saline pounds (Neilson and Lewin,

FIGURE 6.3 (A) Growth, (B) lipid content, and

(C) lipid composition of C. zofingiensis with different йГ initial sugar concentrations. (△) specific growth rate; (□) dry weight; (white column) lipid content; (light gray column) neutral lipids; (gray column) phospho­lipids; (black column) glycolipids. The horizontal line inside the neutral lipids column marks the por­tion of TAG in this fraction. Adapted from Liu et al. (2012a) and the permission for reprint requested.

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1974), due possibly to that glycerol having the capability to raise the osmotic strength of the solution and consequently keep the osmotic equilibrium in cells (Perez-Garcia et al., 2011).

Nitrogen is the second main component of algal biomass. In autotrophic cultures, nitrogen is an important factor influencing intracellular lipid accumulation, and nitrogen limitation/ starvation is generally associated with the enhanced synthesis of lipids, in particular NL (Illman et al., 2000; Hsieh and Wu, 2009; Lacour et al., 2012). In heterotrophic cultures, nitro­gen availability also plays an important role in the profiles of lipids and fatty acids. A low level of nitrogen favors the accumulation of intracellular lipids (Scarsella et al., 2009; Xiong et al., 2010a). The heterotrophically grown Chlorella protothecoides produced 53.8% of lipids
(on a dry-weight basis) under nitrogen-limiting conditions—over two times of that under nitrogen-sufficient conditions (Xiong et al., 2010a). Nitrogen limitation also promoted carbo­hydrate synthesis but at the same time lowered the algal growth and protein level as well as the biomass growth yield coefficient on a glucose basis (Xiong et al., 2010a). The authors also analyzed the carbon flux by using 13C-tracer and GC-MS and indicated that C. protothecoides utilized considerably more acety-CoA for lipid synthesis under nitrogen-limiting conditions than under nitrogen-sufficient conditions (Xiong et al., 2010a). Considering that organic car­bons are used in heterotrophic cultures, the carbon/nitrogen (C/N) ratio, controlling the switch between protein and lipid syntheses, is usually employed to show the combined effect of carbon and nitrogen on lipid synthesis. Thus, it is the higher C/N ratios (corresponding to higher carbon concentrations when the initial nitrogen is fixed or lower nitrogen concentra­tions when the initial carbon is fixed) that trigger the accumulation of lipids, in particular the NLs. The NLs are likely from the excess carbon in the form of acetyl-CoA that enters the lipid synthetic pathway (Liu et al., 2012b) or from the transformation of chloroplast membrane lipids when nitrogen is depleted (Garcfa-Ferris et al., 1996). Up-regulation of enzymes in­volved in lipid biosynthesis, including acetyl-CoA carboxylase (ACCase), stearoyl-acyl car­rier protein desaturase (SAD), acyl-CoA:diacylglycerol acyltransferase (DGAT), and phospholipid:diacylglycerol acyltransferase (PDAT), was observed to be associated with lipid accumulation (Miller et al., 2010; Guarnieri et al., 2011; Boyle et al., 2012; Msanne et al 2012; Liu et al., 2012b). The enhanced lipid synthesis may be not only related to up- regulation of lipid-synthesizing enzymes under nitrogen limitation/starvation but also to the possible cessation of other enzymes associated with cell growth and proliferation (Ratledge and Wynn, 2002). For those reports that culture age affects lipid accumulation in algae (Liu et al., 2010; Liu et al., 2011b), the underlying reason maybe the nitrogen availability in that the aged cultures are accompanied by the depletion of nitrogen, which triggers the accumulation of lipids.

In addition to nitrogen availability, nitrogen sources have been demonstrated to influence the growth and biochemical composition of heterotrophic algae. Algae can utilize various forms of nitrogen, e. g., nitrate, ammonia, urea, glycine, yeast extract, and tryptone (Vogel and Todaro, 1997; Shi et al., 2000; Hsieh and Wu, 2009; Yan et al., 2011). Both nitrate-N and urea-N cannot be directly incorporated into organic compounds but have to be first re­duced to ammonia-N. Ammonia and urea are economically more favorable as nitrogen sources than nitrate in that the latter is more expensive per unit N. The uptake of ammonia results in acidification of the medium, and nitrate causes alkalinization, whereas urea leads to only minor pH changes (Goldman and Brewer, 1980). In this context, urea is the better choice of nitrogen source for avoiding large pH shifts of unbuffered medium. Shi et al (2000) reported the severe drop in culture pH (below 4) of heterotrophic C. protothecoides with am­monia, which resulted in much lower biomass yield compared to with urea or nitrate. Differ­ent algal species may favor different nitrogen sources for growth. For example, Chlorella pyrenoidosa preferred urea to nitrate or glycine for growth, whereas C. protothecoides gave a higher biomass yield when fed nitrate rather than urea (Davis et al., 1964; Shen et al.,

2010) . Those mutants deficient in nitrate/nitrite reductases have to use ammonia for growth (Dawson et al., 1997; Burhenne and Tischner, 2000).

Nitrogen limitation is not always linked to lipid accumulation in algae, e. g., the diatoms Achnanthes brevipes and Tetraselmis spp. accumulated carbohydrates rather than lipids upon nitrogen starvation (Gladue and Maxey, 1994; Guerrini et al., 2000). Diatoms need silicate for growth, and silicate metabolism in diatoms has been reviewed by Martin-Jezequel et al. (2000). In general, silicate limitation/starvation is associated with the enhanced synthesis of lipid in diatoms (Lombardi and Wangersky, 1991; Wen and Chen, 2000). In addition, the content of polyunsaturated fatty acids (e. g., EPA) increased with the depleted silicate (Wen and Chen, 2000). This may be explained by the finding that the silicate-limited diatom cells divert the energy allocated for silicate uptake when silicate is replete into energy storage lipids. Phosphorus plays an important role in the energy transfer of the algal cells as well as in the syntheses of phospholipids and nucleic acids. It was also reported that phosphorus de­ficiency promoted the accumulation of lipids in certain algae (Lombardi and Wangersky, 1991; Scarsella et al., 2009).

Aside from the medium nutrients, environmental factors play an important role in influencing the heterotrophic growth and lipid profile of algae, including but not restricted to temperature, pH, salinity, dissolved oxygen level, dilution rates, and turbulence (Chen and Johns, 1991; Jiang and Chen, 2000a, b; Chen, et al., 2008; Pahl et al., 2010; Ethier et al., 2011). When temperature shifts, the algae need to alter the thermal responses of membrane lipids to maintain the normal function of membranes (Somerville, 1995). Many studies have proved that in heterotrophic mode, a low temperature can induce the generation of unsaturated fatty acids, and vice versa (Wen and Chen, 2001a; Jiang and Chen, 2000a). There are two possible explanations: (1) a reduction in temperature leads to the decreased membrane fluidness; as a result, the algae need to speed up the desaturation of lipids as a compensation to maintain the proper cell membrane fluidity via the up-regulation of desaturase genes (Perez-Garcia et al.,

2011) ; and (2) the low temperature gives rise to more intracellular molecular oxygen and con­sequently improves the activities of desaturases and elongases that are involved in the bio­synthesis of unsaturated fatty acids (Chen and Chen, 2006). The high salinity was found to enhance the lipid accumulation in Nitzschia laevis in heterotrophic mode. Upon changing the concentration of NaCl in the medium from 10 to 20 g L-1, an increase in EPA and polar lipids was observed, accompanied by a slight decline of NLs (Chen et al., 2008). The sufficient oxygen supply is important for algal growth, especially in high cell density fermentation. Chen and Johns (1991) reported that in the heterotrophic culture of Chlorella sorokiniana, a high concentration of dissolved oxygen improved the cell growth as well as the fatty acid yield. The effect of pH on growth and lipids of Crypthecodinium cohnii was reported by Jiang and Chen (2000b), where the highest DHA content was obtained at pH 7.2.

As such, the optimization of nutritional and environmental factors is of great importance to the development of a high-yield lipid production system by heterotrophic algae. The com­monly used approaches for the production optimization are one-at-a-time and statistical methods (Kennedy and Krouse, 1999). The one-at-a-time strategy involves variation of one factor within a desired range while keeping other factors constant (Wen and Chen, 2000; Pahl et al., 2010; Liu et al., 2012a). This strategy is simple and easy to conduct and thus has been widely used for optimizing the production of biomass and desired products. However, the one-at-a-time method has its intrinsic disadvantages, e. g., failing to consider the interactions among factors and requiring a relatively large number of experiments. To overcome these problems, a good choice is the statistical approach-based optimization, which requires three steps: design, optimization, and verification (Kennedy and Krouse, 1999). The raw data obtained after experimental design can be transformed to models or

three-dimensional plots, based on which the optimal factors can be predicted. A verification experiment needs to be conducted to validate the predication. The statistical approach-based optimization has been applied to microalgae for the heterotrophic production of biomass and desired products, e. g., polyunsaturated fatty acid production by N. laevis (Wen and Chen, 2001a), biomass production by Tetraselmis suecica (Azma et al., 2011), and lipid production by Chlorella saccharophila (Isleten-Hosoglu et al., 2012).