Although heterotrophy of algae shows its potential for oil production, the overall produc­tion cost of heterotrophic oils remains relatively high, restricting the commercialization of heterotrophic algal oils. From an estimation of Yan et al. (2011) using heterotrophic C. protothecoides for oil production, the unit production cost of algal oils was still much higher than that of plant oils. Glucose represents a major share of the cost of heterotrophic oil pro­duction. Using alternative low-cost carbon sources may represent a promising approach to bring down the cost of heterotrophic algal oils. Recently, it has been reported that low-cost sugars were used to grow algae for heterotrophic oil production, e. g., hydrolyzed carbohy­drates (Xu et al., 2006; Cheng et al., 2009; Gao et al., 2010) and waste molasses (Yan et al., 2011; Liu et al., 2012a). All these reports suggested the potential of producing algal oils for less cost, such that the algal oils from C. protothecoides based on waste molasses cost approx­imately half those based on glucose (Yan et al., 2011).

The heterotrophic utilization of sugars for biomass by algae remains at a relatively low level, namely, below 0.5 (Cheng et al., 2009; Liu et al., 2010; Yan et al., 2011), which means that more than 50% of sugars were wasted in the form of CO2. To increase the sugar-to- biomass conversion, a photosynthesis-fermentation mode was proposed and resulted in a high sugar-to-biomass conversion of 0.62 (Xiong et al., 2010b). The increased sugar — conversion efficiency may be attributed to the refixation of partial CO2 released from sugar catabolism by the enzyme RuBisCO, which maintained its carboxylation activity in the fer­mentation stage (Xiong et al., 2010b). In a fermentation system, productivity is greatly related to the medium nutrients as well as fermentation parameters. The manipulation of these factors to achieve a maximized output/input ratio may have great potential for improving production economics of heterotrophic algal oils.

Heterotrophic algal biomass contains not only oils but also substantial amounts of proteins and carbohydrates as well as high-value components such as pigments and vitamins. From a biorefinery’s point of view, the residual biomass after oil extraction can be potentially used as food additives, nutraceuticals, and animal feed (Figure 6.8). Also, carbohydrates may be uti­lized for producing the bio-gas methane by anaerobic digestion. The integrated production of oils and other value-added production, coupled with the possible recycling of water and nu­trients, remains a potential strategy to reduce the production cost of algal oils.

Strain improvement by genetic engineering is another feasible and complementary approach to enhancing algal productivity and improving the economics of algal oil produc­tion. Introduction of a bacterial hemoglobin in various hosts has been shown to contribute to growth improvement in oxygen-limited conditions (Zhang et al., 2007). This strategy is par­ticularly suitable for heterotrophic growth of algae to achieve the ultrahigh cell density that may be restricted by the lowered dissolved oxygen associated with cell mass buildup. The­oretically, enhanced oil content can be achieved by the direct genetic engineering of oil bio­synthetic pathways, e. g., overexpression of the genes involved in fatty acid/lipid synthesis (Madoka et al., 2002; Lardizabal et al 2008); the manipulation of transcriptional factors

related to lipid biosynthesis regulation (Courchesne et al., 2009); or the blocking of compet­ing metabolic pathways that share the common carbon precursors such as starch synthesis (Li et al., 2010). Genetic engineering can also be employed to alter fatty acid compositions of oils for improving biofuel quality, e. g., heterologous expression of thioesterases to accumu­late shorter-chain-length fatty acids (Radakovits et al., 2011) or inactivation of the A12 desaturase gene to produce more oleic acid (Graef et al., 2009). In addition, genetic engineer­ing may confer on algae the possibly improved characteristics of tolerance of temperature, salinity, and pH, which will allow cost reduction in algal biomass production and be ben­eficial for growing selected algae under extreme conditions that limit the proliferation of invasive species. Although genetic engineering of algal oils is currently restricted to certain model algae such as Chlamydomonas, the rapid advances in the development of genetic ma­nipulation tools, plus the better understanding of lipid biosynthesis and regulation, will be extended to industrially important algal species for improving the economics of algal oil production.

6.5 CONCLUSIONS

Heterotrophic production has substantial advantages, including rapid growth, ultrahigh cell density, high oil content, and substantial oil productivity. These merits allow significantly lower downstream process costs, though so far the overall oil production from heterotrophic algae is considered not as economically viable as phototrophic production of algal oils. The relatively high cost of heterotrophic algal oils is mainly attributed to the use of expensive or­ganic carbon—in particular, glucose. Advances in the exploration of using low-cost raw ma­terials such as hydrolyzed carbohydrates and waste sugars have enabled potential cost reductions in heterotrophic production of algal oils. Finding ways to further improve the production economics still remains the major challenge ahead for commercialization of heterotrophic algal oils, which will depend to a large extend on significant advancements in culture systems, biorefinery-based integrated production, and algal strain improvement. Breakthroughs and innovations occurring in these areas will greatly expand production capacity and lower production costs, driving heterotrophic algae from today’s high-value market into the low-cost commodity product pipelines.

Thanks to the increasing interest of using Chlorella biomass as the feedstock for oils, great achievements have been made in heterotrophic culture systems and production models for the algae of this genus, allowing ultrahigh cell density comparable to oleaginous yeasts. To this end, sequencing both the genomes and transcriptomes of several typical Chlo — rella strains is currently underway, which will benefit the development of a new molecular toolbox to successfully manipulate Chlorella for more economically feasible industrial production.

Acknowledgments

This study was partially supported by a grant from the 985 Project of Peking University and by the State Oceanic Administration of China.