Microalgal Downstream Processing: Harvesting, Drying, Extraction, Separation, and Purification 105
in the algal biomass. Similarly, Nguyen et al. (2009) have investigated the pretreatment with sulfuric
acid (3% at 110ºC for 30 min) of Chlamydomonas reinhardtii UTEX 90 biomass for ethanol production.
This treatment afforded a maximum glucose release of 58% (w/w) from the biomass polysaccharides
and oligosaccharides. The effect of sulfuric acid pretreatment on Chlorococcum sp. stock cultures was
investigated (Halim et al. 2012) under different conditions: temperature (120 or 160ºC), acid concentration
(3 or 8 vol%), and treatment duration (15 or 45 min) were evaluated not in terms of yields of a given
compound extracted but in terms of cell disruption as observed by microscopy. The disruption efficiency
was highest (66%) when using the higher acid concentration and temperature levels. Disruption could be
as low as 3% when using the lower temperature and acid concentration with 45 min treatment.
Acid treatment was also used to remove chlorophyll from the astaxanthin SCE-prepared extract of
the microalgae Monoraphidium sp. GK12. Fujii (2012) showed that the extract still contained chlorophyll
and therefore investigated the effect of treatment with H 2 SO 4 , HCl, H 3 PO 4, and CH 3 COOH. The two later
acids could not remove the chlorophyll. Treatment with 0.1 N H 2 SO 4 or HCl was suitable for the removal
of more than 80% chlorophyll with no loss of astaxanthin. Miranda et al. (2012) also demonstrated
the efficiency of acidic treatment with H 2 SO 4 (2N, at 120ºC for 30 min) resulting in highest yields of
extracted sugars from S. obliquus (up to 95.6% compared to harsh quantitative acid hydrolysis).
Surfactant pretreatment
Due to growing concerns regarding the use of organic solvents for lysis and extraction of metabolites
from natural sources, non-ionic surfactants have been investigated as potential cell disrupters combined
with salting out agents such as sodium inorganic and organic salts in aqueous solutions (Ulloa et al.
2012). The microalgae used for this work was T. suecica and the metabolites monitored to evaluate the
extraction process were α-tocopherol, β-carotene, and gallic acid. Tween 20, 40, and 80 as well as Triton
X-100, X-102, and X-114 were employed as cell lysis agents, and sodium carbonate, sodium citrate, and
sodium tartrate as salting out agents. The algal biomass was stirred for two days in a shaker at 150 rpm
with the surfactant. Results showed that Tween 20 and Triton-X102 were capable of forming effective
phase segregation in the presence of the three salts. Tween 20 and sodium citrate afforded high extraction
yields, higher than 99% for α-tocopherol and around 60% for β-carotene and gallic acid. The method
using Triton X114 was evaluated against a conventional ultrasound-based method and found out to be
more effective as the antioxidant yield was increased by a factor 1.59, 3.69, and 32.5 for gallic acid,
α-tocopherol, and β-carotene, respectively.
Extraction, separation, and purification methods
Solvent extraction
Extraction of lipids from microalgae has usually been carried out with a mixture of chloroform-methanol
or chloroform-methanol-water, as is the case in the conventional lipids extraction method first described
by Bligh and Dyer (1959). This lipid extraction method is quite often used in the literature as a reference
method compared to various other described methods. However, the use of chloroform in large scale is
precluded by environmental and health risks. The use of other solvents has therefore been investigated.
Talukder et al. (2012) used hexane to extract lipids from dried biomass of N. salina following an acid
hydrolysis pretreatment (5% sulfuric acid at 120ºC for 1 h). Incubation of the hydrolysate with hexane
(at 40ºC and mixing at 200 rpm up to 24 h) was followed by centrifugation (4,000 rpm) allowing separation
of the hexane phase from the hydrolysate. The hexane phase afforded a lipid yield of 85.6%. Hexane was
also used to extract lipids from the microalga Chlorococcum sp. (Halim et al. 2011) with a very low yield
(1.5%), though it should be noted that this alga was found to have a maximum lipid yield of 7.1%.
Using various concentrations (50 to 95%) of isopropanol, Yao et al. (2012) performed the extraction
of lipids from the microalga Nannochloropsis sp. It was concluded that the 70, 88, and 95% IPA
concentrations extracted 82–92% of total oil.
Using the marine microalgae Isochrysis aff. galbana, the extraction of the carotenoid fucoxanthin
with different solvents (hexane, acetone, ethyl acetate, methanol, and ethanol) and various extraction
in the algal biomass. Similarly, Nguyen et al. (2009) have investigated the pretreatment with sulfuric
acid (3% at 110ºC for 30 min) of Chlamydomonas reinhardtii UTEX 90 biomass for ethanol production.
This treatment afforded a maximum glucose release of 58% (w/w) from the biomass polysaccharides
and oligosaccharides. The effect of sulfuric acid pretreatment on Chlorococcum sp. stock cultures was
investigated (Halim et al. 2012) under different conditions: temperature (120 or 160ºC), acid concentration
(3 or 8 vol%), and treatment duration (15 or 45 min) were evaluated not in terms of yields of a given
compound extracted but in terms of cell disruption as observed by microscopy. The disruption efficiency
was highest (66%) when using the higher acid concentration and temperature levels. Disruption could be
as low as 3% when using the lower temperature and acid concentration with 45 min treatment.
Acid treatment was also used to remove chlorophyll from the astaxanthin SCE-prepared extract of
the microalgae Monoraphidium sp. GK12. Fujii (2012) showed that the extract still contained chlorophyll
and therefore investigated the effect of treatment with H 2 SO 4 , HCl, H 3 PO 4, and CH 3 COOH. The two later
acids could not remove the chlorophyll. Treatment with 0.1 N H 2 SO 4 or HCl was suitable for the removal
of more than 80% chlorophyll with no loss of astaxanthin. Miranda et al. (2012) also demonstrated
the efficiency of acidic treatment with H 2 SO 4 (2N, at 120ºC for 30 min) resulting in highest yields of
extracted sugars from S. obliquus (up to 95.6% compared to harsh quantitative acid hydrolysis).
Surfactant pretreatment
Due to growing concerns regarding the use of organic solvents for lysis and extraction of metabolites
from natural sources, non-ionic surfactants have been investigated as potential cell disrupters combined
with salting out agents such as sodium inorganic and organic salts in aqueous solutions (Ulloa et al.
2012). The microalgae used for this work was T. suecica and the metabolites monitored to evaluate the
extraction process were α-tocopherol, β-carotene, and gallic acid. Tween 20, 40, and 80 as well as Triton
X-100, X-102, and X-114 were employed as cell lysis agents, and sodium carbonate, sodium citrate, and
sodium tartrate as salting out agents. The algal biomass was stirred for two days in a shaker at 150 rpm
with the surfactant. Results showed that Tween 20 and Triton-X102 were capable of forming effective
phase segregation in the presence of the three salts. Tween 20 and sodium citrate afforded high extraction
yields, higher than 99% for α-tocopherol and around 60% for β-carotene and gallic acid. The method
using Triton X114 was evaluated against a conventional ultrasound-based method and found out to be
more effective as the antioxidant yield was increased by a factor 1.59, 3.69, and 32.5 for gallic acid,
α-tocopherol, and β-carotene, respectively.
Extraction, separation, and purification methods
Solvent extraction
Extraction of lipids from microalgae has usually been carried out with a mixture of chloroform-methanol
or chloroform-methanol-water, as is the case in the conventional lipids extraction method first described
by Bligh and Dyer (1959). This lipid extraction method is quite often used in the literature as a reference
method compared to various other described methods. However, the use of chloroform in large scale is
precluded by environmental and health risks. The use of other solvents has therefore been investigated.
Talukder et al. (2012) used hexane to extract lipids from dried biomass of N. salina following an acid
hydrolysis pretreatment (5% sulfuric acid at 120ºC for 1 h). Incubation of the hydrolysate with hexane
(at 40ºC and mixing at 200 rpm up to 24 h) was followed by centrifugation (4,000 rpm) allowing separation
of the hexane phase from the hydrolysate. The hexane phase afforded a lipid yield of 85.6%. Hexane was
also used to extract lipids from the microalga Chlorococcum sp. (Halim et al. 2011) with a very low yield
(1.5%), though it should be noted that this alga was found to have a maximum lipid yield of 7.1%.
Using various concentrations (50 to 95%) of isopropanol, Yao et al. (2012) performed the extraction
of lipids from the microalga Nannochloropsis sp. It was concluded that the 70, 88, and 95% IPA
concentrations extracted 82–92% of total oil.
Using the marine microalgae Isochrysis aff. galbana, the extraction of the carotenoid fucoxanthin
with different solvents (hexane, acetone, ethyl acetate, methanol, and ethanol) and various extraction