108 Marine Macro- and Microalgae: An Overview
Milking
Milking is a method for simultaneous production and extraction of target compounds directly from live
cells without harvesting or killing them. The extraction relies on an aqueous-organic biphasic system
where the organic solvent is in direct contact with the cells and therefore must be biocompatible. Zhang
et al. (2011) performed a screening of biocompatible solvents for enhancement of lipid milking from
Nannochloropsis sp. The solvents tested were hexanol, heptanol, octanol, hexane, heptane, octane,
nonane, decane, dodecane, tetradecane, and hexadecane, with partition coefficient (Log P oct ), ranging
from 2.03 for hexanol to 8.80 for hexadecane. This study concluded that on the one hand hydrophilic
solvents with Log P oct > 5.5 (the alcohols and the alkanes with C-6 to C-8) were not biocompatible
with the microalgae Nannochloropsis sp. due to deactivated dehydrogenase and increased cell membrane
permeability. On the other hand, dodecane, tetradecane, and hexadecane (10% v/v) were biocompatible
with the microalgae investigated, with hexadecane allowing extraction (22%) of the biomass total lipids.
Furthermore, it was possible to recover 89% of the solvent used in the milking process.
Similarly, milking of D. salina cultures with various solvents to extract β-carotene showed that
solvents having Log P oct > 5 or having a molecular weight above 150 g/mol were considered biocompatible
for this microalga (Mojaat et al. 2008). This study showed that mixing a toxic polar solvent (such as
dichloromethane or methyl ethyl ketone) to the biocompatible solvent decane in appropriate ratio could
achieve efficient extraction of β-carotene while preserving the microalgal cell membrane integrity.
Applying this result to centrifugal partition chromatography, this group investigated the use of ethyl
oleate with 5% dichloromethane (v/v) and compared it with the solvent mixture decane:dichloromethane
(95:5 and 90:10 v/v) for the extraction of β-carotene from D. salina cultures (Marchal et al. 2013).
With the addition of 10% dichloromethane, the cell viability was below 5%. With the addition of 5%
dichloromethane in decane and ethyl oleate, the cell viability reached 80 and 65% while extraction yields
of β-carotene were 37 and 65%, respectively.
Recently the use of nonionic surfactants (among others Triton X-114) has been reported (Glembin
et al. 2013) to extract in situ the fatty acids from S. obliquus. Cell viability was performed showing
that cell growth remained unaffected after exposure to Triton X-114 and that relative photosynthetic
activity recovered from 82 (start) to 100% (after 10 days). This study showed that the algae cells were
concentrated in the aqueous phase, whereas the fatty acids were enriched in the micellar phase.
The milking process seems to be very promising as it can be carried out in situ directly from the
culture medium without affecting the algal culture which can then be cultivated continuously. This
technological concept is so promising that it has been patented by OriginOil (USA) (Guedes et al. 2011).
Precipitation
Extraction of phycobilliproteins from microalgae usually resorts to ammonium sulfate precipitation
(Duerring et al. 1991; Ficner et al. 1992; Minkova et al. 2003). This can be exemplified by the extraction of
β-phycoerythrin from the red alga Rhodosorus marinus which was performed by successive precipitation
steps with ammonium sulfate (Básaca-Loya et al. 2009). The algal cells were first saturated with 40%
ammonium sulfate and the precipitate discarded. The supernatant was then saturated with 60% ammonium
sulfate to precipitate the β-phycoerythrin. Further purification was performed by chromatography.
Chromatography
Purification of a specific metabolite may require techniques with higher selectivity. Chromatography
is a well-known and established technique used to separate individual components from a complex
mixture. Belarbi et al. (2000) employed column chromatography to extract selectively eicosapentaenoic
acid (EPA) methyl ester, which is a ω-3 C20-polyunsaturated fatty acid methyl ester, from cultures of
P. tricornutum. For that purpose, the fatty acid esters were first chromatographed on an argentated silica
gel chromatography yielding 70% of the EPA present in the biomass with a purity of 83%, followed by
a second chromatographic step on silica gel to remove pigments from the EPA ester fraction and thus
leading to highly purified EPA ester (purity up to 96%).
Milking
Milking is a method for simultaneous production and extraction of target compounds directly from live
cells without harvesting or killing them. The extraction relies on an aqueous-organic biphasic system
where the organic solvent is in direct contact with the cells and therefore must be biocompatible. Zhang
et al. (2011) performed a screening of biocompatible solvents for enhancement of lipid milking from
Nannochloropsis sp. The solvents tested were hexanol, heptanol, octanol, hexane, heptane, octane,
nonane, decane, dodecane, tetradecane, and hexadecane, with partition coefficient (Log P oct ), ranging
from 2.03 for hexanol to 8.80 for hexadecane. This study concluded that on the one hand hydrophilic
solvents with Log P oct > 5.5 (the alcohols and the alkanes with C-6 to C-8) were not biocompatible
with the microalgae Nannochloropsis sp. due to deactivated dehydrogenase and increased cell membrane
permeability. On the other hand, dodecane, tetradecane, and hexadecane (10% v/v) were biocompatible
with the microalgae investigated, with hexadecane allowing extraction (22%) of the biomass total lipids.
Furthermore, it was possible to recover 89% of the solvent used in the milking process.
Similarly, milking of D. salina cultures with various solvents to extract β-carotene showed that
solvents having Log P oct > 5 or having a molecular weight above 150 g/mol were considered biocompatible
for this microalga (Mojaat et al. 2008). This study showed that mixing a toxic polar solvent (such as
dichloromethane or methyl ethyl ketone) to the biocompatible solvent decane in appropriate ratio could
achieve efficient extraction of β-carotene while preserving the microalgal cell membrane integrity.
Applying this result to centrifugal partition chromatography, this group investigated the use of ethyl
oleate with 5% dichloromethane (v/v) and compared it with the solvent mixture decane:dichloromethane
(95:5 and 90:10 v/v) for the extraction of β-carotene from D. salina cultures (Marchal et al. 2013).
With the addition of 10% dichloromethane, the cell viability was below 5%. With the addition of 5%
dichloromethane in decane and ethyl oleate, the cell viability reached 80 and 65% while extraction yields
of β-carotene were 37 and 65%, respectively.
Recently the use of nonionic surfactants (among others Triton X-114) has been reported (Glembin
et al. 2013) to extract in situ the fatty acids from S. obliquus. Cell viability was performed showing
that cell growth remained unaffected after exposure to Triton X-114 and that relative photosynthetic
activity recovered from 82 (start) to 100% (after 10 days). This study showed that the algae cells were
concentrated in the aqueous phase, whereas the fatty acids were enriched in the micellar phase.
The milking process seems to be very promising as it can be carried out in situ directly from the
culture medium without affecting the algal culture which can then be cultivated continuously. This
technological concept is so promising that it has been patented by OriginOil (USA) (Guedes et al. 2011).
Precipitation
Extraction of phycobilliproteins from microalgae usually resorts to ammonium sulfate precipitation
(Duerring et al. 1991; Ficner et al. 1992; Minkova et al. 2003). This can be exemplified by the extraction of
β-phycoerythrin from the red alga Rhodosorus marinus which was performed by successive precipitation
steps with ammonium sulfate (Básaca-Loya et al. 2009). The algal cells were first saturated with 40%
ammonium sulfate and the precipitate discarded. The supernatant was then saturated with 60% ammonium
sulfate to precipitate the β-phycoerythrin. Further purification was performed by chromatography.
Chromatography
Purification of a specific metabolite may require techniques with higher selectivity. Chromatography
is a well-known and established technique used to separate individual components from a complex
mixture. Belarbi et al. (2000) employed column chromatography to extract selectively eicosapentaenoic
acid (EPA) methyl ester, which is a ω-3 C20-polyunsaturated fatty acid methyl ester, from cultures of
P. tricornutum. For that purpose, the fatty acid esters were first chromatographed on an argentated silica
gel chromatography yielding 70% of the EPA present in the biomass with a purity of 83%, followed by
a second chromatographic step on silica gel to remove pigments from the EPA ester fraction and thus
leading to highly purified EPA ester (purity up to 96%).