Microalgal Downstream Processing: Harvesting, Drying, Extraction, Separation, and Purification 107
extraction time of 20 min, an extraction pressure of 4,500 psi, a volume of ethanol modifier of 9.23 mL/g,
an extraction temperature of 50
o
C, and a modifier composition of 99.5%. Astaxanthin was also extracted
using supercritical carbon dioxide (60ºC and 20 MPa for 1 h) from the vegetative green microalga
Monoraphidium sp. GK12 (Fujii 2012) affording 83% recovery of the carotenoid. Addition of ethanol to
the algal biomass (20/1 v/w) prior to SFE could increase the recovery of astaxanthin to 101%.
Supercritical fluid extraction of lipids from Crypthecodinium cohnii was also investigated (Couto
et al. 2010). This work found out that 50% of total oil contained in the raw material was extracted
after 3 h at optimum extraction conditions 30.0 MPa and 323 K, and that the docosahexaenoic acid
(DHA) attained 72% w/w of total fatty acids. Assessment of bio-oil extraction from Tetraselmis chui with
SFE-CO 2 (Grierson et al. 2012) without and with methanol or ethanol as co-solvents resulted in low
recoveries (0.01 to 4.3% wt) of natural oil compared to the amount of lipids as recorded for this species
in the literature (17% wt). Similarly, extraction of carotenoids from the marine strain Synechococcus sp.
with supercritical CO 2 could not compare with the extraction of carotenoids with dimethylformamide
used as a reference, as it only afforded 50% of the total carotenoids at a working pressure of 500 bar
(Montero et al. 2005).
Pressurized fluid extraction
Pressurized fluid extraction (PFE) allows efficient extraction of compounds with solvents mainly due to
the use of high temperatures which increase compounds solubility. PFE could be compared to a Soxhlet
extraction but with the difference that the extraction is performed at high pressure, thus keeping the
solvent in the liquid state, even if the temperature applied is above the boiling point of the extracting
solvent. The high pressure also favors the penetration of the solvent into the biological matrix. Pieber et
al. (2012) investigated the use of pressurized fluid extraction on N. oculata for the recovery of lipids, and
more specifically the PUFA yield. For his extraction solvents applicable in the food and pharmaceutical
industries, hexane, hexane/isopropanol (2:1 vol.%) and ethanol (96 vol.%) were tested. The highest
extraction yield was obtained from ethanol extraction (36 mass%), while hexane afforded the lowest
yield (6.1 mass%). The use of ethanol also resulted in the highest PUFA yield (5.7 mass%), total FA yield
(ca. 17 mass%), and EPA yield (3.7 mass%).
Direct transesterification
When the microalgae biomass is mixed with alcohol and a catalyst at high temperature, lipid extraction,
and transesterification occur simultaneously, thus reducing the operational cost of biodiesel production.
Patil et al. (2011) carried out a response surface methodology (RSM) of the direct conversion of wet
algae (Nannochloropsis sp.) to biodiesel under supercritical methanol conditions. The optimal conditions
were then reported as wet algae to methanol ratio of around 1:9 (w/v), reaction temperature, and time
of about 255ºC and 25 min, respectively. During this process, fatty acid methyl esters (FAME) were
produced from polar phospholipids, free fatty acids, and triglycerides. In their subsequent investigation
(Patil et al. 2012), a comparison of direct transesterification of algal biomass with supercritical methanol
(SCM) and microwave irradiation (MW) conditions was performed. Maximum yields of FAME were
obtained with optimum parameters as 25 and 8, and 6 and 9 for time of reaction (min) and biomass to
methanol ratio (w/v), respectively, as well as a temperature of 250ºC for the SCM process and 2% KOH
(wt.%) for the MW process. Both processes resulted comparable extraction yields of FAME (84.15 and
80.13% for SCM and MW, respectively), though with higher energy requirements for the supercritical
methanol process. Koberg et al. (2011) also investigated the direct transesterification of the crude dried
solid of Nannochloropsis sp. microalgae. To perform the transesterification, the dried algal biomass was
suspended in methanol:chloroform (1:2 v/v) with a SrO catalyst and heated using either sonication (50ºC)
or microwave irradiation (60
o
C). For comparison purposes, the reaction was also carried out by reflux
using the conventional protocol. Both the yield and conversion of biodiesel (conversion of triglycerides
to biodiesel) were 37.1 and 99.9%, and 20.9 and close to 95% for microwave and sonication heating,
respectively. Compared to these results the reflux technique yielded 6.9% biodiesel with only 74.6%
conversion.
extraction time of 20 min, an extraction pressure of 4,500 psi, a volume of ethanol modifier of 9.23 mL/g,
an extraction temperature of 50
o
C, and a modifier composition of 99.5%. Astaxanthin was also extracted
using supercritical carbon dioxide (60ºC and 20 MPa for 1 h) from the vegetative green microalga
Monoraphidium sp. GK12 (Fujii 2012) affording 83% recovery of the carotenoid. Addition of ethanol to
the algal biomass (20/1 v/w) prior to SFE could increase the recovery of astaxanthin to 101%.
Supercritical fluid extraction of lipids from Crypthecodinium cohnii was also investigated (Couto
et al. 2010). This work found out that 50% of total oil contained in the raw material was extracted
after 3 h at optimum extraction conditions 30.0 MPa and 323 K, and that the docosahexaenoic acid
(DHA) attained 72% w/w of total fatty acids. Assessment of bio-oil extraction from Tetraselmis chui with
SFE-CO 2 (Grierson et al. 2012) without and with methanol or ethanol as co-solvents resulted in low
recoveries (0.01 to 4.3% wt) of natural oil compared to the amount of lipids as recorded for this species
in the literature (17% wt). Similarly, extraction of carotenoids from the marine strain Synechococcus sp.
with supercritical CO 2 could not compare with the extraction of carotenoids with dimethylformamide
used as a reference, as it only afforded 50% of the total carotenoids at a working pressure of 500 bar
(Montero et al. 2005).
Pressurized fluid extraction
Pressurized fluid extraction (PFE) allows efficient extraction of compounds with solvents mainly due to
the use of high temperatures which increase compounds solubility. PFE could be compared to a Soxhlet
extraction but with the difference that the extraction is performed at high pressure, thus keeping the
solvent in the liquid state, even if the temperature applied is above the boiling point of the extracting
solvent. The high pressure also favors the penetration of the solvent into the biological matrix. Pieber et
al. (2012) investigated the use of pressurized fluid extraction on N. oculata for the recovery of lipids, and
more specifically the PUFA yield. For his extraction solvents applicable in the food and pharmaceutical
industries, hexane, hexane/isopropanol (2:1 vol.%) and ethanol (96 vol.%) were tested. The highest
extraction yield was obtained from ethanol extraction (36 mass%), while hexane afforded the lowest
yield (6.1 mass%). The use of ethanol also resulted in the highest PUFA yield (5.7 mass%), total FA yield
(ca. 17 mass%), and EPA yield (3.7 mass%).
Direct transesterification
When the microalgae biomass is mixed with alcohol and a catalyst at high temperature, lipid extraction,
and transesterification occur simultaneously, thus reducing the operational cost of biodiesel production.
Patil et al. (2011) carried out a response surface methodology (RSM) of the direct conversion of wet
algae (Nannochloropsis sp.) to biodiesel under supercritical methanol conditions. The optimal conditions
were then reported as wet algae to methanol ratio of around 1:9 (w/v), reaction temperature, and time
of about 255ºC and 25 min, respectively. During this process, fatty acid methyl esters (FAME) were
produced from polar phospholipids, free fatty acids, and triglycerides. In their subsequent investigation
(Patil et al. 2012), a comparison of direct transesterification of algal biomass with supercritical methanol
(SCM) and microwave irradiation (MW) conditions was performed. Maximum yields of FAME were
obtained with optimum parameters as 25 and 8, and 6 and 9 for time of reaction (min) and biomass to
methanol ratio (w/v), respectively, as well as a temperature of 250ºC for the SCM process and 2% KOH
(wt.%) for the MW process. Both processes resulted comparable extraction yields of FAME (84.15 and
80.13% for SCM and MW, respectively), though with higher energy requirements for the supercritical
methanol process. Koberg et al. (2011) also investigated the direct transesterification of the crude dried
solid of Nannochloropsis sp. microalgae. To perform the transesterification, the dried algal biomass was
suspended in methanol:chloroform (1:2 v/v) with a SrO catalyst and heated using either sonication (50ºC)
or microwave irradiation (60
o
C). For comparison purposes, the reaction was also carried out by reflux
using the conventional protocol. Both the yield and conversion of biodiesel (conversion of triglycerides
to biodiesel) were 37.1 and 99.9%, and 20.9 and close to 95% for microwave and sonication heating,
respectively. Compared to these results the reflux technique yielded 6.9% biodiesel with only 74.6%
conversion.