106 Marine Macro- and Microalgae: An Overview
times was investigated (Kim et al. 2012a). Results showed that maximum recovery could be achieved
with acetone, ethyl acetate, and ethanol. Using ethanol as extraction solvent, it was found out that 5 min
extraction time was sufficient to obtain maximum recovery of fucoxanthin. The fucoxanthin degraded
gradually with longer extraction times (loss of 14% after 24 h extraction time compared to 5 min).
This study also optimized a process for the simultaneous preparation of crude fucoxanthin and lipids
from this algae, where the crude ethanolic extract can be partitioned with hexane, yielding crude lipids
in the hexane phase and crude fucoxanthin in the hydroalcoholic phase. This same author reports in
another work (Kim et al. 2012b) that extraction of fucoxanthin with ethanol from the marine microalga
P. tricornutum gives the maximum yield (15.71 mg/g DW) compared to acetone and ethyl acetate (4.60
and 2.26 mg/g DW, respectively), and that this maximum yield could only be achieved with an extraction
time of 60 min. From these two studies it is clear that the extraction solvent is to be selected and optimized
for each type of algae to be extracted. It is widely accepted that carotenoids are best extracted with
acetone, for example, Zhu and Jiang (2008) for the extraction of β-carotene from D. salina, and while
this solvent would have allowed 100% recovery of fucoxanthin from Isochrysis aff. galbana, this same
choice of solvent would have reduced the yield to 29% for P. tricornutum.
Solvent extraction is often performed in a Soxhlet extractor as the solvent in contact with the
biomass is constantly evaporated and recondensed into the sample container, thus enabling continuous
re-establishment of mass transfer equilibria. In a comparative study of static hexane lipid extraction
with Soxhlet hexane lipid extraction, Halim et al. (2011) found that the lipid yield increased from 1.5
to 5.7%, respectively. Wu et al. (2012a) investigated Soxhlet extraction of the carotenoid zeaxanthin
from N. oculata with dichloromethane, hexane, ethanol, and acetone. Highest yields of zeaxanthin were
obtained with dichloromethane and ethanol (100 and 98.3% respectively) with an extraction time of 16 h.
Increasing the extraction time to 24 h caused a reduction of the yield of extraction of zeaxanthin down to
71.7% probably due to thermal decomposition of zeaxanthin during prolonged heat extraction.
Alternatively, bio-solvents, such as the terpenes d-limonene, α-pinene, and p-cymene, recognized
as environmentally safer have also been investigated to extract lipids from the microalgae N. oculata
and D. salina (Dejoye Tanzi et al. 2013) by distillation. Lipid yields from N. oculata extracted with
p-cymene was highest (21.45%) compared to the two other solvents α-pinene and d-limonene (18.75
and 18.73%, respectively). For D. salina the best solvent was α-pinene (3.29%) compared to p-cymene
and d-limonene (2.99 and 2.94% respectively). This process also proved to be effective as it allowed
elimination of water from the microalgae biomass, extraction of the lipids, and recycling of the terpene
solvent (100% recovery).
Supercritical carbon dioxide extraction
Supercritical carbon dioxide fluid extraction (SFE-CO 2 ) is a technology with low environmental impact
due to the absence of harmful residual solvents. Furthermore, supercritical fluids have high diffusivity,
high compressibility, low viscosity, and low surface tension, which allow them to diffuse easily through
the solid biomass matrix and thus, achieve higher extraction yields.
There are several factors affecting the extraction efficiency of SFE-CO2 among others the type of
microalgae and the type of component to be extracted. Furthermore, various operational parameters are
also known to significantly affect the extraction efficiency of SFE-CO2, such as load of biomass, flow
rate, temperature, pressure, duration of extraction, and addition of modifiers.
Guedes et al. (2013) described how the pressure, temperature, flow rate and the use of a
co-solvent could impact the extraction of two types of components, the carotenoids and chlorophylls
from S. obliquus, and showed that highest yield of chlorophylls was obtained at 250 bar and 40ºC while
carotenoids required 200 bar and 60ºC. The yields of chlorophylls and carotenoids were reduced when
the CO 2 flow rate was doubled from 2 to 4 g/min and the addition of 7.7% (v/v) of ethanol afforded a
maximum yield for both classes of compounds. Pan et al. (2012) investigated the extraction of astaxanthin
from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier and found that
the conditions for optimum extraction of astaxanthin were a loading of biomass of 21.67 g/L, a CO 2 flow
rate of 6 NL/min (where NL/min is the flow rate in L/min measured under normal air conditions), an
times was investigated (Kim et al. 2012a). Results showed that maximum recovery could be achieved
with acetone, ethyl acetate, and ethanol. Using ethanol as extraction solvent, it was found out that 5 min
extraction time was sufficient to obtain maximum recovery of fucoxanthin. The fucoxanthin degraded
gradually with longer extraction times (loss of 14% after 24 h extraction time compared to 5 min).
This study also optimized a process for the simultaneous preparation of crude fucoxanthin and lipids
from this algae, where the crude ethanolic extract can be partitioned with hexane, yielding crude lipids
in the hexane phase and crude fucoxanthin in the hydroalcoholic phase. This same author reports in
another work (Kim et al. 2012b) that extraction of fucoxanthin with ethanol from the marine microalga
P. tricornutum gives the maximum yield (15.71 mg/g DW) compared to acetone and ethyl acetate (4.60
and 2.26 mg/g DW, respectively), and that this maximum yield could only be achieved with an extraction
time of 60 min. From these two studies it is clear that the extraction solvent is to be selected and optimized
for each type of algae to be extracted. It is widely accepted that carotenoids are best extracted with
acetone, for example, Zhu and Jiang (2008) for the extraction of β-carotene from D. salina, and while
this solvent would have allowed 100% recovery of fucoxanthin from Isochrysis aff. galbana, this same
choice of solvent would have reduced the yield to 29% for P. tricornutum.
Solvent extraction is often performed in a Soxhlet extractor as the solvent in contact with the
biomass is constantly evaporated and recondensed into the sample container, thus enabling continuous
re-establishment of mass transfer equilibria. In a comparative study of static hexane lipid extraction
with Soxhlet hexane lipid extraction, Halim et al. (2011) found that the lipid yield increased from 1.5
to 5.7%, respectively. Wu et al. (2012a) investigated Soxhlet extraction of the carotenoid zeaxanthin
from N. oculata with dichloromethane, hexane, ethanol, and acetone. Highest yields of zeaxanthin were
obtained with dichloromethane and ethanol (100 and 98.3% respectively) with an extraction time of 16 h.
Increasing the extraction time to 24 h caused a reduction of the yield of extraction of zeaxanthin down to
71.7% probably due to thermal decomposition of zeaxanthin during prolonged heat extraction.
Alternatively, bio-solvents, such as the terpenes d-limonene, α-pinene, and p-cymene, recognized
as environmentally safer have also been investigated to extract lipids from the microalgae N. oculata
and D. salina (Dejoye Tanzi et al. 2013) by distillation. Lipid yields from N. oculata extracted with
p-cymene was highest (21.45%) compared to the two other solvents α-pinene and d-limonene (18.75
and 18.73%, respectively). For D. salina the best solvent was α-pinene (3.29%) compared to p-cymene
and d-limonene (2.99 and 2.94% respectively). This process also proved to be effective as it allowed
elimination of water from the microalgae biomass, extraction of the lipids, and recycling of the terpene
solvent (100% recovery).
Supercritical carbon dioxide extraction
Supercritical carbon dioxide fluid extraction (SFE-CO 2 ) is a technology with low environmental impact
due to the absence of harmful residual solvents. Furthermore, supercritical fluids have high diffusivity,
high compressibility, low viscosity, and low surface tension, which allow them to diffuse easily through
the solid biomass matrix and thus, achieve higher extraction yields.
There are several factors affecting the extraction efficiency of SFE-CO2 among others the type of
microalgae and the type of component to be extracted. Furthermore, various operational parameters are
also known to significantly affect the extraction efficiency of SFE-CO2, such as load of biomass, flow
rate, temperature, pressure, duration of extraction, and addition of modifiers.
Guedes et al. (2013) described how the pressure, temperature, flow rate and the use of a
co-solvent could impact the extraction of two types of components, the carotenoids and chlorophylls
from S. obliquus, and showed that highest yield of chlorophylls was obtained at 250 bar and 40ºC while
carotenoids required 200 bar and 60ºC. The yields of chlorophylls and carotenoids were reduced when
the CO 2 flow rate was doubled from 2 to 4 g/min and the addition of 7.7% (v/v) of ethanol afforded a
maximum yield for both classes of compounds. Pan et al. (2012) investigated the extraction of astaxanthin
from Haematococcus pluvialis by supercritical carbon dioxide fluid with ethanol modifier and found that
the conditions for optimum extraction of astaxanthin were a loading of biomass of 21.67 g/L, a CO 2 flow
rate of 6 NL/min (where NL/min is the flow rate in L/min measured under normal air conditions), an