Alternative Green Biofuel from Microalgae: A Promising Renewable Resource 259
Ultrasonic extraction methods were most effective at disrupting cell walls (Cravotto et al. 2008;
Wei et al. 2008), increasing oil production from Crypthecodinium cohnii and S. obliquus using Soxhlet
extraction in hexane (Cravotto et al. 2008; Balasubramanian et al. 2010). Both ultrasonication and
microwave-assisted methods improve oil extraction of microalgae significantly, with higher efficiency,
reduced extraction times and increased yields, as well as low to moderate costs and negligible added
toxicity. This technology, however, may negatively impact oil quality and/or stability of polyunsaturated
fatty acid-rich oils, as it is difficult to scale up.
Solvent extraction entails extracting oil from microalgae by repeated washing or percolation with an
organic solvent. Extraction methods used should be fast, effective, and non-damaging to oils extracted
and easily scaled up (Medina et al. 1998). The choice of solvent for oil extraction, as with the harvesting
process, will depend on the type of the microalgae selected. Lipids have different types of associations
which need to be disrupted for effective extraction. Pre-treatment of samples may be required for oil
extraction of certain types of biomass. This is generally not necessary for extraction from wet biomass,
as solvents generally rupture cells by disassembling microalgal cell membranes and cell walls as well
(Mercer and Armenta 2011; Gangadhar et al. 2016a). In addition, a suitable organic solvent should be
commercially available, inexpensive, and insoluble in water, have a low boiling point to facilitate its
removal after extraction, and have a considerably different density than water, enabling its re-use. Hexane
is typically the solvent of choice for large scale extractions, as it is inexpensive, has high extraction
efficiency, and is less dense than water (Benerjee et al. 2002). However, if the solvent is not harmful
to the cells it is also possible to isolate the oils from microalgae such as Botryococcus braunii without
breaking the cell walls (Benerjee et al. 2002). To optimise efficiency, cell mechanical rupture is, however,
usually needed before exposure to the organic solvent (Cooney et al. 2009). Solvent extraction is most
effective at recovering the oil from Scenedesmus sp. (Shen et al. 2009) and Nannocloropsis sp. using
Soxhlet extraction (Wiyarno et al. 2011). With Soxhlet extraction, higher solvent concentration led to
higher FFA levels and saponification. Normally, lipids are extracted from microalgal biomass using a
mixture of solvents like chloroform, methanol and water (Bligh and Dyer 1959) and a modified Bligh
and Dyer (Mutanda et al. 2011) method is most commonly used. Disadvantages of this method is that
it produces waste solvent that is costly to recycle at large scale, raising safety concerns due to handling
of large amounts of organic solvents (Sahena et al. 2009). However, the Bligh and Dyer method has
been proven moderately effective when used on wet material. In addition, organic solvents can lead to
contamination in the form of solvent residues being present in the final product. A mixture of hexane and
isopropanol was used to extract oil from Chlorococcum sp., because is an alternative set of solvents with
lower toxicity as compared to a mixture of chloroform and methanol (Halim et al. 2011).
Enzymatic treatment of microbial biomass has the potential to partially or fully disrupt cells with
minimal damage to the desired biomaterials (i.e., oil). It has proven to be successful extracting oil from
plant seeds using sonication and cold pressing (Shah et al. 2004; Soto et al. 2007). One distinct challenge
with enzymes is that in order to design an effective enzymatic procedure for hydrolysing microbial cells,
it is necessary to determine their composition. Therefore, the most appropriate enzymes can be chosen to
optimize extraction conditions.
Supercritical CO 2 extraction is a promising green technology that can potentially be used for largescale microalgal lipid extraction (Ronald et al. 2012). It is rapid, non-toxic, has high selectivity towards
triacylglycerols, and produces solvent-free lipids. Since CO 2 is a gas at room temperature, it is safe
for food applications, and it is thus being favoured with respect to other solvents due to its relatively
low critical temperature and pressure, and the possibility of being recycled upon extraction completion
(Sahena et al. 2009; Wiyarno et al. 2011). Supercritical carbon dioxide extraction is regarded with interest
as an industrial process, due to its shorter extraction time and the ability of obtaining pure compounds
without solvent contamination, being also deemed as safe for thermally sensitive products (Mendes et
al. 2006; Sahena et al. 2009). Moreover, isolation of important compounds such as anti-oxidants (e.g.,
canthaxanthin and astaxanthin) from Chlorella vulgaris and Haematococcus pluvial and β-carotene from
Dunaliella salina are feasible, which may reduce separation costs, as well as possibly counteracting
greenhouse gas effects by using CO 2 waste from industry (e.g., Cyanotech and Aquasearch) (Perrut 2001;
Guerin et al. 2003; Herrero et al. 2006; Mendiola et al. 2007). In addition, the reported methods have been
Ultrasonic extraction methods were most effective at disrupting cell walls (Cravotto et al. 2008;
Wei et al. 2008), increasing oil production from Crypthecodinium cohnii and S. obliquus using Soxhlet
extraction in hexane (Cravotto et al. 2008; Balasubramanian et al. 2010). Both ultrasonication and
microwave-assisted methods improve oil extraction of microalgae significantly, with higher efficiency,
reduced extraction times and increased yields, as well as low to moderate costs and negligible added
toxicity. This technology, however, may negatively impact oil quality and/or stability of polyunsaturated
fatty acid-rich oils, as it is difficult to scale up.
Solvent extraction entails extracting oil from microalgae by repeated washing or percolation with an
organic solvent. Extraction methods used should be fast, effective, and non-damaging to oils extracted
and easily scaled up (Medina et al. 1998). The choice of solvent for oil extraction, as with the harvesting
process, will depend on the type of the microalgae selected. Lipids have different types of associations
which need to be disrupted for effective extraction. Pre-treatment of samples may be required for oil
extraction of certain types of biomass. This is generally not necessary for extraction from wet biomass,
as solvents generally rupture cells by disassembling microalgal cell membranes and cell walls as well
(Mercer and Armenta 2011; Gangadhar et al. 2016a). In addition, a suitable organic solvent should be
commercially available, inexpensive, and insoluble in water, have a low boiling point to facilitate its
removal after extraction, and have a considerably different density than water, enabling its re-use. Hexane
is typically the solvent of choice for large scale extractions, as it is inexpensive, has high extraction
efficiency, and is less dense than water (Benerjee et al. 2002). However, if the solvent is not harmful
to the cells it is also possible to isolate the oils from microalgae such as Botryococcus braunii without
breaking the cell walls (Benerjee et al. 2002). To optimise efficiency, cell mechanical rupture is, however,
usually needed before exposure to the organic solvent (Cooney et al. 2009). Solvent extraction is most
effective at recovering the oil from Scenedesmus sp. (Shen et al. 2009) and Nannocloropsis sp. using
Soxhlet extraction (Wiyarno et al. 2011). With Soxhlet extraction, higher solvent concentration led to
higher FFA levels and saponification. Normally, lipids are extracted from microalgal biomass using a
mixture of solvents like chloroform, methanol and water (Bligh and Dyer 1959) and a modified Bligh
and Dyer (Mutanda et al. 2011) method is most commonly used. Disadvantages of this method is that
it produces waste solvent that is costly to recycle at large scale, raising safety concerns due to handling
of large amounts of organic solvents (Sahena et al. 2009). However, the Bligh and Dyer method has
been proven moderately effective when used on wet material. In addition, organic solvents can lead to
contamination in the form of solvent residues being present in the final product. A mixture of hexane and
isopropanol was used to extract oil from Chlorococcum sp., because is an alternative set of solvents with
lower toxicity as compared to a mixture of chloroform and methanol (Halim et al. 2011).
Enzymatic treatment of microbial biomass has the potential to partially or fully disrupt cells with
minimal damage to the desired biomaterials (i.e., oil). It has proven to be successful extracting oil from
plant seeds using sonication and cold pressing (Shah et al. 2004; Soto et al. 2007). One distinct challenge
with enzymes is that in order to design an effective enzymatic procedure for hydrolysing microbial cells,
it is necessary to determine their composition. Therefore, the most appropriate enzymes can be chosen to
optimize extraction conditions.
Supercritical CO 2 extraction is a promising green technology that can potentially be used for largescale microalgal lipid extraction (Ronald et al. 2012). It is rapid, non-toxic, has high selectivity towards
triacylglycerols, and produces solvent-free lipids. Since CO 2 is a gas at room temperature, it is safe
for food applications, and it is thus being favoured with respect to other solvents due to its relatively
low critical temperature and pressure, and the possibility of being recycled upon extraction completion
(Sahena et al. 2009; Wiyarno et al. 2011). Supercritical carbon dioxide extraction is regarded with interest
as an industrial process, due to its shorter extraction time and the ability of obtaining pure compounds
without solvent contamination, being also deemed as safe for thermally sensitive products (Mendes et
al. 2006; Sahena et al. 2009). Moreover, isolation of important compounds such as anti-oxidants (e.g.,
canthaxanthin and astaxanthin) from Chlorella vulgaris and Haematococcus pluvial and β-carotene from
Dunaliella salina are feasible, which may reduce separation costs, as well as possibly counteracting
greenhouse gas effects by using CO 2 waste from industry (e.g., Cyanotech and Aquasearch) (Perrut 2001;
Guerin et al. 2003; Herrero et al. 2006; Mendiola et al. 2007). In addition, the reported methods have been