One of the areas of microalgae biofuels that must be optimized or re-engineered to make production cost-effective is oil extraction. It is estimated that up to 60 % of the cost of algae biodeisel production involves solvent emulsification and recovery (Molina-Grima et al. 2003). The best studied approach to biodiesel production, first developed for oil seed plants, is the extraction and TAGs or triglycerides into FAMEs. The spent biomass can be used for a variety of applications including biogas, feed, and fertilizers. In the past ten years, a number of alternative forms of catalysis have been developed that use lipases (Ranganathan et al. 2008) or make use of solid bases (hydroxyl groups added to mineral crystals) or catalysts that form biodiesel under high pressure. However, the expense of harvesting, drying, and breaking cell walls remains problematic. In addition, organic solvents for oil extraction are expensive and generate hazardous wastes that must be disposed of at further cost (Williams and Laurens 2010).

HTL has recently been adapted to circumvent many of these problems inherent in lipid extraction processes. HTL is a thermal process that heats a wet slurry of intact algae to 250-350 °C at 1500-3000 psi, converting the biomass to several products including an oil portion ranging from 29 to 52 % yield (See Frank et al. 2013 for a recent review). While TLC produces more oil from algae than lipid extraction, there are several issues with the quality of the oil produced based on the inclusion of other cell components (proteins, nucleic acids, carbohydrates) in the thermal process. A life cycle analysis of TLC of several algal strains (Frank et al. 2012) indicated that the lipid fraction had high levels of N (Williams and Laurens

2010) , leaving questions on combustion emissions for fuel use.

Another recent advancement in the field, supercritical fluid extraction (SFE) of algal biomass, may be an efficient means to extract oils that avoids the use of toxic organic solvents, eliminates the need for the energy-intensive drying of biomass, and avoids high N content in the oil. In addition, SFE allows for the co-extraction of high-value chemicals and leaves a residual biomass that is solvent free and could be marketed as a livestock feed supplement or fertilizer. A carbon dioxide supercritical fluid extraction (CO2-SFE) apparatus for oil extraction using water as a co-solvent would avoid the high cost of drying algae while extracting triglycerides and the co-extraction of valuable nutraceuticals using wet algal biomass.

SFE technology is well developed for processes such as decaffeination and dry­cleaning and is now widely accepted for extraction, purification, and fractionation operations in many industries, especially in the nutraceutical and other “green” industries. SFE is far more efficient than traditional solvent separation methods and is selective, providing high purity of specific products. Additionally, there are no organic solvent residues in the extract or spent biomass. Extraction is efficient at modest operating temperatures, for example, at less than 50 °C, thus ensuring product stability (Herrero et al. 2010). CO2-SFE has been shown to be an efficient solvent for the extraction of a valuable nutraceutical docosahexaenoic acid (DHA) (Couto et al. 2010), for which there is a large growing market.

Based on the literature (Patil and Gude 2011; Choi et al. 1987; Couto et al. 2010; Herrero et al. 2010), there are reasonable starting parameters of temperature, chamber, and release pressures for maximum lipid extraction using dry algal bio­mass. The development of CO2-SFE lipid extraction from wet algae cultures has been explored but is still in its infancy. Adjustments of parameters must be made to use water and methanol co-solvents that alter the overall behavior of the extraction process. Slight variation in temperature will significantly alter the density of the solvent, and therefore the efficiency of the extraction of specific lipoid compounds. An increase in temperature also reduces yield of specific fractions due to product degradation (Patil and Gude 2011; Choi et al. 1987). Cell disruption is a major factor in lipid yields independent of extraction process. Using SFE, cell disruption of wet algae is based on chamber and release pressures, water content, and tem­perature treatment to determine the need for cell lysis prior to CO2-SFE. Halim et al. (2011) have demonstrated the efficiency of SFE extraction of triglyceride fractions from intact wet algal biomass. The extraction efficiency for total lipid extraction including valuable co-products, specifically the marketable nutraceuti — cals, DHA, and luteins, makes the cost-benefit analysis of the whole process favorable. The fractionated products of CO2-SFE, ranging in size from free fatty acids, DHA, triglycerides (the feedstock for biodiesel), and carotenoid compounds such as lutein may be fractionated further using liquid chromatography and the triglycerides transesterified to FAMEs. The parameters for extraction of specific lipids from algae and developing methods that balance cost with production of each specific lipid product that can be scaled up for industrial use is of paramount importance. The CO2 used for extraction can also be recycled to support algal photosynthetic growth. The process outlined here has the potential to be entirely renewable and recyclable as well as cost competitive with liquid fuels.