Recent developments in supercritical

fluid extraction of bioactive compounds from

microalgae: role of key parameters,
technological achievements and challenges.

● Introduction
Microalgae are biological species consisting of more than 7000
organisms growing in different habitats and environment conditions like freshwater,
brackish water and sea water. Microalgae can also be grown in close reactors, as
photobioreactors or open ponds or marine environments, that can be located or
installed in marginal or un-productive lands.

Recently, research on microalgae has rapidly increased due to growing interest in

exploiting their potential in terms of CO2 mitigation and wastewater treatment. In
addition, microalgae biomass is a rich source of natural compounds like carotenoids,
astaxanthin, lutein, and fatty acids. These bioactive compounds have positive and
important properties for all living creatures. In fact, they are being used in
pharmaceutical, nutraceutical, cosmetic, and food industries as natural medicines,
food additives and UV-light protective agents.

 What is Supercritical Fluid ?

A supercritical fluid is a substance that is heated and compressed to a state
beyond its critical point where it exhibits properties of both a gas and a liquid.
At this state, the fluid is in a single phase, and its density is similar to that of a
liquid, while its viscosity and diffusivity are similar to those of a gas.

Supercritical fluids are used in a wide range of industries, including

pharmaceuticals, food processing, and materials science, due to their ability to
dissolve a variety of substances and their gentle, environmentally friendly
nature. They are also used in supercritical fluid extraction, a technique used to
separate and purify different components from a mixture.

 Which compounds are extracted? What is their market utilisation?

Microalgae are known to produce a wide variety of bioactive molecules such
as pigments, polysaccharides, lipids, proteins, vitamins, and antioxidants.
Some of the major bioactive molecules present in microalgae are:
 Chlorophylls: Chlorophylls are the green pigments found in microalgae and
are important for photosynthesis.

Carotenoids: Carotenoids are pigments that give microalgae their

characteristic red, orange, and yellow colors. They have antioxidant properties
and are also important for photosynthesis.

Phycobiliproteins: Phycobiliproteins are water-soluble proteins that are

responsible for the blue and red colors of some microalgae. They are used as
fluorescent probes and are also used in biotechnology and medicine.

Omega-3 fatty acids: Microalgae are rich in omega-3 fatty acids such as
eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have
numerous health benefits.

Polysaccharides: Microalgae produce various polysaccharides such as agar,

carrageenan, and alginate, which have applications in the food,
pharmaceutical, and cosmetic industries.

Vitamins: Microalgae are a rich source of vitamins such as vitamin A,

vitamin B12, and vitamin C.

Antioxidants: Microalgae produce various antioxidants such as phenolic

compounds, flavonoids, and carotenoids, which have health benefits and can
protect against oxidative stress.

 Conventional techniques for extraction and its drawbacks.

Different conventional and non- conventional extraction technologies such as

maceration, Soxhlet extraction, ultrasound-assissted extraction, microwave-
assisted extraction are used. The choice of the suitable extraction method
depends on aspects like biomass properties, physical-chemical properties of
the extractable molecules and their perspective end use - pharmaceutical,
cosmetic, food. Additionally, because of their oxidative properties,
the extraction of these bioactive compounds requires strict control of
temperature, pressure, presence of light, and time. Moreover, bioactive
compounds have different polarity, which makes their simultaneous
extraction difficult with a single solvent. Usually, non-polar solvents are used
to extract non-polar bioactive compounds, while polar solvents are
better suited for polar compounds . Conventional techniques
such as squeezing, maceration, infusion, percolation, steam distillation
and solvent extraction are used to extract bioactive molecules from
different biomass matrices. These techniques often present
several disadvantages, e.g. thermal degradation of molecules due to the
high temperature of extraction, or solvent residues in the extracts that
 can compromise their end use. These bioactive molecules have numerous
applications in various industries such as food, pharmaceutical, and cosmetic
industries, and also have potential as nutraceuticals and functional foods.

● Why to opt for Supercritical Fluid Extraction ?

Advanced extraction with SCF allows for the preservation of the
natural qualities of bioactive compounds, reducing the environmental
impact and minimizing energy costs at the same time.
Furthermore, because of the structural complexity of the microalgae
biomass composition, the rate of intracellular compounds recovery is
low - three-layer or two-layer cell walls impede the mass transfer of
intracellular molecules during extraction. Therefore, microalgae bio-
mass requires appropriate pre-treatments (biological/chemical/phy-
sical) before the extraction step, which facilitates mass transfer of in-
tracellular compounds from microalgae chloroplast.

● Pre-treatments of microalgae biomass before extraction

Before the extraction of these compounds, it is important to carry out some pre-
treatment steps to improve the efficiency of the extraction process and the quality of
the final product.

Here are some common pre-treatment methods for microalgae biomass:

Harvesting: The first step is to harvest the microalgae biomass from the culture
media. Harvesting can be done by different methods, such as centrifugation, filtration,
and flocculation.

Drying: Microalgae biomass contains a high amount of water, which can interfere
with the extraction process. Therefore, it is important to dry the biomass to remove
the excess water. Different drying methods can be used, such as spray drying, freeze
drying, and sun drying.

Grinding: After drying, the microalgae biomass can be ground into a fine powder to
increase the surface area, which can improve the efficiency of the extraction process.

Cell disruption: Microalgae cells have a tough cell wall that can limit the release of
bioactive compounds during extraction. Therefore, it is important to disrupt the cell
wall to release the intracellular contents. Different cell disruption methods can be
used, such as sonication, homogenization, and high-pressure homogenization.

Solvent extraction: After pre-treatment, the microalgae biomass can be extracted

using solvents, such as ethanol, methanol, or chloroform, depending on the target
compounds.
 Overall, pre-treatment steps are critical for the successful extraction of bioactive
compounds from microalgae biomass. The specific pre-treatment method chosen will
depend on the type of microalgae and the target compounds.

● Important parameters to be regulated in SCFE process

Supercritical fluid extraction (SFE) technology is a method of extracting bioactive
compounds from natural sources using supercritical fluids. In SFE, a supercritical
fluid (usually carbon dioxide) is used as the solvent to extract the desired compounds
from the matrix. The supercritical fluid is a state of matter that occurs when a
substance is above its critical temperature and critical pressure, resulting in a unique
combination of liquid and gas-like properties.

SFE technology is particularly useful for extracting bioactive compounds from plant
materials, as it can be used to extract both lipophilic and hydrophilic compounds. The
process is also gentle and selective, allowing for the extraction of specific compounds
while leaving behind unwanted substances.

SFE technology has several advantages over other extraction methods, including:

High selectivity: SFE technology can selectively extract specific bioactive

compounds, leaving behind unwanted substances.

Mild processing conditions: The process can be carried out at low temperatures,
which helps to preserve the integrity of the bioactive compounds.

No solvent residues: SFE uses supercritical fluids as the solvent, which evaporate
completely after extraction, leaving no residue.

Environmentally friendly: Supercritical CO2 is a non-toxic and non-flammable

solvent, making it a safer and more environmentally friendly alternative to traditional
organic solvents.

High yield: SFE technology can extract a high yield of bioactive compounds from
natural sources.

Overall, SFE technology is a promising technique for the extraction of bioactive

compounds from natural sources, offering several advantages over traditional
extraction methods.
 Temperature and pressure
Extraction temperature and pressure play a significant role on so-
lubility of solutes in the solvent, which mainly depends on the chemical

properties of extractive target compounds. In the SCF-CO2

extraction of bioactive compounds, the extraction efficiency increases
with CO2 pressure and temperature up to optimal level. However, a
higher temperature causes thermal degradation of compounds, while
 greater pressure can obstruct the diffusivity of supercritical fluid into
the matrix, which leads to a decreased extraction yield [18,73,87,88].

Also, greater temperature and pressure imply the production of ex-

traction waxes [75]. Pressure and temperature influence the solvation

power of fluid due to their actions on the solvent density and can be

controlled/modified by acting on the operating pressure and tempera-

ture. However, at constant temperature, increasing pressure causes an

increase in the CO2 density and solvation power of the fluids, which
enhance the solubility of the bioactive compounds and extraction yield.
At constant pressure, an increase in temperature reduces the solvent
density and solvation power. Therefore, solubility is closely related to
the SCF-CO2 density and the properties of the solute, such as molecular
mass, polarity, and vapour pressure [18,75,86].

 Co-solvent
The addition of polarity modifiers (co-solvents) to CO2
can improve the extraction efficiency by increasing the solubility
of the
polar and non-polar bioactive compounds. Since supercritical CO2
is a
non-polar solvent, the addition of polar co-solvent improves the
extraction ability of polar bioactive compounds. The use of
acetone,

butanol, dichloromethane, ethanol, methanol, propanol, toluene,

ve-
getable oil, and water was reported by several authors with the aim
to

enhance the recovery efficiency of carotenoids and FAs

[18,20,27,73,85,91,97–99]. The co-solvent addition allows
swelling of

microalgae biomass, thus increasing the internal volume and the

sur-
face area for the contact, enhancing the mass transfer by creating
hy-
drogen bonding with the intracellular biomass compounds [88],
and

increasing solvent polarity [3,18,73,75]. The main effect on the ex-

traction depends on the co-solvent type, biomass composition, and

target extractable compound [18]. Furthermore, the appropriate

choice and
concentration of co-solvent is another major factor that can affect
the

extraction efficiency and selectivity [75,86,97]. Among the several

co-
solvents, ethanol has been broadly used as a co-solvent in
extraction of

bioactive compounds due to less toxicity, since it is considered as

food
grade solvent, which can be used in cosmeceutical, food,
nutraceutical,
and pharmaceutical industries [3,19,22,34,38,78,89–92,97,99–
101].

The choice of the co-solvent should be determined by the affinity

be-
tween the co-solvent and the extractable target bioactive
compounds. The flow rate of CO2, co-solvents concentration and
extraction time

have various effects on selectivity of bioactive compounds and

extrac-
tion efficiency. There exist only few reports in literature on the role
of

CO2 and co-solvents on selectivity of bioactive compounds and ex-

traction efficiency [24,27,73,86,91,97]. Flow rate has a significant
ef-
fect on the residence time for the contact between the solute and
the
 solvent. For example, the increase in CO2 flow rate reduces the re-
sidence time and subsequently decreases the solute dissolution.
 CO2 flow rate and co-solvent concentration
 extraction time on extraction efficiency and purity
Short extraction time and high purity are the two main advantages
of SCF-CO2 technology. Extraction ef-
ficiency is correlated with pressure, temperature and solvent flow rate.

● Environmental aspects and technological achievements

Supercritical fluid extraction (SFE) is a method that uses supercritical fluids, such as
carbon dioxide, as a solvent to extract various compounds from solid or liquid
matrices. SFE has gained significant attention in recent years due to its many
advantages over traditional extraction methods, including higher selectivity, faster
extraction times, and reduced environmental impact.

One of the main environmental advantages of SFE is that it eliminates the need for
harmful solvents, such as chlorinated and aromatic hydrocarbons, which can have
adverse effects on human health and the environment. Carbon dioxide is the most
commonly used solvent in SFE because it is non-toxic, non-flammable, and readily
available. Additionally, carbon dioxide can be easily recovered and recycled, which
reduces waste and lowers the overall environmental footprint of the extraction
process.

Technological advancements have also played a crucial role in the development of

SFE. For example, the use of co-solvents, such as ethanol or water, can enhance the
solubility of certain compounds and improve the efficiency of the extraction process.
The development of more sophisticated equipment, such as high-pressure pumps and
reactors, has also enabled the precise control of temperature, pressure, and flow rate,
which has further improved the selectivity and efficiency of SFE.

Another important technological advancement in SFE is the use of modified

supercritical fluids. These modified fluids, such as supercritical water or supercritical
ammonia, can be used to extract compounds that are not soluble in carbon dioxide.
Furthermore, the use of modified fluids can also lead to higher yields and improved
selectivity, which can be especially useful in industrial applications.
● Global Scenario
● Conclusion