In order to produce biodiesel from microalgae lipids, the later must be priory extracted. The main lipid extraction techniques are the use of chemical solvents, supercritical CO2, physicochemical, biochemical and direct transesterification.
Chemical solvents extraction
Chemical solvents method is by far the most commonly used, but less effective when microalgae are still wet (Samorì et al., 2010). Consequently, for laboratory scale extraction of lipids, freeze-drying (J. Lee et al., 2010) is a popular method, but spray-drying (Koberg et al., 2011), oven-drying (Cooney et al., 2009) or vacuum-evaporation (Umdu et al., 2009) have also been used to dry microalgae. However, drying microalgae prior to lipid extraction could require 2.5 times more energy than a process without drying, which makes a process using a prior drying unprofitable (negative balance) (Lardon et al., 2009).
In laboratory scale studies, even if chloroform-methanol blends have been extensively used with high extraction yields up to 83% (g lipid/g dry weight) (Yaguchi et al., 1997), less polar solvent like hexane are often preferred because of their lower toxicity and affinity for nonlipid contaminants (less polar) (Halim et al., 2010). As an example, hexane was used to obtained lipids content up to 55% (g lipid/g dry weight) from a heterotrophic microalgae, Chlorella protothecoides (Miao & Wu, 2006). For microalgae lipid extraction on an industrial scale, Soxhlet extraction is not recommended due to high energy requirement (Halim et al.,2010).
Other less toxic solvents like alcohols (ethanol, octanol) or 1,8-diazabicyclo-[5.4.0]-undec-7- ene (DBU) have been tested but the yield of fatty acid methyl ester (FAME) obtained was up to 5 times lower than with n-hexane extraction (Samorì et al., 2010) even if the hydrocarbon (lipid) yield was more than twice higher.
Supercritical carbon dioxide extraction
Supercritical CO2 (Dhepe et al., 2003) has the advantages of being not toxic, easy to recover and usable at low temperatures (less than 40˚C) (Andrich et al., 2005). However, this technique requires expensive equipments (Perrut, 2000) and a huge amount of energy to reach high pressures (Tan & Lee, 2011). Few studies used supercritical CO2 extraction to recover microalgae lipids and transformed them into biodiesel (Halim et al., 2010) even if some studies obtained lipid content up to 26% (g lipid/g dry weight) from Nannocloropsis sp. (Andrich et al., 2005). Using supercritical CO2 extraction at operating temperature of 60˚C and pressure of 30 MPa to extract lipids from Chlorococcum sp. microalgae, Halim et al. (2010) obtained a higher extraction yield of lipids with supercritical CO2 than hexane Soxhlet extraction (5.8 and 3.2% (g lipid/g dry weight), respectively). Moreover, using supercritical CO2 extraction with wet microalgae, Halim et al. (2010) obtained a maximum yield of lipids of 7.1% (g lipid/g dry weight) for the same experimental conditions, which was a relatively low lipid yield compared to other species such as Botryococcus sp. (28.6% g lipid/g dry weight) (J. Lee et al., 2010). Consequently, in opposition to chemical solvent extraction, supercritical CO2 lipid extraction can be stimulated by the presence of water in the blend of microalgae.
Some physicochemical techniques like microwave, autoclaving, osmotic shock, beadbeating, homogenization, freeze-drying, French press, grinding and sonication can be used or microalgae cell disrupting in order to recover lipids (Cooney et al., 2009; J. Lee et al., 2010; S. Lee et al., 1998). Using microwave or bead-beating seems to be the most promising techniques to increase the lipid yield. As an example, J. Lee et al. (2010) increased the lipid extraction yield of Botryococcus sp. microalgae in water phase from 7.7 to 28.6% (g lipid/g dry weight) using a 5 min microwave pretreatment.
Few studies have used biochemical extraction to extract lipids from microalgae. Using a 72 h cellulase hydrolysis pretreatment of the Chlorella sp. microalgae, Fu et al. (2010) have obtained a hydrolysis yield of sugars of 70% (concentration reducing sugar/concentration total sugar), although the lipids yield has increased only from 52 to 54% (g lipid/g dry weight).
Direct (in situ) transesterification
Direct transesterification is a process that blends the microalgae with an alcohol and a catalyst without prior extraction. Number of acid catalysts have been investigated for heterotrophic microalgae biomass including hydrochloric (HCl) or sulphuric acid (H2SO4) but acetyl chloride (CH3COCl) remains the catalyst producing the higher FAME yield of 56% (g FAME /g dry weight) (Cooney et al., 2009). A less polar solvent, like hexane or chloroform, can be added to increase the yield of biodiesel production (M. B. Johnson & Wen, 2009). Direct transesterification using a heterogeneous catalyst could be more effective coupled with microwaves heating. As an example, using microwave with direct transesterification of Nannochloropsis in presence of a heterogeneous catalyst (SrO), Koberg et al. (2011) reported an increase in the FAME yield from 7 to 37% (g FAME /g dry weight). However, direct transesterification requires a dry biomass, increasing the cost of harvesting.