In comparison to photoautotrophy, heterotrophic growth mode offers substantial advan­tages, e. g., elimination of the light requirement, ease of control for monoculture, high cell density, and great biomass productivity (Chen, 1996). Lab-scale heterotrophic production of algae has been reported in recent decades, either in shaking flasks or in small-volume fermen­ters (Cheng et al., 2009; Liang et al., 2009; Liu et al., 2010, 2011b; Yan et al., 2011). Liang et al (2009) examined the growth of Chlorella vulgaris under both phototrophic and heterotrophic conditions and indicated heterotrophic C. vulgaris had around threefold higher biomass yield than a phototrophic one. Liu et al (2011b) investigated the growth of Chlorella zofingiensis; the alga achieved 10.1 g L-1 of cell density under heterotrophic conditions compared to 1.9 g L-1 under phototrophic conditions. Chlorella protothecoides, another well-studied green alga, was reported to achieve as high as up to 17 g L-1 of cell density in heterotrophic batch cultures (Cheng et al., 2009). This may be further improved through using culture techniques such as fed-batch, chemostat, and cell recycling, which have been widely used for fermentation of bacteria or yeasts. For example, the fed-batch C. protothecoides achieved a high cell density of 97 g L-1 in a 5-L fermenter (Yan et al., 2011), much higher than that obtained in photoau­totrophic culture systems (open ponds or photobioreactors) and close to the yeast yield

TABLE 6.1 Algae Reported with Heterotrophic Growth. Algae Carbon Sources References Green algae Chlamydomonas reinhardtii Acetate Chen and Johns, 1994, 1996; Zhang et al., 1999a Chlorella minutissima Glucose, starch, sucrose, glycine, acetate, glycerin Li et al., 2011 Chlorella protothecoides Glucose, glycerol, hydrolyzed carbohydrates, molasses, municipal wastewater Zhang et al., 1999b; Miao and Wu, 2006; Cheng et al., 2009; Ruiz et al., 2009; Gao et al., 2010; O’Grady and Morgan, 2011; Yan et al., 2011; Chen and walker, 2012; Zhou et al. 2012 Chlorella pyrenoidosa Glucose Running et al., 1994 Chlorella regularis Glucose, acetate Endo et al., 1977; Sansawa and Endo, 2004 Chlorella saccharophila Glucose, glycerol Tan and Johns, 1991; Isleten-Hosoglu et al., 2012 Chlorella sorokiniana Glucose Chen and Johns, 1991; Zheng et al., 2012 Chlorella vulgaris Agro-industrial co-products, glucose, sucrose, acetate, glycerol Rattanapoltee et al., 2008; Mitra et al., 2012; Liang et al., 2009; Scarsella et al., 2009 Chlorella zofingiensis Glucose, fructose, mannose, sucrose, molasses Ip and Chen, 2005; Liu et al., 2010, 2011b, 2012a Haematococcus lacustris Glucose Chen et al., 1997 Haematococcus pluvialis Acetate Kobayashi et al., 1992 Micractinium pusillum Glucose, acetate Bouarab et al., 2004 Pseudococcomyxa chodatii Glucose Kiseleva and Kotlova, 2007 Tetraselmis suecica Glucose, acetate Day and Tsavalos, 1996; Azma et al., 2011 Diatom Cyclotella cryptica Glucose Pahl et al., 2010 Nitzschia laevis Glucose Wen and Chen, 2001a, b; Chen et al., 2008 Others Aphanothece microscopica Fish processing wastewater Queiroz et al., 2011 Crypthecodinium Cohnii Glucose Couto et al., 2010; Jiang et al., 1999; Jiang and Chen, 2000a, b Galdieria sulphuraria Glucose Schmidt et al., 2005; Sloth et al., 2006 Ochromonas danica Phenolic mixtures Semple, 1998 Schizochytrium limacinum Glycerol Ethier et al., 2011 Schizochytrium mangrovei Glucose Fan et al., 2007 Schizochytrium sp. Glucose Ganuza et al., 2008 Spirulina sp. Glucose Chojnacka and Noworyta, 2004 Spongiococcum exetricicum Glucose Hilaly et al., 1994 Synechocystis sp. Glucose Kong et al., 2003

FIGURE 6.1 Central carbon metabolism of microalgae in heterotrophic cultures based on glucose. Glu, Glucose; G6P, Glucose-6-Phosphate; F6P, Fructose-6-Phosphate; GAP, Glyceraldehyde-3-Phosphate; G3P, 3-Phosphoglycerate; PEP, Phosphoenolpyruvate; Pyr, Pyruvate; AcCoA, Acetyl — CoA; ICT, Isocitrate; AKG, a-Ketoglutarate; Suc, Succinyl — CoA; Fum, Fumarate; Mal, Malate; OAA, Oxalacetate; Ru5P, Ribulose-5-Phosphate; R5P, Ribose-5-Phosphate; X5P, Xyluose-5-Phosphate; E4P, Erythrose-4-Phosphate; S7P, Sedoheptulose-7-Phosphate; Glu, Glutamate; Gln, Glutamine. For details of the reactions with numbers, see Table 6.2.

Stoichiometric Reactions.

Glycolytic pathway

Glc + ATP => G6P + ADP + H 1

G6P <=> F6P 2

F6P + ATP => 2GAP + ADP + H 3

2GAP + H2O => F6P + Pi 4

GAP + NAD + Pi + ADP <=> G3P + ATP + NADH + H 5

G3P <=> PEP + H2O 6

PEP + ADP => Pyr + ATP 7

Pyr + NAD + CoA => AcCoA + NADH + CO2 + H 8

PEP + CO2 + ADP => OAA + ATP 9

Stoichiometric Reactions—Cont’d Tricarboxylic acid cycle

OAA + AcCOA + H2O <=> ICT + CoA + H 10

ICT + NAD <=> AKG + NADH + CO2 11

AKG + CoA + NAD => Suc + NADH + CO2 + H 12

Suc + ADP + P; + FAD <=> Fum + FADH2 +ATP + CoA 13

Fum <=> Mal 14

Fum + NAD + H2O <=> OAA + NADH + H 15

Pentose phosphate pathway

G6P + 2NADP + H2O => Ru5P + CO2 + 2NADPH + 2H 16

Ru5P <=> R5P 17

Ru5P <=> X5P 18

R5P + X5P <=> S7P + GAP 19

S7P + GAP <=> F6P + E4P 20

X5P + E4P <=> F6P + GAP 21

Utilization of nitrogen

AKG + NADPH + Gln => 2Glu + NADP 22

Glu + NH3 + ATP => Gln + ADP + Pi 23

(Li et al., 2007b; Kurosawa et al., 2010; Zhang et al., 2011). Although the growth and biomass production of algae are species/strain dependent and may vary greatly, the overall bio­mass yield and productivity of heterotrophic algae are significantly higher than those of phototrophic ones, as illustrated by Figures 6.2a and 6.2b.

Heterotrophic culture of algae offers not only high cell density but also high level of oils. The lipid contents of alga cultured heterotrophically were shown in Table 6.3. The lipid content varies from 4.8% to 60% of dry weight, depending on the algal species/strains and culture con­ditions. Commonly, stresses such as high light intensity and/or nitrogen starvation are re­quired to induce intracellular oil accumulation of algae under photoautotrophic conditions. These stresses, however, are unfavorable for algal growth and biomass production, causing the contradiction between growth and oil synthesis. In contrast, the heterotrophic algae are able to accumulate oil while simultaneously building up biomass; for example, the intracellular oil content of C. zofingiensis increased from 0.25 to 0.5 g g-1 (on a dry-weight basis) when the cell density increased from 5 to 42 g L-1 (Liu et al., 2010). The accumulated oil contains mainly neu­tral lipids, in particular triacylglycerol (TAG). The TAG may account for up to 80% of neutral lipids or 71% of total lipids (Liu et al., 2011b). TAG is regarded as superior to polar lipids (phos­pholipids and glycolipids) for biodiesel production due to its higher content of fatty acids. Taking into account the rapid growth and abundance of oils, heterotrophic algae usually allow

FIGURE 6.2 Biomass (a, b) and oil (c, d) productivities of phototrophic (open) and heterotrophic (filled) algae, based on the data of research articles published in the past decade. The differences in biomass and oil productivities between cultures under phototrophic and heterotrophic growth conditions were statistically significant using Duncan’s multiple-range test with the ANOVA procedure.

TABLE 6.3 Oil Content of Heterotrophic Algae. Algae Oil Content (% Dry Weight) References Green algae Chlorella minutissima 16.1 Li et al., 2011 Chlorella protothecoides 44.3-48.7 Li et al., 2007a Chlorella protothecoides 44 Cheng et al., 2009 Chlorella protothecoides 52.5 Gao et al., 2010 Chlorella protothecoides 58.9 O’Grady and Morgan, 2011 Chlorella protothecoides 32 Chen and Walker, 2012 Chlorella protothecoides 49.4 De la Hoz Siegler et al., 2012 Chlorella protothecoides 28.9 Zhou et al., 2012 Chlorella saccharophila 26.7-36.3 Isleten-Hosoglu et al., 2012 Chlorella sorokiniana 20.1-46 Chen and Johns, 1991 Chlorella sorokiniana 23.3 Zheng et al., 2012

TABLE 6.3 Oil Content of Heterotrophic Algae—Cont’d Algae Oil Content (% Dry Weight) References Chlorella vulgaris 23-34 Liang et al., 2009 Chlorella vulgaris 32.9 Rattanapoltee et al., 2008 Chlorella vulgaris 35-58.9 Scarsella et al., 2009 Chlorella vulgaris 11-43 Mitra et al., 2012 Chlorella zofingiensis 52 Liu et al., 2010 Chlorella zofingiensis 51.1 Liu et al., 2011b Chlorella zofingiensis 48.9 Liu et al., 2012a Diatoms Cyclotella cryptica 4.8-7.4 Pahl et al., 2010 Nitzschia laevis 12.8 Chen et al., 2008 Others Aphanothece microscopica 7.1-15.3 Queiroz et al., 2011 Crypthecodinium Cohnii 19.9 Couto et al., 2010 Schizochytrium limacinum 50.3 Ethier et al., 2011 Schizochytrium mangrovei 68 Fan et al., 2007 Schizochytrium sp. 35 Ganuza et al., 2008

a high volumetric oil productivity (Figures 6.2c and 6.2d), e. g., 7.3 g L-1 day-1 in the case of C. protothecoides under fed-batch culture conditions (Yan et al., 2011). The fatty acid character­istics of oils, e. g., carbon chain length and unsaturation degree, largely determine the properties of biodiesel such as cetane number, viscosity, cold flow, and oxidative stability (Knothe, 2005). Although the fatty acid species of algae grown heterotrophically may show few differences in comparison to photoautotrophy, the proportions of individual fatty acid vary greatly. Liu et al. (2011b) investigated the fatty acid profiles of C. zofingiensis and indicated that heterotrophic cells contained low levels of C16:0, C16:3, C18:0, and C18:3 but much higher content of C18:1 than autotrophic cells. The proportion of C18:1 is regarded as an important factor for bio­diesel quality because it can provide a compromise solution between oxidative stability and low-temperature properties (Knothe, 2009). The higher the C18:1 content, the better the biodie­sel quality. The biodiesel derived from heterotrophic algae was analyzed with respect to the key properties (e. g., energy density, viscosity, flash point, cold filter plugging point, and acid value), and the results showed that most properties complied with the specifications established by the American Society for Testing and Materials (Xu et al., 2006).

In addition to the lab-scale cultures, many attempts have been made to develop industrial — scale processes for the heterotrophic cultivation of algae. The heterotrophic Chlorella cultures have long been initiated in Japan and Taiwan in the late 1970s; Chlorella species were cultured in stainless steel tanks using glucose and/or acetate as carbon and energy sources, with an annual production of 1,100 tons biomass (Lin, 2005). Thereafter, large-scale heterotrophic 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), and Spongiococcum exetriccium fed-batch cultured in 450-L fermen­ters (Hilaly et al., 1994), though these cultures were used not for oils but for high-value prod­ucts. Recently, a scale-up heterotrophic cultivation of C. protothecoides was reported for oil production in 11,000-L fermenters, where the daily biomass production of 20 kg and oil pro­duction 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 rel­atively 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 or­ganic 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 an­alyses. For example, Vazhappilly and Chen (1998) intensively studied the heterotrophic poten­tial 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 poten­tial. In some cases, the obligate photoautotrophic algae can be metabolic engineered to grow heterotrophically. Zaslavskaia et al (2001) reported that a genetically modified Phaeodactylum tricornutum, through introducing a gene encoding a glucose transporter, was capable of thriv­ing on exogenous glucose in the absence of light, suggesting an alternative approach to increas­ing 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 oper­ation 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 attrib­uted to the high initial concentration of substrates (e. g., sugars) or the possible buildup of cer­tain inhibitory substances produced by algae during culture periods. For example, the sugar concentration of over 20 g L-1 was reported to inhibit the growth of C. 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 mo­lasses (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).