cultivation of several other algal strains were reported, for example,Tetraselmis suecica in 50,000-L fermenters (Day et al., 1991), Crypthecodinium cohnii with a capacity of 150,000 L (Radmer and Fisher, 1996), andSpongiococcum exetricciumfed-batch cultured in 450-L fermen- ters (Hilaly et al., 1994), though these cultures were used not for oils but for high-value prod- ucts. Recently, a scale-up heterotrophic cultivation ofC. protothecoideswas reported for oil production in 11,000-L fermenters, where the daily biomass production of 20 kg and oil pro- duction of 8.8 kg were achieved (Li et al., 2007a).
Because of the elimination of light requirements and sophisticated fermentation systems that have developed, the scale-up of heterotrophic cultures for high cell density and oil yield is rel- atively easier to achieve than that of autotrophic cultures. The production of heterotrophic algal cultures, however, is restricted, due largely to (1) the limited number of available heterotrophic species, (2) possible contamination by bacteria or fungi, (3) inhibition of growth by soluble or- ganic substrates (e.g., sugar) at high concentrations, and (4) the relatively high cost of organic carbon sources. The first limitation might be overcome by performing extensive screening an- alyses. For example,Vazhappilly and Chen (1998)intensively studied the heterotrophic poten- tial of 20 algal strains and suggested that 6 of them showed good heterotrophic growth. As the screening expands, increasing algal species/strains will be identified with heterotrophic poten- tial. In some cases, the obligate photoautotrophic algae can be metabolic engineered to grow heterotrophically.Zaslavskaia et al (2001)reported that a genetically modifiedPhaeodactylum tricornutum,through introducing a gene encoding a glucose transporter, was capable of thriv- ing on exogenous glucose in the absence of light, suggesting an alternative approach to increas- ing the available number of heterotrophically grown algae. The second problem is due mainly to the relatively slow growth of algae compared with other microorganisms such as bacteria or yeast that grow fast and finally dominate the cultures. Rigorous sterilization and aseptic oper- ation are necessary and considered to be effective to circumvent such possible contamination.
Growth inhibition is a common problem occurring in batch cultures, which has restricted the use of batch cultures in commercial production processes. The growth inhibition may be attrib- uted to the high initial concentration of substrates (e.g., sugars) or the possible buildup of cer- tain inhibitory substances produced by algae during culture periods. For example, the sugar concentration of over 20 g L–1was reported to inhibit the growth ofC. zofingiensis(Liu et al., 2010, 2012a). Advances in heterotrophic culture systems may eliminate or reduce the growth-inhibition problems, where fed-batch, chemostat, and cell recycle have been intensively investigated (Wen and Chen, 2002a; De la Hoz Siegler et al., 2011; Liu et al., 2012a). The organic carbon sources—in particular, glucose—account for the major cost of a culture medium and contribute to the relatively high cost of heterotrophic production, which makes the algal oils from heterotrophic cultures less economically viable than those from autotrophic cultures.
Cheap alternatives are sought with the goal of bringing down production costs, e.g., waste mo- lasses (Yan et al., 2011; Liu et al., 2012a), carbohydrate hydrolysate (Cheng et al., 2009; Gao et al., 2010), and biodiesel byproduct glycerol (O’Grady and Morgan, 2011).
6.4 FACTORS AFFECTING HETEROTROPHIC
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 heterotro- phic 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 ofC. zofingiensisand found that glucose, fructose, mannose, and sucrose were effi- ciently consumed by the cells for rapid growth, whereas lactose and galactose were poorly assimilated and hardly supported the algal growth. In contrast,C.protothecoidesmay be un- able to directly assimilate sucrose, and pretreatment using invertase is required to release glu- cose and fructose (Yan et al., 2011). The growth, lipid content, and fatty acid profile of heterotrophically grownC. zofingiensiswere 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 con- centrations (5 to 50 g L–1), higher sugar concentrations gaveC. zofingiensishigher 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–1sugar was 0.5 g g–1, 79% greater than that at 5 g L–1sugar (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 re- sponse to the increased sugar concentrations (Figure 6.3c). The fatty acid profiles of hetero- trophic 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 het- erotrophic mode. For example, feeding pure acetic acid enabledCrypthecodinium cohnii to yield much higher productivity of docosahexaenoic acid (DHA) of 1,152 mg L–1d–1; the su- periority 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,
120 6. HETEROTROPHIC PRODUCTION OF ALGAL OILS
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, nitro- gen 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 protothecoidesproduced 53.8% of lipids
12
1.0
0.8
0.6 8
4
A 0
B
C 0.6
0.4
0.2
0.0 100 80 60 40 20 0
5 10 15 20 30 40 50
Sugar concentration (g L-1)
Lipid distribution (% total lipdis)Lipid content (g g-1)Dry weight (g L-1) Specific growth rate (day-1) FIGURE 6.3 (A) Growth, (B) lipid content, and
(C) lipid composition ofC. zofingiensiswith different initial sugar concentrations. (△) specific growth rate;
(□) dry weight; (white column) lipid content; (light gray column) neutral lipids; (gray column) phospho- lipids; (black column) glycolipids. The horizontal line inside the neutral lipids column marks the por- tion of TAG in this fraction.Adapted from Liuet al.
(2012a) and the permission for reprint requested.
121
6.4 FACTORS AFFECTING HETEROTROPHIC PRODUCTION OF ALGAL OILS
(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 carbo- hydrate 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 using13C-tracer and GC-MS and indicated thatC. 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 car- bons 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 concentra- tions 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 (Garcı´a-Ferris et al., 1996). Up-regulation of enzymes in- volved in lipid biosynthesis, including acetyl-CoA carboxylase (ACCase), stearoyl-acyl car- rier 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 may be 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 re- duced 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 heterotrophicC. protothecoideswith am- monia, which resulted in much lower biomass yield compared to with urea or nitrate. Differ- ent algal species may favor different nitrogen sources for growth. For example,Chlorella pyrenoidosapreferred urea to nitrate or glycine for growth, whereasC. 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 brevipesandTetraselmisspp. accumulated carbohydrates rather than lipids upon
122 6. HETEROTROPHIC PRODUCTION OF ALGAL OILS
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 de- ficiency 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 con- sequently improves the activities of desaturases and elongases that are involved in the bio- synthesis 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 ofChlorella 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 ofCrypthecodinium cohniiwas reported byJiang 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 com- monly 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 123
6.4 FACTORS AFFECTING HETEROTROPHIC PRODUCTION OF ALGAL OILS
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 byN. laevis(Wen and Chen, 2001a), biomass production byTetraselmis suecica(Azma et al., 2011), and lipid production byChlorella saccharophila(Isleten-Hosoglu et al., 2012).