Journal of CO₂ Utilization 36 (2020) 196–209

Contents lists available at ScienceDirect

Journal of CO2 Utilization

journal homepage: www.elsevier.com/locate/jcou

Recent developments in supercritical fluid extraction of bioactive T

compounds from microalgae: Role of key parameters, technological
achievements and challenges
Antonio Molinoa,*, Sanjeet Mehariyaa,d, Giuseppe Di Sanzob, Vincenzo Laroccab, Maria Martinob,
Gian Paolo Leonec, Tiziana Marinod, Simeone Chianesed, Roberto Balducchib, Dino Musmarrad
a
ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability - CR Portici, P. Enrico Fermi, 1,
80055 Portici, NA, Italy
b
ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability - CR Trisaia, SS Jonica 106, 75026
Rotondella, MT, Italy
c
ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Sustainability - CR Casaccia, Via Angullarese 301,
00123 Roma, RM, Italy
d
Department of Engineering, University of Campania “Luigi Vanvitelli”, Real Casa dell’Annunziata, Via Roma 29, 81031 Aversa, CE, Italy

A R T I C LE I N FO A B S T R A C T

Keywords: Microalgae are a rich source of natural bioactive compounds, e.g. astaxanthin, β-carotene, lutein, and fatty acids
Microalgae (FAs), that are currently in high demand in the market. Conventional extraction methods often produce adverse
Carotenoids effects on some of these compounds. Replacing conventional extraction methods with more efficient advanced
Fatty acids green technologies that offer greater extracts purity and low environmental impact is therefore a challenging and
Lipids
sought-for target. This review is a comprehensive overview of supercritical fluid (SCF) extraction processes,
Pre-treatment
Supercritical-CO2 fluid extraction
including the latest research on the extraction of bioactive compounds from microalgae biomass and their
Selectivity benefits on human health. In addition, the role of key operating parameters on the selectivity of various com-
pounds is discussed. This study provides useful knowledge that can productively contribute to the future de-
velopment of SCF-based extraction technologies on an industrial scale.

1. Introduction in pharmaceutical, nutraceutical, cosmetic, and food industries as nat-

ural medicines, food additives and UV-light protective agents
Microalgae are photosynthetic organisms including more than 7000 [2,13–17]. In this context microalgae can be an interesting starting
species that grow in a wide variety of habitats. They can develop in point to obtain these natural compounds with a sustainable approach
such diverse environment conditions as freshwater, brackish water and [18].
sea water [1]. Interestingly, microalgae can be grown in various sys- In recent years research has focused on the extraction of carotenoids
tems i.e. close reactors, as photobioreactors or open ponds or marine and FAs, in high demand as food additives. Different conventional and
environments, that can be located or installed in marginal or un- non- conventional extraction technologies such as maceration, soxhlet
productive lands with no use of herbicides/pesticides, thus lowering the extraction, ultrasound-assissted extraction, microwave-assisted extrac-
impact on environment. In the presence of sun light, microalgae are tion are used. The choice of the suitable extraction method depends on
able to convert carbon dioxide (CO2) into oxygen (O2) and biomass aspects like biomass properties, physical-chemical properties of the
[2–4]. Nowadays, research on microalgae has rapidly increased as there extractable molecules and their perspective end use - pharmaceutical,
is interest in exploiting their potential in terms of CO2 mitigation and cosmetic, food [18]. Additionally, because of their oxidative properties,
wastewater treatment [5–8]. In addition, microalgae biomass is a rich the extraction of these bioactive compounds requires strict control of
source of natural compounds like carotenoids, astaxanthin, lutein, and temperature, pressure, presence of light, and time. Moreover, bioactive
fatty acids (FAs) [9–12]. These bioactive compounds have positive and compounds have different polarity, which makes their simultaneous
important properties for all living creatures. In fact, they are being used extraction difficult with a single solvent. Usually, non-polar solvents are

⁎
Corresponding author.
E-mail address: [email protected] (A. Molino).

https://doi.org/10.1016/j.jcou.2019.11.014
Received 6 September 2019; Received in revised form 8 November 2019; Accepted 10 November 2019
2212-9820/ © 2019 Elsevier Ltd. All rights reserved.
A. Molino, et al. Journal of CO₂ Utilization 36 (2020) 196–209

used to extract non-polar bioactive compounds, while polar solvents are carotenoids [41]. Among them, the astaxanthin market demand was
better suited for polar compounds [19–21]. Conventional techniques equal to 550 million USD (M$) in 2017 and is estimated to reach
such as squeezing, maceration, infusion, percolation, steam distillation around 800 M$ in 2023 with a CAGR of 4.8 % [42]. In 2015, the β-
and solvent extraction are used to extract bioactive molecules from carotene market demand was estimated to be around 425 M$ with a
different biomass matrices [18,19,21]. These techniques often present forecast of 500 M$ in 2023 (Fig. 1b) [40]. In addition, the lutein market
several disadvantages, e.g. thermal degradation of molecules due to the demand was 135 M$ in 2015 and may be get around 200 M$ in 2024,
high temperature of extraction, or solvent residues in the extracts that with a CAGR of 5.3 % thanks to the growing demand of lutein-rich
can compromise their end use [18]. dietary supplements [43].
Advanced extraction with SCF allows for the preservation of the Lipids are generally liposoluble substances composed of carbon and
natural qualities of bioactive compounds, reducing the environmental hydrogen atoms binds with covalent bonds composed by FAs molecules.
impact and minimizing energy costs at the same time [19,21–30]. This kind of lipids is also called triglycerides. An important class of
Furthermore, because of the structural complexity of the microalgae lipids is composed of the phospholipids that make up cell membranes.
biomass composition, the rate of intracellular compounds recovery is FAs are composed of linear chains of hydrocarbons with 12–24
low - three-layer or two-layer cell walls impede the mass transfer of carbon atoms that can be saturated, mono unsaturated and poly-
intracellular molecules during extraction. Therefore, microalgae bio- unsaturated, depending on the kind of bonds in their chain [44]. FAs
mass requires appropriate pre-treatments (biological/chemical/phy- represent a fraction of lipids that can be produced by animals, vege-
sical) before the extraction step, which facilitates mass transfer of in- tables and microorganisms; they are contained into the cells and are
tracellular compounds from microalgae chloroplast [31–35]. linked to glycerol molecules to constitute glycolipids that have struc-
In this review the latest applications of bioactive compounds for tural functions for the cellular membrane [3]. FAs can be classified as
human health are illustrated as well as the advancement of many pre- ω-3 FAs as docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19), eicosa-
treatment techniques for microalgae biomass and their significant role pentaenoic acid (EPA, 20:5Δ5,8,11,14,17), and α-linolenic acid (ALA,
on extraction efficiency of bioactive compounds are summarized. 18:3Δ9,12,15) as well as ω-6 FAs as arachidonic acid (ARA, 20:4Δ5,8,11,14)
Extensively, detailed examination of the application of SCF extraction [45]. These ω-3 and ω-6 FAs have several health benefits, especially for
to recover bioactive compounds from microalgae biomass is put for- the prevention of cardiovascular and inflammatory diseases, and con-
ward in this study. tribute to the mental health of adults and to the development of the
brain of the foetus and of children in the early years of life [46]. Reg-
2. Classification of bioactive compounds and market demand ulatory agencies and the Food and Agriculture Organization/the World
Health Organization (FAO/WHO) have established that the adequate
Bioactive compounds can be classified as carotenoids and FAs and consumption of polyunsaturated fatty acids (PUFA) should be between
chemical structure, polarity, density and chemical formula of each 6 % and 11 % of the total caloric value of the diet in adult humans [47].
bioactive compound showed in as shown in Table 1. Microalgae-derived PUFAs, such as ARA and DHA, are added as sup-
Carotenoids are derivatives of the C40 tetraterpenoid pigment plements to infant food that is worth 10 B$ per annum [2,45]. There-
phytoene, and synthesized by two chains of C20 geranylgeranyl di- fore, the global demand of PUFAs market is projected to achieve a
phosphate molecules. Around 600 carotenoids have been identified promising CAGR of 10.7 % in 2016–2026 [48].
mainly in plants and microorganisms such as bacteria, fungi, and mi-
croalgae [36]. Carotenoids are natural, yellow-orange lipo-soluble 3. Selection of microalgae for bioactive compounds and its
pigments and can be divided into two major groups: (i) hydrocarbon application
carotenoids and (ii) oxygenated carotenoids. Hydrocarbon carotenoids
are known as α-carotene, β-carotene and lycopene, while oxygenated Microalgae species can accumulate significant amount of natural
carotenoids are derivatives of hydrocarbon carotenoids, also known as bioactive compounds having different applications in pharmaceuticals,
xanthophylls, such as lutein, canthaxanthin, astaxanthin, and fucox- nutraceuticals, cosmeceuticals, food, and feed supplements. Among the
anthin) [1,36–39]. The market demand of carotenoids was calculated to several microalgae species, fresh water microalgae Haematococcus plu-
be 1.24 billion USD (B$) in 2015 and is expected to grow to 1.6 B$ in vialis can be cultivated for the production of natural astaxanthin, whose
2023 with a compound annual growth rate (CAGR) of 3.5 %, as shown content can reach up to 5 % of dry biomass weight (DBW), corre-
in Fig. 1a [40]. The most used carotenoids are astaxanthin, β-carotene, sponding to 80–99 % of total carotenoids. Natural astaxanthin has
and lutein, accounting for more than 70 % of the total market of several health benefits and is considered as a “super anti-oxidant”. In

Table 1
Classification of microalgae derived bioactive compounds and their chemical structure/properties.
Major source Bioactive compound Chemical structure Polarity Density (g/cm3) Chemical formula

Haematococcus pluvialis Astaxanthin Polar 1.071 C40H52O4

Dunaliella salina β-carotene Non-polar 1.00 C40H56

Scenedesmus almeriensis Lutein Polar 1.071 C40H56O2

Nannochloropsis gaditiana Docosahexaenoic acid (DHA) Non-polar 0.943 C22H32O2

Eicosapentaenoic acid (EPA) Non-polar 0.943 C20H30O2

α-linolenic acid (ALA) Non-polar 0.916 C18H30O2

Arachidonic acid (ARA) Non-polar 0.922 C20H32O2

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Fig. 1. Projected global demand of carotenoids. [a] Predictable global carotenoids market revenue in 2023, with respect to the carotenoid type as described by Global
Market Insights, published in 2018; [b] Approximate growing global market revenue of β-carotenoids from all sources, both synthetic and natural.

2016, Shah et al. [1] reviewed more than 50 publications to summarize powder on antitumor activity in an in-vivo model of mammary carci-
the potential health benefits of Haematococcus derived astaxanthin. nogenesis and resulted in up to 83.4 % reduction of tumor size. Re-
Their review suggests that natural astaxanthin is a superior antioxidant, cently, Saini and Keum [36] summarized the main carotenoid-based
able to reduce inflammation, improve gastrointestinal health and im- commercial products available on the market - derived from different
mune-system, potentially act against atherosclerotic cardiovascular sources - as reported in Fig. 3.
disease, reduce the chance of heart attacks, Alzheimer's disease and Several microalgae species from different genera can accumulate
other neurological diseases, inhibit the growth of cancer cells and tu- significant amounts of FAs, mainly EPA and DHA, as reported by
mours, protect from UV-radiations and decrease the chance of idio- Adarme-Vega et al. [2]. Phaeodactylum tricornutum and Nannochloropsis
pathic infertility. sp. can accumulate up to 39 % of EPA of the total FA content, while up
Some microalgae species, such as Muriellopsis sp., Scenedesmus al- to 30–40 % of DHA content of total FAs can be produced using
meriensis, Chlorella protothecoides, C. zofingiensis, Botryococcus braunii, Thraustochytrium and Schizochytrium limacinum microalgae strains [2].
Neospongiococcus gelatinosum, and Chlorococcum citriforme, can accu- Camacho-Rodríguez et al. [55] reported that N. gaditiana is a rich
mulate significant amounts of lutein [49]. However, among them, S. source of FAs, with an EPA content of up to 4.3 % of DBW. Several
almeriensis is one of the most suitable sources, since it can accumulate studies showed that ω-3 and ω-6 rich FAs have various health benefits
lutein in the range of 0.2-0.5 % on dry weight that is 10–15 times such as prevention of cardiovascular, nervous system and inflammatory
greater than the lutein content in Tagetes erecta flower [49,50]. S. al- diseases [56]. Horrocks and Yeo [57] reported that deficiencies of ω-3
meriensis has proven to be a potential source of lutein, which finds uses can cause foetal alcohol syndrome, deficit hyperactivity disorder, cystic
in food and pharmaceutical industries [51]. In Europe lutein is been fibrosis, phenylketonuria, unipolar depression, aggressive hostility, and
classified and authorized as a food additive, as in Regulation No 231/ adrenoleukodystrophy, while a regular intake of DHA rich ω-3 FAs can
2012 with E161b classification [52]. In addition, lutein offers several help reduce the risk of asthma, hypertension, arthritis, thrombosis,
benefits for human health as it plays a role in the prevention of age- myocardial infarction and cardiac arrhythmias, depression, adult-onset
related macular degeneration (AMD) and cataracts, amelioration of the diabetes mellitus, thrombosis, and some cancers [58]. In addition, ω-3
first stages of atherosclerosis, protection of the retina from oxidative exhibits several positive effects on brain function and the nervous
stress and some types of cancer [53]. system. Particularly, an adequate intake of EPA and DHA in pregnant
The unicellular halophilic microalgae Dunaliella salina and Dunalielle women is essential for healthy brain development of the foetus [59].
bardawil are the most common sources of β-carotene, producing up to Whereas, ω-6, (ARA), DHA are necessary for normal growth and
10–12 % of β-carotene of DBW [38]. D. salina derived β-carotene functional development of infants [2].
manifests strong antioxidant activity and is a nutrition supplement for
its natural antimicrobial and antioxidant properties; it can also reduce
the risk of several reactive oxygen species (ROS)-associated diseases 4. Safety regulation and dose/intake recommendation of
such as cancer [16]. The molecular mechanism involving bioactive bioactive compounds
compounds induced ROS-associated cell death in a cancer cell is shown
in Fig. 2. US Food and Drug Administration (FDA) classified solvents in three
Atasever-Arslan et al. [17] studied the cytotoxic effect of D. salina different classes: class 1, class 2 and class 3, and also recommended the
extract against SH-SY5Y neuroblastoma cells and reported that D. salina permitted daily exposure (PDE) limit of each solvent for their uses in
extract manifested anticancer properties. β-carotene can inhibit carci- pharmaceuticals/nutraceuticals. Table 2 showed the list of solvents,
nogenesis in a murine model of squamous cell cancer by increasing cell which falls into different class of solvents as per FDA classification. The
immunity [16]. β-carotene is the precursor of vitamin A, which is im- solvents in class 1 should not be employed in the pharmaceutical/food
portant for the function of retina. Singh et al. [14] cultivated D. salina industry for manufacturing of human consumable products because of
under different stress conditions, which enhance β-carotene con- their unacceptable toxicity or their deleterious environmental effect.
centration. Furthermore, the extract was tested against cancer cell The use of class 2 solvents should be limited with PDE because of their
growth and resulted in a significant antioxidant and cytotoxic effect in inherent toxicity. The PDE limit of class 2 solvents depends on each
breast cancer cell lines (MCF-7). The extracts of D. salina are able to individual compound; for example, for chloroform PDE limit is equal to
induce apoptosis and G0/G1 arrest in the A549 cancer cell line [54]. 0.6 mg/day, while hexane can be used up to 2.9 mg/day. Solvents in
Srinivasan et al. [15] investigated the effect of D. salina lyophilized class 3 are regarded as less toxic as they have a lower risk to human
health and can be used in pharmaceuticals/nutraceutical at specific

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Fig. 2. The molecular mechanism of bioactive compounds induced ROS-associated cell death in a cancer cell as proposed by Centella et al. [37].

levels. However, long-term toxicity or carcinogenicity studies for many bioactive compounds. Microalgae cells can be disrupted/pre-treated by
solvents of class 3 have not been carried out. It is considered that different means, which could be broadly divided into three different
amounts of class 3 residual solvents should be 50 mg/day or less. categories, applied individually or in various combinations, i.e. che-
Therefore, class 3 solvents are generally used in formulation of drugs mical, enzymatic/biological and mechanical/physical, as shown in
and preparation of nutraceutical products. Fig. 4. The choice of the most suitable cell disruption method depends
Furthermore, a specific/controlled dose of bioactive compounds has on hardness of cell walls, physical and chemical properties of micro-
a positive effect on human health for preventing several diseases. On algae cells, and extractable intracellular compounds as well as se-
the contrary, an over dose of bioactive compounds causes adverse im- lectivity of the selected method. A detailed discussion of various ap-
pact on human health [60,61]. Specific doses or tolerable upper intake plied/used pre-treatment methods on microalgae biomass for effective
levels for bioactive compounds were proposed by the European Food extraction of bioactive compounds is presented in the following sub-
Safety Authority (EFSA), FAO, and WHO. EFSA established the dietary sections.
recommendations for EPA and DHA, which should be in the range of
250−500 mg/day for adults. The dietary recommendation was based 5.1. Chemical pre-treatments of microalgae biomass
on cardiovascular risk considerations for European adults. The high
intake of EPA and DHA can cause several negative effects on human Chemical pre-treatment methods are easy and offer several ad-
health, including bleeding episodes, impaired immune function, in- vantages, as low energy input and consumption, and good scalability,
creased lipid peroxidation, and impaired lipid and glucose metabolism but are also characterized by some drawbacks, such as chemical cost,
[61]. However, no tolerable upper intake level (UIL) for EPA, DHA or toxicity and challenging recovery of chemicals in the final products.
DPA was set [61]. EFSA indicated as a supplemental intake of 20 mg/ Different chemicals, such as organic solvents, acids, bases, hydrogen
day of β- carotene could be appropriate [62]. However, there are in- peroxide, ozone, and ionic liquids could be used for the chemical pre-
adequate scientific data to establish a precise limit for an UIL of isolated treatment of microalgae biomass to enhance the recovery of bioactive
β-carotene as no dose-response relationship for β-carotene effects is compounds [33]. Organic solvents can favour the pre-treatment step
available either from the intervention trials in humans or from appro- and increase the extraction yield of carotenoids in comparison to that
priate animal models. Moreover, it is not possible to be more specific in obtained with acids [38]. Singh et al. [64] used various chemicals to
distinguishing different isomeric forms of β-carotene or specific for- pre-treat Thraustochytrids for the acetone-based extraction of astax-
mulations. The consumption of astaxanthin is generally safe through anthin. They freeze dried Thraustochytrids biomass with organic or in-
supplemented food. According to Ambati et al. [60] the recommended organic acids and observed a significant increase in the astaxanthin
dose of astaxanthin could be around 2−4 mg/day. Another study re- extraction yield. Results indicated that the extraction yield was greater
ported that no adverse effects were found with the assimilation of 6 mg/ for inorganic acids than organic ones. A greater astaxanthin extraction
day of astaxanthin in adult human [63]. Although, astaxanthin UIL recovery was obtained with HCl and H2SO4, which was 5- and 4-fold
remains still unknown; literature reports do not shown significant side greater in comparison to the extraction with untreated biomass, re-
effects of astaxanthin consumption in animals and humans, hence en- spectively. Authors reported that astaxanthin yield was directly pro-
couraging its applications. portional to the strength of the used acid, but also caused astaxanthin
degradation. On the contrary, the pre-treatment with dimethyl sulf-
5. Pre-treatments of microalgae biomass before extraction oxide (DMSO) improved the astaxanthin yield by 2.5-fold, as compared
with direct extraction. Nevertheless, on an industrial scale the use of
Microalgae cells are composed of rigid, complex and multi-layer DMSO is not desirable, because of its potential toxicity and product
walls, which cause obstacle during the extraction of intracellular mo- contamination [38,64].
lecules. The rigid cell wall prevents the solvent from coming into con-
tact with intracellular compounds and blocks mass transfer of mole- 5.2. Enzymatic or biological pre-treatments of microalgae biomass
cules during the extraction process. Therefore, cell disruption
constitutes an important step of the extraction of various intracellular Enzymatic/biological hydrolysis is a promising, non-destructive

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Fig. 3. Major carotenoids-based commercial products available on the global [36].

pre-treatment and can prevent thermal degradation of thermolabile and high enzyme costs that need additional process optimization [33].
carotenoids. Enzymatic/biological pre-treatment is based on the use of Praveenkumar et al. [31] suggested energy efficient biological pre-
cell-wall degrading enzymes, such as cellulose and amylase, aid cell treatment using germination based on the life cycle of H. pluvialis for
disruption and the recovery of intracellular compounds [18,65]. The astaxanthin extraction. The natural germination resulted in damage and
enzymatic cell-wall degradation is very limited for H. pluvialis sp., due deconstruction of the cyst cell-wall, while the controlled germination of
to rigid cell wall properties, which demand greater concentration of red cysts for around 12−18 hours led to 1.2-fold increase of the as-
lysing enzymes. However, enzymatic pre-treatment has some issues taxanthin yield respect to the initial mature cysts. The supplementation
such as prolonged reaction time, causes contamination of the extract, with nitrate, which promotes cyst germination, was reported for the

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Table 2
FDA classification of different solvents in varied class.
Class of solvents Solvents

Class 1 Benzene, Carbon tetrachloride, 1,2-Dichloroethane, 1,1-Dichloroethene, 1,1,1-Trichloroethane

Class 2 Acetonitrile, Chlorobenzene, Chloroform, Cumene, Cyclohexane, 1,2-Dichloroethene, Dichloromethane, 1,2-Dimethoxyethane, N,N- Dimethylacetamide, N,N-
Dimethylformamide, 1,4-Dioxane, 2-Ethoxyethanol, Ethyleneglycol, Formamide, Hexane, Methanol, 2-Methoxyethanol, Methylbutyl ketone,
Methylcyclohexane, Methylisobutyl ketone, N-Methylpyrrolidone, Nitromethane, Pyridine, Sulfolane, Tetrahydrofuran, Tetralin, Toluene, 1,1,2-
Trichloroethene, Xylene
Class 3 Acetic acid, Acetone, Anisole, 1-Butanol, 2-Butanol, Butyl acetate, tert-Butylmethyl ether, Dimethyl sulfoxide, Ethanol, Ethyl acetate, Ethyl ether, Ethyl
formate, Formic acid, Heptane, Isobutyl acetate, Isopropyl acetate, Methyl acetate, 3-Methyl-1-butanol, Methylethyl ketone, 2-Methyl-1-propanol, Pentane, 1-
Pentanol 1-Propanol, 2-Propanol, Propyl acetate, Triethylamine

reduction. Singh et al. [64] reported that bead-beating pre-treatment of

Thraustochytrium sp. S7 resulted in a 5-fold greater astaxanthin ex-
traction yield as compared to the untreated biomass, and, in particular,
it increased from 26.77 ± 1.23 μg/g to 138.53 ± 4.27 μg/g. Molino
et al. [67] demonstrated that ball milling mechanical disruption of H.
pluvialis red cyst cell for 5 min at 400 rpm can enhance the astaxanthin
recovery up to 98 % during the extraction. Another physical pre-
treatment method is ultrasonication, which works by generating ul-
trasonic waves having different intensities. Zou et al. [68] reported that
ultrasound irradiation at 200 W with a frequency of 40 kHz for 16 min
at 41.1 °C resulted in an optimum recovery of astaxanthin (27.58 mg/g)
from H. pluvialis. However, the authors also observed that higher tem-
peratures and longer irradiation times led to astaxanthin thermal de-
gradation. In addition, sonicated cell disruption of the Botryococcus sp.,
Chlorella vulgaris, and Scenedesmus sp. at 600 W for 5 min resulted in
lower enzyme release, which was around 10-fold lower as compared to
Fig. 4. Commonly used pre-treatment methods for enhance the extraction re- HPH pretreatment [69]. Halim et al. [70] reported that average cell
covery of bioactive compounds from microalgae biomass. disruption was around 4.5 % during the ultrasonication pre-treatment,
while cell disruption using HPH was approximately 16-fold greater than
astaxanthin extraction by ionic liquids (ILs) as green solvents, by Pra- that for ultrasonication. Results showed that 73.8 % cell disruption was
veenkumar et al. [31]. They reported that natural germination can be a obtained using HPH, while chemical treatment using sulfuric acid re-
uniquely simple pre-treatment procedure for robust microalgae cyst sulted in an average cell disruption of 33.2 % and with bead beating
cell, able to enhance the extraction of astaxanthin at room temperature corresponded to 17.5 %. Singh et al. [64] compared different physical/
with ILs. Furthermore, a greater germination efficiency could be mechanical methods for the extraction of astaxanthin from Thraus-
achieved by manipulating the nutritional composition of the germina- tochytrids and found that astaxanthin extraction yield was greater with
tion medium. This method has several advantages, such as less-energy ultrasonication rather than with chemical and other physical cell dis-
input, absence of toxic solvents, recovery of the natural astaxanthin ruption methods. Steam explosion is another efficient and cost-effective
without thermal degradation and reduction of the downstream costs. pre-treatment method. Nurra et al. [71] used steam explosion for lipid
However, several issues including germination rate improvement recovery from N. gaditana and found that the steam explosion sig-
(longer germination time), synchronized weakening of the cyst cells nificantly improved the lipid extraction yield from 0.3 % to 3.6 % (at
and recovery and recycling of expensive IL should be properly con- 120 °C) and to 8.8 % (at 180 °C). The enhancement in lipid extraction
sidered for further applications [31,33,38]. efficiency is due to the disruption of the microalgae cell wall caused by
the steam explosion pre-treatment. Lorente et al. [32] compared several
pre-treatment methods including autoclaving, ultrasound, microwave
5.3. Mechanical or physical pre-treatments of microalgae biomass and steam explosion to determine the most effective method for the
extraction of lipids. In this case, N. gaditana, C. sorokiniana and
Mechanical/physical cell disruption methods are promising and Phaeodactylum tricornutum were used for lipid recovery and among the
preferred thanks to their ability to avoid product contamination, faster, pre-treatment methods, steam explosion showed the highest lipid ex-
and ease of scaling up [66]. The optimum degree of cell disruption traction yields. Recently, the effect of acid catalysed steam explosion
presupposes that cell wall is broken down in order to facilitate the entry pre-treatment was reported as an efficient pre-treatment method for
of solvents to solubilize intracellular compounds. Mechanical pre- lipid extraction [72]. However, steam explosion may be unsuitable for
treatment before extraction can be exploited to disrupt the cell wall of the extraction of thermolabile bioactive compounds due to the high
microalgae, promoting the recovery of carotenoids and FAs. In fact, to temperatures [18,34,73,74]. Kim et al. [33] reported that mechanical/
achieve high recovery, efficient cell disruption and extraction steps are physical pre-treatments usually can be realistic for concentrating the
required. Mechanical pre-treatment of microalgae cell can be carried biomass in short processing times. However, these methods are highly
out using different approaches, such as ball milling, ultrasonication, energy demanding and need to be scaled-up to become cost-effective.
high pressure homogenization (HPH), and steam explosion [33,38]. In As reported, various cell-wall disruption methods could be used
ball milling pre-treatment, microalgae cells are broken down by ap- including chemical, biological, and physical techniques, but many cri-
plying high intensity fluid-mechanical stress through a specifically de- tical issues, such as energy consumption, toxicity, chemical cost, me-
signed homogenization. Ball milling is a promising technique, in par- tabolite stabilities and scale-up, still need to be investigated and solved.
ticular on microalgae, where the extraction process is facilitated by the A flawlessly optimized pre-treatment method can help in the cost-ef-
mechanical disruption of the cell wall and cell membranes, enabling the fective extraction of bioactive compounds from microalgae biomass.
non-selective release of the intracellular compounds. Several factors
associated with milling can contribute to cell wall disruption and size

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6. Supercritical fluid extraction technology for bioactive especially in the food supplementation with bioactive compounds,
compounds natural oils as peanuts, soybean, olive, and sunflower oils are used as
co-solvents [30,85]. However, extract composition is greatly affected
Extraction technologies are often based on the temperature and by extraction operational conditions such as temperature, pressure, co-
pressure strength using organic or aqueous solvents. Bioactive com- solvents, solvent flow rate, and extraction time and need to be opti-
pounds exhibit varying polarity, solubility and chemical stability, thus mized for an efficient recovery of bioactive compounds
appropriate pressure as well as solvent type should be selected for the [18,21,24,27,73,85,86].
extraction of targeted bioactive compounds. Temperature is another
parameter that influences the extraction process. SCF extraction could 6.1. Role of temperature and pressure
be a promising technique for the optimization of recovery of molecules
with potential applications in several industrial sectors. In fact, through Extraction temperature and pressure play a significant role on so-
SCF extraction, it is possible to reach greater extraction selectivity and lubility of solutes in the solvent, which mainly depends on the chemical
short processing times [21,75] in comparison with traditional extrac- properties of extractive target compounds. For example, astaxanthin
tion methods. Moreover, the use of SCF allows to prevent the presence and lutein show thermal degradation at high temperature, therefore its
of solvent traces in the final extracts with the possibility at large scale of value needs to be optimized for extraction [13,24,67]. In the SCF-CO2
CO2-recovery in a close loop with economical advantage respect to the extraction of bioactive compounds, the extraction efficiency increases
use of other solvents with CO2 pressure and temperature up to optimal level. However, a
This last issue is especially felt when the extracts find applications as higher temperature causes thermal degradation of compounds, while
food additives in the nutraceutical field [21,24,37,67,75,76]. Several greater pressure can obstruct the diffusivity of supercritical fluid into
solvents can be used in supercritical fluid extraction due to their critical the matrix, which leads to a decreased extraction yield [18,73,87,88].
property such as ethanol, hexane, methanol, pentane, butane, nitrous Also, greater temperature and pressure imply the production of ex-
oxide, sulfur hexafluoride and fluorinated hydrocarbons, but CO2 is traction waxes [75]. Pressure and temperature influence the solvation
used for over the 90 % of SCF extraction of natural compounds [77]. power of fluid due to their actions on the solvent density and can be
The thermodynamics and heat transfer properties of CO2 makes it a controlled/modified by acting on the operating pressure and tempera-
preferred solvent for SCF based extraction processes. This technology is ture. However, at constant temperature, increasing pressure causes an
based on the use of an extraction fluid used over its critical conditions increase in the CO2 density and solvation power of the fluids, which
to improve the extraction capacity. The critical conditions of some enhance the solubility of the bioactive compounds and extraction yield.
solvents are reported in Table 3 [76]. Data in Table 3 show that SCF- At constant pressure, an increase in temperature reduces the solvent
CO2 represents a superior solvent for extraction of thermolabile mole- density and solvation power. Therefore, solubility is closely related to
cules without any degradation or change of the chemical composition the SCF-CO2 density and the properties of the solute, such as molecular
thanks to lower extraction temperature and pressure [77]. Also, CO2 mass, polarity, and vapour pressure [18,75,86]. Di Sanzo et al. [24]
has similar physicochemical properties like a gas and a liquid, showing reported that increasing extraction temperature from 50 to 80 °C at
similar viscosities, intermediate diffusivities, and high density that en- constant pressure caused a decrease in extraction yield of astaxanthin
hance both the penetration into microalgae biomass and the solubili- and lutein, while by increasing pressure from 10 to 55 MPa at constant
sation of intracellular compounds [78]. In addition, CO2 is not-flam- temperature the extraction yield was enhanced and showed 98.6 % and
mable, less toxic, cost-effective as compared to organic solvents [76]. In 52 % recovery of astaxanthin and lutein at 50 °C and 55 MPa, respec-
SCF−CO2 extraction, solvent is in gas phase at atmospheric conditions, tively (Table 4). In addition, 93.25 % recovery of FAs was achieved at
so a substantial removal of CO2 is achieved in extracts and leads to a moderate temperature, i.e. 65 °C and a pressure of 55 MPa. Yen et al.
solvent-free extract. In addition, if employed on an industrial scale, CO2 [89] studied the effect of temperature (35−80 °C) at 40 MPa and the
could be recycled [76]. effect of pressure (20−40 MPa) at 47.5 °C, observing that the gradual
In supercritical conditions CO2 easily penetrates the cell wall due to increase in the extraction temperature and pressure enhanced lutein
its high permeability and diffusivity parameters [18,21,24,38,75,77]. recovery from Scenedesmus biomass and similar phenomena was ob-
The main disadvantage related to the use of SCF-CO2 is that CO2 shows served by Macías-Sánchez et al. [87] during the extraction of lutein and
a chemical behaviour similar to that of lipophilic solvents. In fact, it is carotenoids from S. almeriensis. Even though, the increasing tempera-
able to extract non-polar compounds because CO2 is non-polar solvent. ture was correlated also to a greater impurity content as observed in the
In order to overcome this obstacle, substances with opposite polarity, HPLC analysis profile [89], for instance, Macías-Sánchez et al. [90]
such as water, methanol, and ethanol as co-solvents are often used to observed that carotenoids extraction efficiency increased at tempera-
modified the solvent polarity [18,24,34,38,77,85]. In some cases, tures greater than 60 °C, and 30, 40 and 50 MPa from D. salina. This
behaviour was attributed to the fact that at these pressures the density
of the CO2 was greater and, at the same time, the increase in tem-
Table 3 perature enhanced the solvent diffusivity and the vapour pressures of
Critical properties of different solvents used in SF extraction processes.
the extracted carotenoids, thus supporting their dissolution and
Solvent Critical Critical Critical Critical Reference yielding better extraction yields. Furthermore, several studies high-
temperature (°C) pressure volume (cm3/ lighted similar effects of pressure and temperature on the recovery of
(MPa) mol)
several bioactive compounds [87,88,90–92].
Ammonia 405.4 11.35 72.5 [26] The effects of temperature and pressure during SCF-CO2 extraction
CO2 31.1 7.37 94.1 [23] on bioactive compounds recovery from microalgae are reported in
Dimethyl ether 126.95 5.27 171.0 [79] Table 4.
Ethane 32.15 4.87 145.5 [29]
Ethylene 9.15 5.04 131.0 [80]
Methanol 239.45 8.09 118.0 [81] 6.2. Role of co-solvent
n-Hexane 234.35 3.02 368.0 [82]
Propane 96.65 4.25 200.0 [79] CO2 manifests a limited ability to extract high-polarity compounds
Water 373.95 22.06 55.9 [83] at high densities. The addition of polarity modifiers (co-solvents) to CO2
Xenon 16.95 5.80 118.0 [28]
can improve the extraction efficiency by increasing the solubility of the
Olive oil 720.0 0.33 204.0 [84]
Soybean oil 697.0 0.33 192.0 [84] polar and non-polar bioactive compounds. Since supercritical CO2 is a
non-polar solvent, the addition of polar co-solvent improves the

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Table 4
Effect of temperature and pressure during SCF-CO2 extraction of bioactive compounds from microalgae.
Microalgae Extraction Condition Bioactive compound recovery* (%) Reference

a b c d
CO2 FR (g/min) t (min) P (MPa) T (°C)

H. pluvialis 7.48 120 55 80 13.9 (Astaxanthin), 1.6 (Lutein) [24]

65 36.2 (Astaxanthin), 38.4 (Lutein)
140 50 98.6 (Astaxanthin), 52.4 (Lutein)
120 40 95.8 (Astaxanthin), 46.7 (Lutein)
10 0.5 (Astaxanthin), 1.0 (Lutein)
N. gaditana 14.48 100 25 50 24.0 (EPA) [93]
65 27.2 (EPA)
40 18.5 (EPA)
55 19.2 (EPA)
D. salina 14.48 110 55 50 15.1 (β-carotene) [94]
65 21.5 (β-carotene)
75 17.7 (β-carotene)
10 65 1.7 (β-carotene)
40 25.5(β-carotene)
55 21.5 (β-carotene)
S. almeriensis 14.48 120 25 65 14.2 (Lutein) [95]
40 50.3 (Lutein)
55 97.6 (Lutein)
50 92.3 (Lutein)
Nannochloropsis sp. 14.48 100 10 75 4.79 (EPA) [96]
40 8.74 (EPA)
55 15.59 (EPA)
10 50 11.32 (DHA)
40 39.49 (DHA)
55 79.63 (DHA)

*Recovery of bioactive compound was calculated based on initial content of bioactive compounds, which was extracted using standard extraction methods. a: CO2
flow rate (g/min); b: Total extraction time (h); c: Operative pressure (MPa); and d: Operative Temperature (°C).

Table 5
Role of different co-solvent for recovery of bioactive compounds from several microalgae species.
Microalgae Extraction conditions Carotenoid extaction Yield/ Recovery* Reference

a b c
Pre-tratment method; P (MPa), T (°C), t (h) Co-solvent (% of CO2)

Scenedesmus Freeze drying; 40 MPa, 70 °C, 1 h 40% (mol%) Methanol Lutein recovery of 50.75% [89]
40% (mol%) Ethanol Lutein recovery of 62.20%
40% (mol%) Propanol Lutein recovery of 51.55%
40% (mol%) Butanol Lutein recovery of 38.68%
40% (mol%) Acetone Lutein recovery of 16.91%
Haematococcus pluvialis 40 Mpa, 70 °C, 5 h 10% (v/v) Olive oil Asthaxanthin recovery of 51% [27]
10% (v/v) Soybean oil Asthaxanthin recovery of 36.4%
Nannochloropsis oculata Grinding and freeze drying; 35 MPa, 50 °C, 0.5 h 10 mL Ethanol Zeaxanthin recovery of 63.2% [99]
10 mL Soybean oil Zeaxanthin recovery of 32.2%
10 mL Dichloromethane Zeaxanthin recovery of ∼40%
10 mL Toluene Zeaxanthin recovery of ∼20%
Haematococcus pluvialis Hydrotermal; 8 MPa, 55 °C, 0.25 h 20% (v/v) Ethanol Astaxanthin recovery of 98.3% [85]
20% (v/v) Olive oil Astaxanthin recovery of 98.6%

*Recovery of bioactive compound was calculated based on initial content of bioactive compounds, which was extracted using standard extraction methods. a:
Pressure (MPa); b: Temperature (°C) and c: Total extraction time (h).

extraction ability of polar bioactive compounds. The use of acetone, varies up to a certain value, above which a further addition does not
butanol, dichloromethane, ethanol, methanol, propanol, toluene, ve- show any positive effect. Furthermore, the appropriate choice and
getable oil, and water was reported by several authors with the aim to concentration of co-solvent is another major factor that can affect the
enhance the recovery efficiency of carotenoids and FAs extraction efficiency and selectivity [75,86,97]. Among the several co-
[18,20,27,73,85,91,97–99]. The co-solvent addition allows swelling of solvents, ethanol has been broadly used as a co-solvent in extraction of
microalgae biomass, thus increasing the internal volume and the sur- bioactive compounds due to less toxicity, since it is considered as food
face area for the contact, enhancing the mass transfer by creating hy- grade solvent, which can be used in cosmeceutical, food, nutraceutical,
drogen bonding with the intracellular biomass compounds [88], and and pharmaceutical industries [3,19,22,34,38,78,89–92,97,99–101].
increasing solvent polarity [3,18,73,75]. The main effect on the ex- The choice of the co-solvent should be determined by the affinity be-
traction depends on the co-solvent type, biomass composition, and tween the co-solvent and the extractable target bioactive compounds
target extractable compound [18]. For example, β-carotene is a non- [75]. Kitada et al. [102] optimized the choice of co-solvent, i.e., ethanol
polar compound due to its greater molecular weight, which shows and acetone, and observed that ethanol was a more suitable co-solvent
lower solubility in SCF-CO2. Therefore, addition of polar co-solvent for the extraction of lutein and β-carotene with respect to acetone. SCF-
improves its extraction yield [19,88]. Addition of 5 % (v/v) ethanol as CO2 with ethanol as co-solvent showed ∼6-fold greater lutein recovery
co-solvent allows the extraction of palmitoleic and linolenic acid during with respect to SCF-CO2 without co-solvent at 60 °C and 30 MPa. In
SCF-CO2 use [19]. In general, the amount of co-solvent to be added another study, Yen et al. [89] used different co-solvents with varied

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polarity during SCF-CO2 extraction of lutein from Scenedesmus sp. and et al. [91] assessed the effect of CO2 flow rate (2−4 mL/min) at 50 MPa
reported that ethanol was a more effective co-solvent compared with and 50 °C with and without co-solvent on astaxanthin content in the
methanol, propanol, butanol and acetone. SCF-CO2 extraction with extract. In absence of co-solvent, the amount of total extract could be
ethanol as co-solvent showed ∼62 % recovery of lutein, while SCF-CO2 increased, whereas the amount of astaxanthin in the extract almost
extraction with acetone yielded ∼17 % recovery, with 40 % (mol%) of remained unchanged. By employing ethanol as co-solvent, the amount
co-solvent/CO2 (ratio of co-solvent flow vs. CO2 flow rate) at 70 °C and of astaxanthin extracted was 2 times higher, and effective extraction
40 MPa (Table 5). However, authors proposed that ethanol played a could be achieved at lower pressure (40 MPa). In addition, when the co-
role as an “entrainment’’ in the pressure-composition phase diagram of solvent was used, the CO2 flow rate considerably affected the extracted
CO2, lutein and ethanol, which led to the enhancements of solubility of content of astaxanthin. In fact, larger amounts of astaxanthin were
lutein and improved lutein recovery [89]. Krichnavaruk et al. [27] in- extracted as the CO2 flow rate decreased. Ruen-Ngam et al. [86] in-
vestigated the role of ethanol, olive oil, and soybean oil as co-solvents vestigated the role of CO2, and co-solvent flow rate on lutein extraction
for SCF-CO2 extraction of astaxanthin from H. pluvialis. In the first at 40 °C and 40 MPa. Results showed that extraction of lutein was im-
phase, the experiment was carried out to achieve the optimum con- proved by increasing CO2 flow rate from 2 mL/min up to an optimum
centration of co-solvent as 6, 10, and 12 % (v/v) to CO2. Results showed level and tended to decrease in extraction efficiency at greater CO2 flow
that co-solvent concentration of 10 % (v/v) to CO2 was optimum for rate. Therefore, increasing CO2 flow rate up to optimum level caused an
selectivity of astaxanthin from H. pluvialis, whereas, soybean oil was increase in intermolecular interaction and force between solute and
less effective than olive oil and ethanol as 10 % co-solvent at 70 °C, solvent; it may cause reduction in intra-particle diffusion and could be
40 MPa, and at the solvent flow rate of 3 mL/min. Recently, Saravana dominant in extraction process. Therefore, lutein recovery was lower at
et al. [97], extracted carotenoids from brown algae using different ve- the CO2 flow rate of 5 mL/min compared to at 3 and 4 mL/min. Sub-
getable oils (canola, soybean, sunflower oil), ethanol and water with co- sequently, the role of ethanol as co-solvent flow rate of 0-0.5 mL/min
solvent flow rates [0.50–2.00 (% of CO2, w/w)] at 50.62 °C, 30 MPa. on lutein extraction efficiency was investigated at 40 °C and 40 MPa.
The data indicated that sunflower oil used as a co-solvent with SCF-CO2 The recovery of lutein was drastically increased at co-solvent flow rate
increased the recovery of carotenoids and its efficiency was greater than of 0.4 mL/min, reaching maximum at ∼53 %, which was ∼28-fold
canola, soybean, ethanol, and water. In addition, SCF-CO2-extracted oil greater than extraction without co-solvent. However, increasing the
with sunflower oil as co-solvent had a content rich in FAs, high anti- ethanol flow rate further to 0.5 mL/min caused ∼3-fold decrease of
oxidant activity, and high stability. In addition, co-solvent increases the lutein recovery. Ethanol as co-solvent improved the polar character-
selectivity of extractable bioactive compounds due to diffusion of solute istics of the solvent, which increased the solubility of lutein, while a
and solvent. Vegetable oil could be an appropriate choice of co-solvent very high amount of co-solvent limited the mass transfer diffusion [86].
for supplementation of food products, which can directly be consumed Saravana and co-workers [97] investigated the role of co-solvent flow
as high-quality oil with high content of unsaturated fatty acids. The rate [0.50–2.00 (% of CO2, w/w)] to define the best operative condi-
lutein selectivity was increased up to 2.5-fold with co-solvent flow rate tions in order to enhance the extraction yields. Results demonstrated
of 0.4 mL/min at 40 °C and 40 MPa [86]. Molino et al. [73] found that that by gradually increasing the co-solvent flow rate, the solubilization
the addition of co-solvent in SCF-CO2 reduced the recovery of astax- of bioactive compounds and the extraction yield were enhanced.
anthin and increased the recovery of lutein and therefore, it could be Table 6 summarized the role of co-solvent concentration on extraction
concluded that the co-solvent concentration needs to be optimized on a efficiency of bioactive compounds at optimum extractive pressure and
case-by-case basis considering the different biomass chemical proper- temperature. Recently, Di Sanzo et al. [24] evaluated the SCF-CO2 ex-
ties, co-solvent polarity, target extractable bioactive compounds; it also traction of astaxanthin, lutein, and FAs recovery and purity at different
depends on SCF parameters such as pressure and temperature, extrac- pressures, temperatures and CO2 flow rates. The experimental data
tion time and flow rate. For example, SCF-CO2 without co-solvent was showed that the maximum recovery of astaxanthin and lutein could be
more selective for the extraction of β-carotene than SCF-CO2 with 5 % achieved at lower temperature and higher pressure (50 °C and 55 MPa)
ethanol as co-solvent at 60 °C and 40 MPa. In contrast, selectivity of with the lowest CO2 flow rate investigated (3.62 g/min), while the
palmitic acid, stearic acid, and ω-9 was drastically increased during greater flow rate (14.48 g/min) led to a greater astaxanthin and lutein
SCF-CO2 with 5 % ethanol as co-solvent at 60 °C and 40 MPa. ω-6, and purity. For FAs recovery and intermediate, temperature (65 °C) was
ω-7 were extracted during SCF-CO2 with 5 % ethanol as co-solvent at selected as favourable value at 55 MPa, with a CO2 flow rate of 3.62 g/
60 °C and 40 MPa, while without co-solvent the extraction efficiency min with a greater content of FAs.
was lower, thus might tend to compromise products purity [19]. The Another parameter that strongly influences the extraction process,
choice of co-solvent based on the end application of bioactive com- especially in the case of compounds with similar polarity, is biomass
pound, such as vegetable oils, can be used for direct food supple- loading. In fact, the initial biomass concentration influences the CO2
mentation and ethanol co-solvent can be used for application in cos- and co-solvent flow rate.
meceutical, nutraceutical, and pharmaceutical industries. Thus, the
suitable co-solvent with optimum concentration at promising extraction 6.4. Role of extraction time on extraction efficiency and purity
conditions should be considered industrial scale process.
The role of different co-solvents on the recovery of bioactive com- Short extraction time and high purity are the two main advantages
pounds from several microalgae species is reported in Table 5. of SCF-CO2 technology. Several studies demonstrated the importance of
extraction time for the recovery of target bioactive compounds, but
6.3. Role of CO2 flow rate and co-solvent concentration limited studies discussed its role in determining purity. Extraction ef-
ficiency is correlated with pressure, temperature and solvent flow rate
The flow rate of CO2, co-solvents concentration and extraction time [18,24,91]. Krichnavaruk et al. [27] optimized the effect of extraction
have various effects on selectivity of bioactive compounds and extrac- time on astaxanthin recovery from H. pluvialis using SCF-CO2 at 40 MPa
tion efficiency. There exist only few reports in literature on the role of and 70 °C. The largest amount of astaxanthin was recovered in the first
CO2 and co-solvents on selectivity of bioactive compounds and ex- extract at 60 min, and then tended to decrease steadily over 300 min
traction efficiency [24,27,73,86,91,97]. Flow rate has a significant ef- and this due to the supercritical conditions, in which the solvent is
fect on the residence time for the contact between the solute and the rapidly diffused into the cell matrix and broke down the cells, therefore
solvent. For example, the increase in CO2 flow rate reduces the re- intracellular compounds dissolved in solvent and subsequently re-
sidence time and subsequently decreases the solute dissolution. During covered. Machmudah et al. [91] highlighted that temperature and
investigation of astaxanthin extraction from H. pluvialis, Machmudah pressure had a significant role for the extraction of astaxanthin in a

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Table 6
Effect of co-solvent concentration on recovery of bioactive compounds from several microalgae species.
Microalgae Extraction Condition Bioactive compound recovery (%) Reference

a b c
P (MPa), T (°C), t (h) Co-solvent concentration

Scenedesmus for lutein recovery 40 MPa, 70 °C, 1 h 10% Ethanol/CO2 (mol%) 1.28 [89]
20% Ethanol/CO2 (mol%) 6.04
30% Ethanol/CO2 (mol%) 76.65
40% Ethanol/CO2 (mol%) 62.20
H. pluvialis for astaxanthin recovery 40 MPa 0.5 mL/g sample ∼50 [92]
65 °C, 3.5 h 1.0 mL/g sample ∼60
2.0 mL/g sample ∼78
3.0 mL/g sample ∼80
3.5 mL/g sample ∼81
Chlorella vulgaris for lutein recovery 40 MPa 0.3 mL/min ethanol 53 [86]
40 °C, 2 h 0.4 mL/min ethanol 53
0.5 mL/min ethanol ∼20

*Recovery of bioactive compound was calculated based on initial content of bioactive compounds, which was extracted using standard extraction methods. a:
Pressure (MPa); b: Temperature (°C) and c: Total extraction time (h).

Table 7
Summary of published works at optimum condition on supercritical CO2 extraction with co-solvent of bioactive compounds from several microalgae species.
Algae species Pretreatment Optimum extraction Condition Carotenoid Yield Reference

a b c
Solvent P (MPa) T (°C) t (h)

Haematococcus pluvialis Crushing and then grinding in CO2 and 9.4% (mass) 30 60 na Astaxanthin > 97% recovery [22]
dry ice ethanol
Chlorella vulgaris na CO2 and 7.5% ethanol (v/ 50 80 3 Lutein ∼≥1.8 mg/g algae; β-carotene [102]
v) ∼≥0.2 mg/g
Chlorococcum littorale Freeze drying CO2 and 10% ethanol (m/ 30 60 3 Total carotenoids 0.21% dw [103]
m)
Scenedesmus sp. Freeze drying and milling CO2 and 30% ethanol (m/ 40 70 1 Lutein 2.210 mg/g [89]
m)
Haematococcus pluvialis na CO2 and 5% ethanol (v/v) 55 70 4 Astaxanthin 80.6 % recovery [91]
Haematococcus pluvialis Freeze drying and ball milling CO2 and 10% ethanol (v/v) 30 60 na Carotenoid recovery 92%; [88]
Chlorella vulgaris Crushing CO2 and 5% ethanol (v/v) 30 40 na Carotenoids recovery 69% [104]
Nannochloropsis sp. Ball milling CO2 and 20% ethanol (w/ 30 40 2 45 glipids/100 gdry biomass [3]
w)
Nannochloropsis oculata Freeze drying CO2 and 16.7 wt % ethanol 35 50 0.5 Carotenoids recovery of 74.7% [105]
Nannochloropsis oculata Freeze drying CO2 and 10 mL ethanol 35 50 0.5 Zeaxanthin 63.2% recovery [99]
Monoraphidium sp. Freeze drying CO2 and 20 mL ethanol 20 60 1 Astaxanthin 2.46 ± 0.23 mg/g dw [101]
Haematococcus pluvialis Drying CO2 and 10% olive oil (v/ 40 70 5 Asthaxanthin 51% recovery [27]
v)
Nannochloropsis gaditana Freeze drying CO2 and 5% ethanol 50 60 3 Carotenoids 2.893 mg/galgae dw [106]
Synechococcus sp. (molar) 30 50 Carotenoids 1.860 mg/galgae dw
Dunaliella salina 40 60 carotenoids 9.629 mg/galgae dw
Haematococcus pluvialis Freeze drying CO2 and 2.3 mL/g sample 43 65 3.5 Astaxanthin recovery 87.42% [92]
ethanol
Synechococcus sp. na CO2 and 5% ethanol (v/v) 40 40 3 β-carotene 0.70 mg/galgae dw [19]
Arthrospira platensis Air drying and milling CO2 and 26.7% ethanol (v/ 15 60 0.83 Carotenoids 283 mg/galgae dw [107]
v)
Haematococcus pluvialis Ball milling CO2 and 12.5% (v/v) 55 65 1.33 Astaxanthin recovery 92.4%; [73]
ethanol Lutein recovery 92.9%
Dunaliella salina na CO2 and 5 % mass fraction 20 45 3 25 g carotenoids/kg microalgae biomass [108]

*Recovery of bioactive compound was calculated based on initial content of bioactive compounds, which was extracted using standard extraction methods. a:
Pressure (MPa); b: Temperature (°C), c: Total extraction time (h), and na: not available.

shorter time due to the solvation power of fluid. Di Sanzo et al. [24] and Molino et al. [93] reached a greater purity and recovery of EPA from N.
Molino et al. [93] investigated the effect of extraction time and its in- gaditana using SCF-CO2 at 50 °C and 25 MPa in 20 min observing that by
fluence on purity of astaxanthin, lutein and EPA, respectively. Results increasing the time, the EPA purity decreased. In addition, by in-
demonstrated that 68 % astaxanthin purity was achieved at 50 °C and creasing the extraction pressure and temperature, the EPA selectivity
40 MPa, while 83 % astaxanthin purity was attained at 50 °C and was limited hence both the purity and recovery of EPA decreased.
55 MPa with a CO2 flow rate of 14.48 g/min and an extraction time of Table 7 summarized literature data at optimum conditions for efficient
80 min. Using SCF-CO2 at 50 °C and 55 MPa with a CO2 flow rate of recovery of bioactive compounds. In future, research should be focused
14.48 g/min, astaxanthin purity was increased by increasing the ex- on purity of bioactive compounds and the online coupling of SCF-CO2
traction time up to 80 min, after which purity tended to decrease; while with analytical techniques can be developed and optimized for precise
the maximum recovery was attained at 20 min. In the first extract at analyses of bioactive compounds.
20 min, 20 % purity astaxanthin was obtained, which steadily increased
up to forth extract at 80 min. Moreover, the purity of the extracted
compounds depended on the selectivity and on the amount of extract.

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7. Environmental aspects and technological achievements production from residual biomass could result a self-sustainable and
promising technologies for microalgae biomass processing for high-
Significant advances have been made over the past 20 years towards value added compounds and biofuels.
the commercialization of SCF-CO2 for the extraction of a wide range of
compounds. Recently, SCF-CO2 extraction of bioactive compounds from 9. Conclusions
microalgae biomass has gained tremendous attention due to greater
selectivity and their end uses in different industries. The development Microalgae represent a natural source of bioactive compounds, in-
of SCF-CO2 extraction processes is closely related to environmental cluding carotenoids, astaxanthin, lutein and FAs which find application
sustainability, due to complete extraction using CO2 as green solvent. in pharmaceutical, nutraceutical, cosmetic, medical and food industrial
The CO2 can be produced via different biological routes such as trans- sectors. The extraction of natural pigments as well as lipids from mi-
formation of lignocellulosic waste and could be used as solvent in SCF croalgae biomass represents a crucial step since it affects the final
extraction process. In fact, several developed applications of SCF-CO2 products properties and, consequently determines their end-uses. An
extraction not only tend to replace organic solvents, but also to reduce ideal extraction method should allow to operate at ambient tempera-
the toxic impact on human health [25]. Compared with conventional ture and pressure, preserving, at the same time, the original char-
extraction methods, techniques based on SCF-CO2 can provide wide acteristics of the targeted bioactive compounds. SCF-CO2 seems to
features of dense gases, such as high compressibility and diffusivity, comply with the prerequisites of an environmentally friendly and cost-
very high evaporation rate and the possibility of fine tuning the solvent effective extraction. It is considered sustainable and non-toxic and,
power through density modulation (mainly temperature and pressure). compared with the traditional extraction techniques, allows to obtain
SCF-CO2 process has greater selectivity of carotenoids, which could be greater selectivity and extraction efficiency achieved by tailoring the
directly used for food supplementation [27,97]. Recently, vegetable oils operative parameters. In particular, by varying SCF-CO2 temperature,
have been used as co-solvent to enhance the selectivity of carotenoids pressure and flow rate, both selectivity and purity of the final extract
and FAs, which can directly be consumed as high quality oil with can be modulated. Therefore, SCF-CO2 extraction approach is pro-
bioactive compounds [27,97]. Therefore, SCF-CO2 technology could be mising for recovery of thermolabile molecules, which should not un-
a benchmark in food industry for direct supplementation of food pro- dergo any deterioration and variation of physical-chemical properties
ducts with bioactive compounds. In addition, SCF-CO2 extraction due to the low extraction temperature and pressure. The major lim-
technology showed various environmental benefits such as lower en- itation of SCF extraction is represented by the CO2 chemical behaviour
ergy consumption, use of non-toxic solvent, recycling of solvent and in supercritical conditions, which is similar to that of lipophilic sol-
reduce the further purification cost of extract. Moreover, after the ex- vents. In order to overcome this obstacle, the addition of a suitable
traction, the left-over biomass could be used in biorefinery processes for polar co-solvent is an advantageous strategy for modifying SCF-CO2
biofuel production. For example, SCF-CO2 was used to extract bioactive polarity and increasing the solubility of bio-active compounds, thus
compounds (lipids and carotenoids) from Nannochloropsis sp. micro- enhancing the extraction efficiency. The use of co-solvents deriving
algae, while the residual/leftover biomass was used to produce biohy- from renewable sources as well as the integration with microalgae
drogen [3]. This concept could be further developed, within well-es- biomass pre-treatments procedures should confer a relevant advantage
tablished processes, for integration of biorefinery approach in obtaining to SCF extraction technique.
high-value-added products while keeping the standards required for
environmental sustainability. Declaration of Competing Interest

8. Challenges and future perspectives The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
SCF-CO2 process presents few issues such as lower extraction yield ence the work reported in this paper.
of non-polar carotenoids, not appropriate for samples with greater
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