Extraction of High Value Compounds from Macroalgae using Supercritical … · 1.5 Extraction...

Extraction of H

Extraction of High-Value Compounds

from Macroalgae using Supercritical

CO2

Masters Thesis

2014/ 2015

MEng Chemical Engineering

Extraction of High Value Compounds from Macroalgae using Supercritical CO ₂

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Executive Summary

The aim of this investigation was to examine the best parameters for the extraction of

Fucosterol from Ascophyllum nodosum (a common brown macroalgae species), using

Supercritical Carbon Dioxide. The macroalgae used was a waste by-product of a fertiliser

from macroalgae process. The literature review and experiments show that Fucosterol is

present within Ascophyllum nodosum. Supercritical Carbon Dioxide was used, as not only is

it an example of ‘green chemistry’, but it has also shown to be most effective in research to

date. It is obvious that there are many factors affecting the efficiency of this extraction, such

as temperature and pressure. These were investigated to obtain optimal extraction parameters.

This review explores the procedures of cultivation, processing and utilisation of macroalgae,

and the many high-value compounds present, as well as their high-value uses. These include

compounds such as polysaccharides, polyphenols and lipids.

Results show that using supercritical carbon dioxide yields low amounts of sterols from the

macroalgae samples. Optimal conditions were found to be 40oC at 75 bar. These results

compare well with the literature on sterol extraction from other sources.

Results of total compound mass were also calculated using exhaustive solvent extraction, for

both waste and fresh Ascophyllum nodosum. These were compared with the supercritical

extraction results of waste and raw macroalgae. It was found that there are a lower total

number of fatty acids and sterols within the waste macroalgae samples.

Due to low yields of supercritical runs, subcritical conditions were also explored to

investigate their efficiency. These were found to be more inefficient for sterol extraction than

supercritical.


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Contents

1.1 Macroalgae ........................................................................................................................ 5

1.1.1 Phaeophyceae- Brown Macroalgae ............................................................................ 6

1.2 Common compounds contained within brown macroalgae .............................................. 8

1.2.1 Polysaccharides .......................................................................................................... 9

1.2.2 Lipids ........................................................................................................................ 10

1.3 Higher Value Compounds ............................................................................................... 13

1.3.1 Polyphenols .............................................................................................................. 13

1.3.2 Proteins ..................................................................................................................... 14

1.3.3 Sterols ....................................................................................................................... 15

1.3.4 Fucosterol ................................................................................................................. 16

1.4 Processing ....................................................................................................................... 18

1.5 Extraction Methods ......................................................................................................... 19

1.5.1 Soxhlet Extraction .................................................................................................... 19

1.5.2 Ionic-Liquid Extraction ............................................................................................ 20

1.5.3 Supercritical Carbon Dioxide (CO2) ........................................................................ 21

1.6 Alternative technology to traditional methods for bioactive extraction. ........................ 24

1.7 Characterisation of extracts ............................................................................................. 26

1.8 Ascophyllum nodosum .................................................................................................... 28

2.0 Project Objectives ........................................................................................................... 31

3.0 Experimental ................................................................................................................... 32

3.1 Methodology ................................................................................................................... 32

3.1.1 Preparation of macroalgae sample ........................................................................... 32

3.1.2 Supercritical Rig ....................................................................................................... 33

3.1.3 Analysis of Extracts ................................................................................................. 33

3.1.4 Preparation for Analysis ........................................................................................... 33

3.1.5 Exhaustive Extractions ............................................................................................. 34


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3.1.6 NMR Analysis .......................................................................................................... 34

4.0 Results and discussion .................................................................................................... 37

5.0 Conclusions and further works ....................................................................................... 44

5.1 Further Work ............................................................................................................... 44

6.0 Acknowledgments ........................................................................................................... 46

7.0 References .................................................................................................................. 47

8.0 Appendix ......................................................................................................................... 52

8.1 NMR Calculation ............................................................................................................ 52

8.2 NMR Results ................................................................................................................... 53

8.3 Start-up of Supercritical Rig ........................................................................................... 60

8.4 Shut-down of Supercritical Rig ...................................................................................... 61


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1.1 Macroalgae

Macroalgae are seaweeds, they exhibit immense assortment in size, shape and colour, and

can be found in fresh/ sea water. This review will explore many elements of macroalgae,

including potential utilisations, cultivation, processing and extraction of desirable

compounds.

Although thought to be very similar, and both photosynthetic, macroalgae are not considered

to be plants. Algae comes in many forms, from unicellular or colonial, to multi-cellular.

Whereas terrestrial plants are only multi-cellular, the difference in their cells structures can

be seen in Figure 1.

The main difference between these two cells is the lack of a vacuole in the algal cell. One of

the main functions of this organelle is to hold water that the cell can use. Algal cells have

direct contact with water on a regular basis, this may explain the lack of vacuole present.

Macroalgae is characterised into different categories. Rhodophyta (also known as red algae)

compared to all other macroalgal organisms is least like its plant siblings. Although

chlorophyll is present in the cells, they have a unique coloration. It is due to the presence of

two pigments; phycocyanin and phycoerythrin. They absorb longer, bluish wavelengths of

light and this allows them to grow in deep waters, as longer wavelengths can penetrate a body

of water much deeper, (C. Van Den Hoek, 1995).

Cyanophyta (commonly known as blue/ green algae) are closer than any other kind of macro-

algae to vascular plants. Green algae get their colour from chlorophyll pigments,

predominantly chlorophyll a and b (C. Van Den Hoek, 1995).

Figure 1: (Lea, Lowrie 2000)(Lee 2000)Figure 8: Showing Algal cell

(left) and terrestrial plant cell (right).


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1.1.1 Phaeophyceae- Brown Macroalgae

These forms of macroalgae are mainly marine and attached to rocks around seacoasts, with a

few freshwater exceptions. They prefer cold conditions and as so are in abundance in areas

such as the North Sea. Climate effects the chemistry of macroalgae and so in different

conditions will present different abundances of compounds. All species of brown macroalgae

are multicellular. The cell walls of macroalgae are composed of a network of cellulose

microfibrils, which is strengthened by Calcium Alginate, together with an amorphous

mucilaginous matrix fraction composed of fucoidan and mucilaginous alginates (Reddy

1996).

This class of macroalgae contains in the region of 265 genera and between 1500-2000 species

(Reddy 1996). Brown macroalgae species are of potential economic importance if high-value

compounds can be successfully extracted from them. Currently, it is estimated that globally,

2 million tonnes are cultivated each year, for chemical processing, and nutritional means

(Lucas, Southgat 2012), but these are low-value utilisations.

Long-line cultures are the most usual method of commercial cultivation of brown macroalgae

(Figure 2). This type of technique accounts for 90% of the world’s macroalgae production

(Lucas, Southgat 2012). The culture itself is composed of a 30-60m long fibre rope, secured

by two anchor ropes. The structure is supported by several buoys, which are attached to ropes

to secure it. Each cultivation rope holds on average about 30 plants, and the distance between

each rope is about 6/7 metres. On average between 150- 300 thousand plants can be cultivated

in 1 Hector.

Figure 2: Image showing a typical long-line culture for Kelp seaweeds (Lucas, Southgat

2012).


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During cultivation there are differences observed between the lower and upper plants along

the lines, mainly due to differences in light exposure. The ropes are inverted to help with this

problem, or the lines are adjusted so they are more horizontal.

Environmentally speaking, the farming of brown macroalgae has both positive and negative

assets. Many studies have been done around the world to determine the effects of macroalgae

farming on the biodiversity of the surrounding ecosystems. For example studies on farming

in tropical seas have indicated that macroalgae farming above sand can only have positive

impacts on biodiversity, attracting much fish to the area (Radulovich, Umanzor et al. ). It also

has to be taken into consideration that farms may be set up in areas of an already existing

great biodiversity. These farms may shade lower-laying seagrasses for example, and without

enough sunlight would be killed off. The introduction of a new macroalgae species may lead

to biosecurity issues, and become invasive on local flora and fauna that originally lived there

(Radulovich, Umanzor et al.). Excessive growth of certain algae in a body of water often

results in severe depletion of oxygen in the habitat. This leads to mass mortality of aquatic

animals like fish, due to suffocation. Sometimes in spells of high temperatures and solar in-

tensity, this results in disintegration of algal blooms, releasing into the medium their noxious

components (Kumar, Singh 1979).

Brown macroalgae owe their colour to an accessory pigment fucoxanthin, along with others,

all of which are contained within the chloroplast organelle, responsible for photosynthesis.

Many compounds are produced and stored within the Brown Algae cells. An example of

which is mannitol (Figure 3(1)). Unlike its red and blue/ green counterparts, brown algal

cells are abundant in this material. Mannitol is a low molecular mass sugar alcohol, which in

brown species has an osmoregulatory role, meaning it is active in the regulation of the osmotic

pressure within the cell, in addition to its function as an energy reserve. The concentration

of this compound fluctuates with differences in environmental salinity.


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(1)

Figure 3: Structural diagram of a Mannitol Compound (Best 2008).

The isolation of natural mannitol from plant sources has been researched before. It has been

hypothesised that mannitol can be efficiently extracted using supercritical fluids from the

likes of plane tree leaves (Ghoreishi, Sharifi 2001). However from an engineering

perspective, the scale-up of this would not be economically feasible. Low temperatures

suffice for the extraction of mannitol, however high pressure are required to attain the desired

solubility factors in the supercritical solvent. This coupling would result in a high cost of

operation.

The main commercial source of industrial mannitol is currently from the hydrogenation of

fructose (Kearsley, Deis 2006). This process however only has a conversion rate to mannitol

of 50% (Weymarn 2002). The remaining fructose is converted to sorbitol. The sorbitol-

mannitol mix is very difficult to purify and so there are engineering and purification cost

drawbacks. This also means that the mannitol production rate will be affected by the demand

of sorbitol. Hence the need for efficient alternatives, as mannitol has many applications across

varied industries.

In addition to its industrial applications, mannitol has several clinical uses for example as a

facilitating agent for the transportation of pharmaceuticals directly into a patient’s brain. As

the arteries in the brain are a lot more selective than others, active transport is needed for

diffusion here, as such Mannitol can be used as it is capable of stretching tight junctions

between endothelial cells (Best 2008). Mannitol has also been used as a functional food , with

the purpose of increasing blood glucose to a lesser extent that that of sucrose, so has a use as

a sweetener for people suffering from diabetes (Lawson 2007).

1.2 Common compounds contained within brown macroalgae


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Chemical compounds that have been derived and isolated from a biological source such as

macroalgae are referred to as natural products (Sticher 2008). These compounds extracted

from natural sources have a wide variety of industrial applications, in fields such as cosmetics

or medicine. Having the ability to be readily absorbed and broken down, these products are

expected to continue to be an instrumental part in the finding of for example new drugs in the

future (Sarker, Latif et al. 2006b).

1.2.1 Polysaccharides

Within macroalgae cells polysaccharides come in many forms, for example they can be stored

as starch (a source of glucose for respiration), or they could be in the form of cellulose, which

is an important structural element of the cell walls. The oligosaccharides general formula is

CX(H20)Y where X is a number that can range from between 200 and 2500, depending on the

molecule being formed. Again factors such as the age of macroalgae, climate and time of year

affect the concentration of these compounds within the cells.

The main concentration of polysaccharides in macroalgae is found within the cell wall.

Different types of macroalgae have different types of polysaccharides in varied

concentrations. This also varies with season, and location. Red macro-algae, for example, are

high in Agar and Carrageenan’s, whereas brown macroalgae are high in alginates, fucoidan

and laminates (C. Van Den Hoek 1995).

Commercial starch is a very useful compound. 80% of the world’s starch is produced in the

USA from Maize (Bowsher, Steer 2008). It can be used in foods as a bulking agent or

viscosity modifier; in medicine as a plasma expander, meaning they can increase the volume

of blood plasma, increasing the pressure in patients who have suffered blood loss e.g.,

haemorrhaged (Bowsher, Steer 2008). As starch from grains is needed for foods such as

bread, there is controversy over using it as an energy source. Questions relating to its

sustainability throw up social, economic and political barriers. Utilising macroalgae as a

source of starch for food, again however this may be met by anthropological barriers as with

an increased demand, it may not be feasible to achieve this and also sustain the other

industries which require it.

The polysaccharides within macroalgae constituents have the potential as a fuel source. The

primary process considered for the production of fuel from macroalgae is fermentation. This


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comes in a variety of forms; for instance anaerobic digestion could be utilised to produce

biogas (carbon dioxide and methane) (Equation 1).

𝐶6𝐻12𝑂6 → 3𝐶𝑂2 + 3𝐶𝐻4(Equation 1)

Biogas normal consists of ~60% methane (Lyons, Lerat 2009). Measures can then be taken

to upgrade this to bio-methane, which could then be utilised on the gas grid. Alternatively the

macroalgae could be fermented to produce bio-ethanol (Equation 2).

𝐶6𝐻12𝑂6 → 𝐶2𝐻5𝑂𝐻 + 2𝐶𝑂2 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 2)

From an engineering perspective, however, there are process factors to consider. Being

macroalgae its concentration of salt and polyphenols is high, as well as sulphated

polysaccharides. All of which can be inhibitors to these processes, or cause damage to the

equipment used, for instance, corrosion to pipelines. An example of where macroalgae has

been successfully employed as a fuel includes a plant installed by SOPEX, in Morocco. This

plant was designed to treat waste macroalgae from agar production. 12 tonne of waste/ day

was expected to generate 100,000 m3/year (Lyons, Lerat 2009). From a sustainability point

of view, this is very promising as this is a renewable energy source and can help in prolonging

the global stock of fossil fuels. Additional positive impacts of these processes are that algae

will use up carbon dioxide in the process of photosynthesis, and then once processed, the

waste can be recycled as animal fodder or fertiliser (Kumar, Tuohy 2013).

Alginates are another polysaccharide abundant in brown macroalgae; as an example

application, it again can be used for making gels or gums. It has the ability to absorb 200-300

times its own weight in water, which makes it highly useful for many applications. In this

instance, high absorption capability, it has uses in cosmetics as a thickening agent, or in food

products e.g. ice-cream. It also has uses in materials and textiles, as in to make them

waterproof if desired. (Rowe, Sheskey 2009).

1.2.2 Lipids


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Lipids within macroalgae are important for energy storage and are key components in the

structure of the cell membrane. Macroalgae have low concentrations of lipids, typically in the

range of 1- 5% dry weight (Miyashita, Mikami et al. 2013a). These are subject to changes

and affected by seasons and location. The lipid content in seaweed have been found to directly

relate to solar intensity and temperature they are exposed to (Olofsson, Lamela et al. 2012).

Therefore in areas of high annual temperatures and solar intensity, there would be a higher

lipid content. Seasonally, at times of the year in which conditions are optimal, more lipids

would be present. Seaweeds store these lipids to use in colder conditions to perform their

metabolic function.

Typical lipids that can be located within brown macroalgae include oleic acid, Omega-3

polyunsaturated fatty acids, Omega-6 arachidonic acid and Fucoxanthin. They have many

potential benefits for humans, including antidiabetic and anti-obesity properties as well as

promotion of healthy liver functions, as depicted in Figure 4 (Miyashita, Kazuo, Mikami,

Nana, Hosokawa, Masashi 2013).

Figure 4: Diagram is showing benefits of lipids found in brown macroalgae (Miyashita,

Mikami et al. 2013a).

One of the best-known sources for fatty acids for human consumption is from oily fish. It has

been reported that dry weight of these animals can contain up to 50 wt % fatty acids

(Miyashita,Kazuo, Mikami, Nana, Hosokawa, Masashi 2013), which is significantly lower

than that of macroalgae. From a commercial and engineering viewpoint, macroalgae do not

compare to this, however if an efficient process was formed from fatty acid extraction that


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was economically feasible compared to that of oily fish it would be worthwhile. Further to

this, as the macroalgae lipid concentration varies with time of year, the resulting products of

the process implemented would have to be managed very closely.

Fucoxanthin (C42H5806) (Figure 5 (2)) is a pigment found in the chlorophyll of brown

macroalgae, which contributes to the brown/ olive green colour. It is located in the

mitochondria. Its function within the cells is to promote the burning of lipids for energy.

(2)

Figure 5: Chemical structure of Fucoxanthin, (Ravi Hindupur 2015).

One of the best methods exploited for fucoxanthin from brown macroalgae is solvent

extraction. The literature suggests the use of dimethyl ether and ethanol as a co-solvent is

highly efficient. When this extraction was utilised using enzymatic pre-treatments the yields

also increased; (0.64% g/g of extract pre-treated) biomass showed >50% improvement over

non-treated biomass (0.30%, g/g of extract) (Billakanti, Catchpole 2010). The compound

itself however is fragile and is sensitive to pH, light and temperature. In the scale up of the

operation these would all have to be tightly controlled, so the environment does not change

to reduce the degradation of the compound. This process would be more efficient if

supercritical fluid could be utilised. These fluids are good for thermally liable materials and

so perfect for fucoxanthin. Supercritical fluids are also a lot more environmentally friendly,

factors of which must be considered with scaling to industrial scale. Efficient and friendly

extraction would be most beneficial as it has much clinical application including, but not

limited to, slowing tumor growth and relieving oxidative stress (Miyashita,Kazuo, Mikami,

Nana, Hosokawa, Masashi 2013).


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1.3 Higher Value Compounds

Brown macroalgae is abundant with useful compounds such as polysaccharides and lipids, as

previously discussed. It is also hosted to many higher value compounds, which if extracted

successfully, can be exploited for high-value applications across an array of industries. This

section explores some of these chemicals and their potential uses.

1.3.1 Polyphenols

They are a class of organic compounds which consist of a hydroxyl group bonded to an

aromatic hydrocarbon group (multiples) and example of which is catechin (Figure 6 (4)).

Phenol being the most basic for this class of compound (Figure 6 (3)) is also known as

carbolic acid. In macroalgae cells, polyphenols are found mainly in the cell wall, however,

soluble types do exist and are stored in membrane-bound vesicles known as physodes. A

variety of low, intermediate and high molecular weighted polyphenols have been reported

from marine macroalgae with many potential clinical applications such as active antioxidants,

anti-HIV, and antidiabetic (Front matter. 2013).

(3) (4)

Figure 6: Image is showing Phenol Structure (left) and the polyphenol catechin (right).

One potential medical application of polyphenols is in the treatment of diabetes. It was found

that phenolic-rich extracts from the brown macroalgae species Ascophyllum nodosum

effectively inhibited α-glucosidase, and so can be used to control chronic diseases such as

type-2 diabetes, by controlling the hyperglycemic effect (Pantidos, Boath et al. 2014).

They also showed, using dried samples of Ascophyllum nodosum the total phenol content was

found to differ, as a result of the extractants used. It was observed that the use of aqueous


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solvents improved the extraction of phenolic groups, for example, a twofold improvement in

total phenol content was observed when using acetonitrile over ultra-pure water. Where ultra-

pure what yielded 178 ± 8 µg GAE/mL and the use of 50% aqueous acetonitrile containing

0.2% formic acid resulted in a yield of 363 ± 3 µg GAE/mL (Pantidos, Boath et al. 2014). We

can conclude from this that brown macroalgae are rich in these beneficial compounds, as such

are an excellent source of dietary phenolic compounds.

Other industrial applications apply within the medical and cosmetics industries. Polyphenols

are highly useful compounds for skin damage prevention, protection and repair. An increasing

number of studies on skin suggest that a controlled topical delivery of nano-encapsulated

polyphenols might overcome many limitations observed from the use of bulk polyphenols,

and so have a clinical benefit of helping to target inflammation, skin cancers, premature aging

and skin healing (Menaa, Menaa et al. 2014).

Phlorotannins are polyphenol derivatives, which are naturally occurring in macroalgae as well

as terrestrial plants. With relation to terrestrial plants, their tannin content has been widely

researched in relation to nutrition, in particular their relation to the metabolism of proteins,

as complexes are readily formed between these two compounds. Benefits of these compounds

include improvement of protein utilisation and productivity of animals. However, there is

great diversity in terrestrial plants in terms of molecular weights and structure of the tannins.

In relation to aquatic plants, these compounds have only been found in brown macroalgae.

Structurally they are less complex than those found in terrestrial plants (Wang, Xu et al.

2008). These compounds can be extracted in the same way as polyphenols, my means of

aqueous solvent extraction.

1.3.2 Proteins

Macroalgae are exceptional reservoirs for proteins and amino acids. Their function in algae

includes protection against solar damage, salt stress and thermal stresses. They are found in


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various locations; the cell wall, within pigments and attached to carbohydrate (Front matter.

2013). The amount of these compounds as with others is affected by the conditions of the

algae’s environment; light availability, solar intensity, temperature, wave force, salinity,

nutrient and mineral availability, carb levels, and since these are proteins, nitrogen

availability is very important (Conde, Balboa et al. 2013b). The types of proteins found within

marine macroalgae range from hexapeptides right down to free amino acids.

The seasons will have a great effect on the amount of polypeptides and amino acids being

produced. In terms of amounts per type of macroalgae, red species are said to be the most

abundant in polypeptides, reaching maximum recorded levels of 47.0% dry weight (Front

matter. 2013). Brown macroalgae have the lowest level of polypeptides, however, contain the

largest abundance of amino acids. The market for amino acids is growing, and so the need

for efficient cost effective methods of production are always desired. The most usual method

today for amino acid production is fermentation in terms of volume and value (Ikeda 2003).

For cost effective production many plants utilised high production rates and recovery sys-

tems. The microorganisms used in these types of the process have been specifically developed

for amino acid secretion, and so are very efficient. These microbes can also be chosen to tend

towards specific amino acids. As there are 20 known amino acids in the world today, this

makes fermentation the most efficient option. If brown macroalgae were used for amino acid

extraction, the types of amino acids would vary, and their abundance would change with the

seasons, this is why bio-synthesis is used for industrial production. However, the advantages

of natural product extraction are that the costs of chemicals for synthesis are eradicated.

1.3.3 Sterols

Sterols are an important class of organic molecules also known as steroid alcohols, the basic

structure of which can be seen below (Figure 7 (5)). They have a variety of types including,

cholesterol, fucosterol, and dihydroxy sterol, the most dominant of which in macroalgae is

Cholesterol (Front matter. 2013).


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Figure 7: Image is showing the basic structure of a sterol (Front matter. 2013).

The commercial uses of sterols such as cholesterol are mainly in the medical and cosmetic

industries. Their properties include moisturising, an excellent emulsifier and emollient prop-

erties.

Innovative methods of sterol production are always being explored. One method explores the

use of yeasts to produce the compounds for cultivation. Yeasts can be manipulated genetically

to make various materials, which only occur naturally in eukaryotes, this is known as meta-

bolic engineering. These cultivations are highly reproducible and inexpensive. Advantages

of this process over, for example utilisation of bacteria is that sterol biosynthesis, is better

suited to eukaryotic membranes and so more accommodating (Wriessnegger, Pichler 2013).

This process has the potential to be an excellent commercial source of sterols. An advantage

of macroalgae over this, however, is that the sterols already occur naturally in abundance

within the cells. Therefore, there is no need for genetic manipulation. However, as yeasts are

easily reproducible and quick to grow, they could dominate this market, as they would not

have the competitive demand that brown macroalgae have with the food industry.

1.3.4 Fucosterol

Fucosterol (Figure 8 (6)) composes over 95% of the sterol content within the brown

macroalgae species Ascophyllum nodosum (Cousens 1981). Other sterols are present, but it

is considered to be the result of oxidation. A more detailed study of sterols presented findings

that of 0.10% dry weight Ascophyllum nodosum contained 90% Fucosterol (Cousens 1981).

Fucosterol has the following properties:


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Molecular Formula: C29H48O

Molecular Weight: 412.69

Composition: C (84.40%), H (11.72%), O (3.88%)

Phytosterols incorporate plant sterols and stanols. Stanols are classed as sterols, which are

saturated, as in they have no double bonding in the sterol ring structure.

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In 1934, fucosterol was initially isolated from brown macroalgae (Bouzidi, Viano et al. ).

Fucosterol has exhibited many biological activities, which are of human benefit. An example

of which include its anti-cancer effects. It was found that fucosterol exhibits the cytotoxic

effect on cancer cells, and so was a potential additive for modern cancer treatments. It was

also found that this sterol has antidiabetic activity. Research using mice induced with diabetes

were given does of fucosterol at 100 and 300 mg/kg and discovered that it reduced the

hyperglycemic effect by 25-33% (Bouzidi, Viano et al. ).

Currently a commercial source of fucosterol is a by-product extraction of alginic acid from

Macrocystis pyrifera (a species of giant kelp), of which it can then be purified (Shimadate,

T.,Rosenstein,F.U. 1977) . With the clinical powers of fucosterol, however, there is a push

for more efficient methods of extraction.

Within Ascophyllum nodosum, the sterol content has been the topic of a past investigation

noted in the 1970s journal by Knights. It was stated from this journal that using petroleum

ether as the solvent for extraction; fresh species sample’s sterol content was almost pure

fucosterol. When compared with commercial samples of Ascophyllum nodosum, however,

the sterol content had a varied range. Knights emphasised that while the most likely

Figure 8: Structure image of

Fucosterol (Bouzidi, Viano et al. )


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explanation for this was the formation of other sterols by oxidation of fucosterol, there was

also the possibility that they could have been released due to the degradation through the

drying process for commercialisation (Knights 1970).

1.4 Processing

Once cultivated macroalgae can be processed to make many valuable products. After harvest,

the chemistry of the macroalgae will begin to change. Thus, it is essential that processing is

started as soon as possible. After harvesting, the length of time and the way the macroalgae

is stored, is directly related to the decomposition of desired compounds, this may be my

natural or thermal mechanisms. To combat this, they can be stored in cold rooms for

preservation. Macroalgae is then chopped to increase surface area for processing, so

efficiency is maximised.

Fertiliser production is a common agricultural process for macroalgae. They can be processed

in ways to create a nutrient rich liquor for use. For the purpose of this report, waste

macroalgae from a fertiliser production process are used. The macroalgae are provided by

OGT, an Irish processing company. Their process sees the macroalgae cells being ruptured

by a mechanical press, solids removed and water evaporated off.

Other processes include those mentioned in section 1.2.1. Where the constituents of the

macroalgae cells can be exploited to produce fuels, such as biogas from anaerobic digestions,

the fermentation process of which may take up to three days at 25-30oC. The products of

which will be methane and Carbon Dioxide, usually at an average ratio of 60-40%

respectively (Marine Algae. 2014).


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1.5 Extraction Methods

There are many conventional extraction techniques that can be employed for the cultivation

of materials from natural sources, and hence brown macroalgae. This section will explore

these systems, and their advantages and limitations. Novel techniques for emerging research

will also be reviewed, as tools for enhanced extraction.

1.5.1 Soxhlet Extraction

Extraction by these means is advantageous for desirable products which have limited

solubility in a solvent, as well as the impurities being of the same nature. It is a very

convenient process and so is extensively used in extraction from plant sources (Sarker, Latif

et al. 2006a). Below shows a standard Soxhlet extractor (Figure 9):

Figure 9: Schematic Diagram of Soxhlet Apparatus (Mitra 2003).


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Firstly the material desired for extraction (e.g. brown macroalgae) is ground to a desirable

particle size, then placed in a thimble, designed out of thick filtration paper. This is then

placed into the main chamber of the equipment. The solvent used for the extraction is then

poured into the distillation flask along with the extracto and a condenser.

The mixture is then heated under reflux. The solvent travels up the distillation arm section of

the apparatus, then floods the chamber containing the material being subject to extraction (i.e.

the macroalgae). The purpose of the condenser is to ensure that any solvent vapour is cooled

and drips back down to the solid material chamber. This results in the chamber filling with

warm solvent, and as a result compounds of interest will dissolve into it. When the chamber

fills, it empties speedily through a siphon side arm. This rapid motion also makes sure no

solid materials are carried through (Sarker, Latif et al. 2006a). This process can be repeated

as many times as thought needed. This has advantages as it is less time consuming than other

methods, and little solvent is consumed. It also allows for exhaustive extractions to be

completed so a picture of total amounts of desired materials can be explored. A disadvantage

associated with this technique is that the solvent is repeatedly heated to boiling point, and

may result in damage to compounds that are liable to thermal degradation, or result in the

formation of artefacts. (Sarker, Latif et al. 2006a).

1.5.2 Ionic-Liquid Extraction

Ionic liquids are salt structures in their liquid state at ambient temperatures, and can remain

in this liquid phase at temperatures over 300 K, therefore, as expected they have low vapour

pressures at room temperature. They can conduct electricity and are potent solvents. These

properties suggest that ionic liquids have the potential to replace the volatile organic solvents

used in alternative processes, such as in soxhlet extraction. This has advantages from an

environmental and sustainability point of view, as these volatile solvents can be toxic and

hazardous to the environment.

Ionic liquids have been found to be highly effective in the breakdown of the algal cell wall to

isolate cellulose (Teixeira 2012). As a result when used on, for instance, brown macroalgae

the cell walls will be ruptured, consequently the constituents of the cells to be released and

cultivated.


21 | P a g e

A disadvantage of this form of extraction is that the ionic fluids have a moderate to high

viscosity and, as such, can be hard to fully utilise in processes. This may result in efficiency

problems in the scale up of extraction processes, due to low flow rates, which may even result

in blockages in utilities

One option is to use ionic liquids as pre-treatment to other extraction techniques. As they are

so good at breaking down cellulose, the resulting biomass from this treatment could then go

on to further processing, such as that of supercritical fluid extraction (Section 1.5.3).

1.5.3 Supercritical Carbon Dioxide (CO2)

Supercritical fluids are materials in which their respective pressure and temperature critical

points have been exceeded. A compound in its pure form in the gaseous phase cannot be

liquid regardless of applied pressure if the temperature exceeds the critical point. The vapour

pressure at the critical temperature equals that of the critical pressure, as shown in Figure 10.

Figure 10: Pressure v. Temperature graph showing the supercritical phase (Makhopadhyay

2000).

In the supercritical range only one phase exists. It is here that it is classified as a supercritical

fluid, but cannot be classified as a liquid or a gas. The fluid has the solvent power of liquids


22 | P a g e

as well as the high miscibility, high diffusivity and relatively weak molecular association of

that of gases.

Relating this specifically to carbon dioxide, it has the following critical parameters:

Critical temperature- 304.25 K (Makhopadhyay 2000)

Critical pressure- 72.9 atm (Makhopadhyay 2000)

Supercritical fluids exhibit low toxicity and environmental effects. Other advantages include:

Fresh solvent is passed over the used solid material i.e. brown macroalgae.

Supercritical CO2 exhibits high selectivity powers and can be manipulated by

changing the pressure and/ or temperature. This in itself is very useful for the research

of materials from biomass. The parameters can be adjusted to obtain optimum

extraction factors for the specific compounds desired.

Carbon dioxide becomes critical at a relatively low temperature, which allows for the

extraction of thermally liable compounds. The research is then more valid of actual

compound content within the macroalgae cells. Other undesirable reactions such as

hydrolysis and rearrangement could be successfully prevented.

This extraction can allow for the direct coupling of a chromatographic method, which

is useful for means of directly quantifying highly volatile compounds. This is very

useful in this field of research, as in quantifying extractions from biomass several

characterization techniques should be applied to get the best understanding of the

material and the errors reduced.

Carbon dioxide can be recycled or reused in the process, and so minimises waste

generation, making the process very green, and also minimising costs.

Additionally a co-solvent can be added in small amounts to improve the process efficiency.

The addition of said co-solvents has an effect on the characteristics of the solvent, such as

polarity, without considerably altering the density and compressibility of the original

supercritical fluid. Further to this, a co-solvent can improve the selectivity of the separation

by preferentially interacting with one or more components facilitating selection fractional

separation (Makhopadhyay 2000), which is an advantage for targeted extraction from

biomass.


23 | P a g e

There are limitations, however, to this form of extraction. The use of supercritical fluids is

expensive, and if scaled up to an industrial scales, where high pressures and temperatures are

required, it would require a lot of energies and therefore money to run.

The diffusivity of the supercritical fluid directly related to temperature and pressure.

Likewise, density is directly related to the viscosity of the fluid. The literature suggests that

increasing the viscosity of the supercritical fluid, the diffusivity lowers. This is because the

solvent molecules are more densely packed, stopping the solvent particles from moving as

easily (Yang, Yan et al. 2013).

Other research shows that increasing the pressure has an adverse effect on the diffusivity; this

is linked to the above regarding the viscosity, as increased pressure would increase fluid

density and viscosity. However by increasing the fluids temperature, it was observed that the

diffusivity of the fluid increased. In this situation more thermal energy would give compounds

and materials more kinetic energy in the system, so diffusion from macroalgae to supercritical

fluid is observed (Cheung 1999a)

Further findings saw the number of carbon-to-carbon double bonds, as well as their position,

affect the diffusivity, with the diffusivity being lower for molecules with increasing carbon

to carbon double bonds (Yang, Yan et al. 2013). It is suggested that a complex special

configuration related to these bonds is what causes this diffusion behaviour.

With relation to supercritical fluid extraction and brown macroalgae, there has been limited

research. Supercritical fluids have been successfully employed to extract lipids and other

compounds (Miyashita, Mikami et al. 2013b) from macroalgae. However, research does

suggest that the viability of using supercritical fluids depends on the pre-treatment processes

utilised before extraction if an efficient extraction is to take place (Crampon, Boutin et al.

2011).

Literature suggests that when using supercritical fluids for extraction, the smaller the

molecular weight, the more easily it will dissolve in the solvent. Research undertaken by the

‘Indian Institute of Science’ looked at the solubility of fatty acids in supercritical carbon

dioxide. Looking at their results, it showed that even within the fatty acid family there was

great variance in solubility. Over a range of pressures at a temperature of 308K, the difference

in solubility is very clear.


24 | P a g e

Figure 11: Solubility of various fatty acids in SCCO2 at 308K. Legend: experimental data

for (*) lauric acid,(▿) palmitic acid (□) behenic acid,(○) stearic acid (Madras, Kulkarni et

al. 2003)

From the above graph (Figure 11) it can be seen, lauric acid (C12H2402) which has a molecular

mass of 200.32, has the highest solubility in supercritical carbon dioxide. Comparing that

even with the second largest fatty acid in the group (palmitic acid, molecular weight 256.42)

there is a substantial difference in solubility.

Other literature on the same topic found similar results. The Indian Institute of Science (De-

partment of Chemical Engineering) found that pressure effects the solubility of both stearic

and palmitic acid in supercritical carbon dioxide, however the individual solubilitys are over-

all higher for that of palmitic acid, which has the lower molecular weight, as such the same

trend can be seen (Garlapati, Madras 2010).

1.6 Alternative technology to traditional methods for bioactive extraction.

The use of ultrasonic technology can be employed as an alternative method of extraction from

biomass. One study concentrated on the brown macroalgae species Ascophyllum nodosum

and identified the best process conditions including extraction time and ultrasonic amplitude

to facilitate industrial applications. Advances in this field of technology are needed to enhance


25 | P a g e

the yield of extraction and efficiency, to overtake those of solid-liquid extraction methods.

The results found that utilising ultrasound-assisted extraction has the potential to enhance

bioactive compound yields. Ultrasonic technology has a relatively cheap capital cost, which

is an advantageous factor when considering industrial scale applications. Likewise, the

apparatus set up required is simple (Figure 12). It was found that this type of enhanced

extraction was good for extraction of compounds such as phenolics and uronic acid from

Ascophyllum nodosum (Kadam, Tiwari et al. 2015).

Figure 12: Schematic diagram of ultrasound-assisted extraction assembly (A – ultrasound

generator, B – transducer, C – ultrasound cylinder probe, D – beaker with sample and solvent

of extraction, E – bubble cavitation phenomena, F – thermocouple, G – data recorder)

(Kadam, Tiwari et al. 2015).

Response surface methodology was engaged to examine the effect of process variables

(extraction time, ultrasonic amplitude) to enhanced bioactive yields. Furthermore, ultrasound

was discovered to be efficient in extracting higher molecular weight phenolic compounds

(Kadam, Tiwari et al. 2015). This shows that ultrasound is a potential pre-supercritical

extraction treatment, for enhanced extract recovery.

The use of microwaves is another tool that can be made use of to enhance extraction from

macroalgae. It was established that the use of microwave-assisted extraction under optimal


26 | P a g e

process conditions was an effective method to recover fucoidan from brown macroalgae

species such as F. vesiculosus. This form of enhanced extraction has a short process time and

uses non-corrosive solvents with little waste. This means that is a reduction in costs due to

lower energy consumption compared to conventional technologies (Rodriguez-Jasso,

Mussatto et al. 2011).

Enzyme-assisted extraction is also a viable novel method. Macroalgae cell walls are made up

of a complex matric of polysaccharides. Enzymatic digestion also aids in removing

mechanical barriers for both water soluble and non-soluble bioactive compounds. It can,

therefore, be concluded that this is a valid method for industrial extraction applications

(Wijesinghe, Jeon 2012).

1.7 Characterisation of extracts

Upon completion of extraction, the next step is to characterise the present components of the

extract and their abundance. For this, there are many techniques that have been developed for

the analysis of bioactive compounds.

Nuclear magnetic resonance (NMR) spectroscopy (Figure 13) is a research technique that

involves the interaction of the magnetic properties of certain atomic nuclei. It defines the

chemical and physical makeup of atoms and molecules within the sample being examined.

Utilising the phenomenon of NMR, this equipment can produce comprehensive information

with regards to the structure, reaction state, dynamics and chemical surroundings of the

compounds. The resonance frequency of the equipment alters the intramolecular magnetic

field around an atom with molecules, hence details of the electronic structure of this material

can be determined (Keeler 2007).


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Figure 13: Schematic diagram of NMR Spectroscopy set-up (Jacobsen 2007).

High-performance liquid chromatography (HPLC) is an analytical chemistry technique used

in the separation of mixture constituents, thus identifying each component and quantifying

them. Pumps are used to pressurise liquid solvent (containing the mixture to be analysed) and

pass it through a solid adsorbent material filled column. As the mixture has many different

components, each will interact differently with the adsorbent material resulting in variation

in flow rates for different compounds. This leads to their separation as they flow out of the

column (Lindsay, Kealey 1988).

The solid adsorbent material is typically a granular material made of solid particles such as

silica, polymers, etc. They are usually between 2 and 50 micrometres in size (Lindsay, Kealey

1988). The solvents typically used in the pressurised liquid include water, acetonitrile,

methanol, etc., this is referred to the ‘mobile phase’. The temperature and composition of this

fluid play a major role in the separation process, as this influences the interaction between it

and the adsorbent material. These interactions are physical in nature, such as ionic, dipole-

dipole or hydrophobic, more often than not a combination.

Gas chromatography (GC) is another typical form of characterisation of bioactive

compounds. It is commonly used for separation and analysis of compounds that can be

vapourised without decomposition. Once the mixture is separated by GC respective amounts

of materials can be calculated (Pavia, Donald L., Gary M. Lampman, George S. Kritz,

Randall G. Engel 2006).


28 | P a g e

Similar to HPLC, GC also has a mobile phase, in this instance it is known as a carrier gas.

This gas is usually an inert gas such as helium or an unreactive gas such as nitrogen. The

stationary phase uses an inert solid support coated in a microscopic layer of polymer or liquid

within a piece of metal or glass tubing, known as a column.

The wall of the column is where the gaseous compounds being analysed interact; it is here

the mixture separates at different times, known as the retention time of the compounds. These

retention time analysis is what gives GC its characterisation usefulness.

GC in principle is similar to that of HPLC, but with several notable differences. The first

being that GC separates compounds in a mixture between a liquid stationary phase and a

mobile gas phase, whereas HPLC utilises a solid stationary phase and a liquid is the mobile

stage. Further to this, the column in which the gas phase passes through in GC is located in

an oven where the gas’ temperature is controlled. No such equipment is used in HPLC, which

makes it useful to halt any thermal degradation of compounds (Higson 2004).

1.8 Ascophyllum nodosum

Ascophyllum nodosum is the specific brown macroalgae (Figure 14) species being analysed.

It is a large, common species of the northern Atlantic Ocean. It is a commercially abundant

species native to Ireland and is used as a source of animal feed and fertiliser.

Ascophyllum nodosum has a maximum life span estimation of hundreds of years. It is a slow

growing species, which coupled with its long lifespan makes it perfect for rotational

harvesting, to allow adequate time for regrowth (Berg 1992).

This species of brown macroalgae is easily identified by its ability to float with altered water

surfaces. Vesicles, commonly referred to as air bladders, act as a support for the thallus, thus

allowing the macroalgae to be exposed to sunlight at a maximum (Barsanti, Gualtieri 2014).


29 | P a g e

Figure 14: Image is showing Ascophyllum nodusom macroalgae. A- apical tip; B- basal

shoot; H- holdfast; I- internode; L-lateral Shoot; P-primary Shoot; R- receptacles; S- stump;

V- vesicle. (Cousens 1981)

Pharmacological research continues to uncover potential in this species of macroalgae, along

with other potential useful chemicals. Fucoidan, as extracted from A. nodosum, has shown

excellent metal binding abilities, binding to lead most efficiently (Barsanti, Gualtieri 2014).

The Ascophyllum nodosum used for this research comes from Co. Donegal in Ireland. It is

the waste product of macroalgae processed for the manufacture of fertilisers. The company

is called ‘Oilean Glas Teoranta’, and cultivates the macroalgae from the surrounding

coast0line. The following process is followed to produce the fertiliser product known as ‘al-

gaegreen’ (Figure 15):


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Figure 15: Process flow diagram of OGT Macroalgae to fertiliser process.

The macroalgae go through many operations, as shown in Figure 15 that will affect the

chemistry of the macroalgae cells. It will be important, therefore, to compare the analysis of

the waste Ascophyllum nodosum with fresh samples.

The composition of this macroalgae species is approximately 80% water and 20% dry weight,

of which 40-70% is carbohydrates such as cellulose and mannitol. 3-10% is proteins such as

pigments; 4-8% are polyphenols and their derivatives, and 2-4% is made up of lipids. The

remained is a wide array of other micro/macro-elements (Conde, Balboa et al. 2013a).


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2.0 Project Objectives

The aim of this research is to assess the extraction potential of sterols from waste Ascophyllum

nodosum, using supercritical carbon dioxide (sCO2). As explored in the review of literature

on supercritical fluids, there are many factors, which directly affect the diffusivity of the

solvent. Parameters including temperature, pressure, use of co-solvent and therefore

viscosity.

It was found that the best pressure for these experiments was 75 bar. Experiments will,

therefore, be carried out at various temperatures between 308 and 328 K. Initial experiments

utilised an ethanol co-solvent, however, the experiments that proceed will not use a co-

solvent, as it has been hypothesised that they defer the fluid from dissolving sterols and tend

more towards fatty acids.

Runs using subcritical carbon dioxide (298 K) will also be run, to examine the dissolving

power effects of carbon dioxide as a solvent below critical point, and compare this with

supercritical runs. This parameter was used as CO2 becomes supercritical at 304.25 K.

Fresh samples of Ascophyllum nodosum will also be examined. sCO2 will be used in the same

way as with the waste macroalgae, and the yield of extraction with be compared between the

two. This allows for a better understanding of the reliance on supercritical fluids for this

application.

To understand the abundance of the desired sterols within the macroalgae, exhaustive

extractions will be carried out on both waste and fresh Ascophyllum nodosum. These results

can then be compared to the extractions using supercritical carbon dioxide.

NMR spectroscopy will be used to analysis the amount of sterols present in the extractants.

NMR is being employed as it has a low error of analysis and gives a broad idea of all the

constituents within a mixture. However it must be noted that no truly accurate method exists

to characterise biomass, this is a key challenge faced in this field of research.


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3.0 Experimental

The extraction of fucosterol from Ascophyllum nodosum, a brown macroalgae species, is

being explored. The raw material for these experiments has been provided by Oilean Glas

Teo (OGT), a macro-algae process plant in Co. Donegal, Ireland. They produce liquid

fertiliser from Ascophyllum nodosum which leaves a solid by-product. The method of

extraction used is supercritical carbon dioxide, of which carbon dioxide comes from a bottled

source. Thar, equipment was used with specialised producer of heating and cooling systems.

The Thar equipment was controlled using a computer system called ‘The Process Suite’. This

allowed for control of start-up and shut-down, as well as the temperatures and pressures

desired. Below shows a process flow diagram of the supercritical extraction process used

(Figure 16).

Figure 16: Process flow diagram of the supercritical rig employed for extraction.

3.1 Methodology

3.1.1 Preparation of macroalgae sample

The macroalgae is a brown granular substance, of varying particle sizes. A grinder was used

to ground the macroalgae down to 0.2 mm in size. This increases the surface area of the

macroalgae. A mass of ground Ascophyllum nodosom is then weighed out for extraction. The


33 | P a g e

mass is then placed into the stainless steel extraction vessel (500ml), which fits into the

supercritical rig.

3.1.2 Supercritical Rig

A schematic diagram of the rig and the process is shown in Figure 16. This process

equipment is used to set the temperature and pressure parameters of the process. This was

done through a computer program known as the ‘Process Suite’. A full description of the

start-up and shut-down procedure is shown in the appendix.

3.1.3 Analysis of Extracts

The extractants were then taken directly for analysis. Any unused extracts were stored in a

labelled glass vile so that if desired they could be reanalysed.

3.1.4 Preparation for Analysis

Any solid present in the extracts was dissolved in dichloromethane. This solvent is used as

its polarity allows it to easily dissolve organic materials. 3g of Magnesium Sulphate (MgSO4)

was added to remove excess water from the solution. The solution was then filtered through

a one layer thick sheet of filtration paper. This removed any large impurities as well as the

MgSO4 used to dry the solution.

The filtered mixture was then evaporated down. The extracts were removed when a thick

green liquid was left. The sample left was then transferred to a clean vial.

An amount of extracts was weighed out. Vanillin was then added to it, as this is our internal

standard set point for the analysis. Vanillin was prepared in a chloroform-d solution, where

1ml of solution had 0.01025g of vanillin. Thus, 1 ml was added to each sample being tested.

The mixture was then transferred to a glass NMR tube, and analysed using NMR

spectroscopy.


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3.1.5 Exhaustive Extractions

Exhaustive extractions were done by solvent extraction. Organic solvent, in this case Hexane,

was used to get an idea of total compound masses in both waste and fresh Ascophyllum

nodosum.

5g of macroalgae was placed into a round-bottomed flask along with 100ml of Hexane.

Magnetic stirrers are placed into this flask and it is clamped into place in a water bath over a

heater. Reflux apparatus is the attached to the flask and water allowed to flow through it. A

balloon was filled with nitrogen gas and attached to the apparatus by syringe, this kept the

reaction conditions inert. The heater was then turned to 75oC and left for 4 hours, as Hexane’s

boiling point is 69oC.

The solution was then taken off reflux; the liquid phase drained off and a 100ml of fresh

hexane added to the macroalgae. Reflux was then repeated for a further 4 hours.

The extract liquor was then reduced down, and the samples analysed in the same format as

the supercritical extractants.

3.1.6 NMR Analysis

NMR spectroscopy was used for analysis of the extracts. This was achieved by integration of

the peaks from a NMR spectrum. This resulted in the relative number of protons present for

a particular part of a molecule, By an internal standard, in our case Villain,, a rough estimate

of yields of compounds can be determined.

The sterols and fatty acid peaks were analysed by spiking the sample with fixed concentration

of vanillin; this can be used as a reference point to determine the mass of other compounds.

The steps of this calculation are as follows:

1. Moles of vanillin is calculated using the following equation: 𝑀𝑜𝑙𝑒𝑠 = 𝑀𝑎𝑠𝑠

𝑅𝑀𝑀

2. Peak of vanillin is identified, found at 9.8 ppm, on the x-axis.

3. Peak of desired product is identified and integrated in relation to vanillin, for example,

fatty acids is located at 2.3 ppm, or sterols which are located at 3.5 ppm.

4. The moles of vanillin is the multiplied by the ppm of the desired product.


35 | P a g e

5. This mole ratio can then be used to find the mass of substance present, using the RMM

of the product being identified.

𝑀𝑎𝑠𝑠 = 𝑀𝑜𝑙𝑒𝑠 ∗ 𝑅𝑀𝑀

6. This mass can then be analysed against the mass of extracts originally obtained from

the supercritical rig, to get the yield.

Hydrogen showcases a large peak on the NMR spectrum, and so the solvent used for this

analysis is not a standard organic solvent but Deuterated-Chloroform. This chemical does

not exhibit a large interfering peak.

The peaks of the desired compounds can be found at the following x-axis values:

Sterols- 3.5 ppm (Figure 17)

Fatty Acids- 2.3 ppm (Figure 18)

Vanillin- 9.8 ppm (Figure 27)

The peaks of the desirable compounds appear on the NMR graph as follows:

Figure 17: NMR graph showing sterol peaks.


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Figure 18: NMR graph showing Fatty Acid peaks.


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4.0 Results and discussion

From the beginning of this investigation, it was known from previous research that both fatty

acids and sterols were present in the waste A. Nodosum (Miyashita, Mikami et al. 2013,

Cousens 1981). My investigation looked into what process conditions where best suited to

that of sterol extraction, with Fucosterol being the focus of the research. From the literature,

it is suggested that the best temperature in supercritical extraction for sterols is 40oC (Nyam,

Tan 2010) and this is also the case for fatty acids (Cheung 1999b). Taking this as a basis, the

results obtained will be analysed and comparison to the literature made. It can be assumed

the remaining bulk of extracts would be made up of polysaccharides, polyphenols, lipids, and

other compounds such as pigments.

The experiments were designed using software called ‘Design Expert 8’. It was used so

random experiments would not be done. If a range of parameters is inserted into the software,

it will select points within those parameters and then using the results will calculate the most

probable parameters for the most efficient results.

The first thing researched was the use of supercritical Carbon Dioxide. In literature, it is

suggested to use a co-solvent. Co-solvents increase the polarity of the solvent and, as a result,

make the desired materials dissolve more easily. However, it was decided that we would not

use a co-solvent for the purpose of these experiments. As sterols where the main focus of the

research and optimisation of their extraction was the goal, it was felt that the use of a co-

solvent, like ethanol, would make the solvent tend more toward the fatty acid compounds.

From an economic viewpoint, this would also account for cost saving on an industrial scale.

Analysing the small amounts extracted using NMR spectroscopy, it is also clear that the

amounts of fatty acids outweighs the amount of sterols present, this became evident for all of

the runs completed at the various temperatures and pressures utilised.


38 | P a g e

Figure 19: Graph showing extract amounts at various temperatures.

The trend for both the Sterols and the Fatty acids follows the same general pattern, as shown

in Figure 18. It can be seen the extraction peaks at a temperature of 40oC for both of the

compounds. This trend follows what was expected from the review of literature.

Some anomalous results can be seen here, however, for example, the fatty acid line at 55oC.

These results may be due to human error in calculation, or mechanical error of measurement

tools used when carrying out the analysis, for example, the scales used to measure out the

amount of extract used were not calibrated perfectly.

From analysing the results, it can be seen that the overall the mass of both the sterols and the

fatty acids is small. The total number of extracts from a mass of maroalgae on average was a

small fraction of the original mass utilised within the supercritical rig. Reasons for this may

also be linked to the choice not to use a co-solvent as overall less materials were being

dissolved and carried out of the macroalgae extraction vessel.

The next step in the experiment was to determine if the low product yield was due to

inefficiency of the use of supercritical fluid extraction for this particular need, or if the waste

macroalgae used was the problem. A run on the supercritical rig was completed using a fresh

30 35 40 45 50 55 60

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

Temperature (oC)

Mas

s (g

)

Graph showing amount of extracts from supercritcal extraction using a

variety of temperatures

Total Mass of Sterols (g)

Total Mass of Fatty Acids (g)


39 | P a g e

sample of A. Nodosum. The thought behind this was that the waste macroalgae comes from a

fertiliser production process and that this may have leached sterols out of the macroalgae,

explaining the low product yield. The samples were prepared, in the same way, using a

grinder to get particulate sizes of 0.2mm.

Figure 20: Table is showing results of supercritical carbon dioxide runs completed at 40°C.

Figure 21: Graph showing sterol and fatty acid content from supercritical extraction of both

waste and fresh Ascophyllum nodosum.

As can be seen from the results (Figure 20), there was no increase in yield of extracts when

fresh macroalgae was used in the supercritical rig. From this, it can be determined that the

use of a co-solvent free supercritical extraction is not efficient for the extraction of sterols. It

can be seen however, that the amounts of sterols extracted from the waste was greater than

that of the fresh sample. A possible reason for this could be that during the fertiliser

0.0000

0.0020

0.0040

0.0060

0.0080

0.0100

0.0120

0.0140

Sterols Fatty Acids

Mas

s (g

)

Chart showing Sterol and Fatty Acid content in extracts after

supercritical extraction at 40oC

Waste Sample Fresh sample

Temperature(°C)

Total Mass

of sterols

Total mass

of fatty acids

Total mass

of extract

Mass of

seaweed

used

Waste Sample 40 0.00481 0.00626 0.21 85.0000

Fresh sample 40 0.00113 0.01198 0.0195 67.29


40 | P a g e

production process, many of the more easily dissolvable compounds are taken out, and so

sterols will be favoured more during the sCO2 extraction process.

Possible reasons for the overall low extracted mass of sterols may be explained by the link

between molecular mass and solubility. From Figure 10 in section 1.5.3 it can be seen, lauric

acid (C12H2402) which has a molecular mass of 200.32, has the highest solubility in

supercritical carbon dioxide. Comparing that even with the second largest fatty acid in the

group (palmitic acid, molecular weight 256.42) there is a substantial difference in solubility.

It can be deduced from this the explanation of the low sterol yield from the supercritical

extractions. Sterols, in general, have a much larger molecular mass than that of fatty acids.

For example fucosterols, molecular weight is 412.69. Whereas oleic acid has a mass of

282.46. Taking the above literature into consideration, it explains the low yield of sterols and

the higher yield of fatty acids, in both runs done with fresh and waste macroalgae.

To determine the total amount of sterols and fatty acids within the Ascophyllum nodosum

samples (fresh and waste) exhaustive extractions were carried out. Hexane was used as the

solvent and the macroalgae was heated under reflux. By doing this, an analysing the product

extracts using NMR the totals were estimated.

Figure 22: Table of results showing results of exhaustive extractions.

Total Mass of sterol

(g)

Total mass of fatty acids

(g)

Mass of macroalgae

used (g)

Fresh Sample 0.05 0.25 5.00

Waste Sample 0.03 0.15 5.00


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Figure 23: Bar chart showing sterol and fatty acid content from exhaustive extractions from

waste and fresh Ascophyllum nodosum.

The total amounts of fatty acids significantly lower in the waste as expected. It can also be

seen that the total sterol number has also decreased. It is also evident that the total number of

sterols within the macroalgae whether fresh or waste is very low.

Runs using subcritical carbon dioxide were also run, this was to justify the use of the

supercritical fluid as a solvent. In subcritical conditions the rate of materials dissolving

should, in theory, be lower, however it was worth exploring its use for sterol extraction.

Carbon dioxide becomes supercritical at 31.1oC, therefore, a run where carried out at 25oC to

ensure subcritical conditions, the results in terms of material mass extracted are below:

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Total Mass of sterol (g) Total mass of fatty acids (g)

Chart repesenting the Sterol and Fatty Acid contents from exhaustive

extractions on fresh and waste A. Nodosum

Fresh Sample Waste Sample

Temperature(oC) Pressure (bar)

Total Mass of

sterol (g)

Total mass

of Fatty

Acids (g)

Fatty

acids:

sterol ratio

Total

mass of

extract(g)

Mass of

macroalgae

used (g)

25 75 0.000110 0.00162 0.0649 0.0324 65.8

Figure 24: Table of results for extraction process using sub-critical carbon dioxide.


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As can be seen, the total mass of sterols extracted under subcritical conditions is lower than

that of any of the runs done using supercritical carbon dioxide.

Figure 25: Bar chart of mass of sterols and fatty acids from subcritical carbon dioxide.

Furthermore, it can be seen that similarly to supercritical extraction, fatty acids are favoured

in terms of dissolution into the solvent.

Mass of sterol Mass of Fatty Acids

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

Mas

s (g

)

Extract mass from subcritical extraction using Carbon Dioxide (25oC)


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Run Temperature(oC) Pressure (bar)

Mass of ex-tract used (g)

Mass of sterol (g)

Total Mass of sterol (g)

Mass of Fatty Acid (g)

Total mass of FA (g)

Fatty Acid: Sterol ratio

Total mass of extract (g)

Mass of macroalgae used (g)

1 40 75 0.012 0.007 0.127 0.020 0.361 0.351 0.210 85.000

2 35 76 0.131 0.004 0.018 0.007 0.031 0.568 0.600 95.410

3 55 75 0.065 0.002 0.010 0.013 0.079 0.127 0.390 56.510

4 55 74 0.021 0.001 0.003 0.014 0.033 0.080 0.050 56.860

5 55 74 0.015 0.001 0.002 0.005 0.022 0.108 0.064 85.830

6 45 74 0.240 0.002 0.002 0.004 0.003 0.508 0.180 81.240

7 35 76 0.340 0.004 0.007 0.005 0.001 0.760 0.070 65.780

8 45 75 0.218 0.001 0.005 0.003 0.001 0.325 0.010 71.026

9 45 76 0.590 0.001 0.008 0.028 0.002 0.050 0.036 69.680

10 45 76 0.358 0.001 0.000 0.004 0.001 0.308 0.037 71.270

Figure 26: Table of results of supercritical extraction from waste Ascophyllum nodosum.


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5.0 Conclusions and further works

To summarise, many extractions using supercritical carbon dioxide were carried out on the

brown macroalgae Ascophyllum Nodosum. The macroalgae in question was the waste by-

product from a macroalgae to fertiliser process plant, ‘OGT’. The macroalgae was ground

down to particle sizes of 0.2 mm before extraction. The main focus of the experiments was to

determine the best conditions for the extraction of sterols; organic compounds which have a

broad range of industrial uses from cosmetics to biomedical applications. No co-solvent was

used in conjunction with the supercritical fluid, as this tends to favour the extraction of fatty

acids.

It was found that using supercritical carbon dioxide the yield of sterols were low. The best

temperature for extraction of sterols followed the same trend of that seen in literature, with

40oC being the optimum. Fatty acid extraction was also found to be 40oC, which was also

expected from literature. Experiments utilising subcritical carbon dioxide were also run to

compare with that of supercritical fluids. It was expected to have a lower yield and the results

found reflected that hypothesis. It was found through the process of exhaustive extractions, that

there is also a lower number of sterols within the waste macroalgae by-product than within the

fresh macroalgae. This also helps to evaluate the low sterol yield from the supercritical runs.

5.1 Further Work

The next steps for these experiments are to determine a method of optimum extraction. It is

known that the sterols are present, but the use of supercritical carbon dioxide (without co-

solvent) is not the best format. Suggestions would be to use pre-treatment methods as explored

in this report's literature review. Methods such as ultrasound-assisted extraction, microwave-

assisted extraction or the use of enzymes are all options worth exploring. One of the hurdles to

overcome in extraction from biomaterials is the strong, complex structure of the cell walls.

These are usually made up of an interlinking, sturdy network of polysaccharides. The use of

these suggested treatments would help to break this down and allow for more cell constituents

to be extracted. Further to this, more analysis of the total sterol number within the macroalgae

should be carried out to get a more accurate idea of the total mass of sterols within the cells. It

may be found, however, in whatever format of extraction that is utilised, that the trend of lower


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molecular weighted compound dissolving into the solvent is a priority as seen between fatty

acids and sterols in this report.


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6.0 Acknowledgments

I would like to take this opportunity to thank:

My supervisor for their continued support and guidance throughout my project.

My friends and family who have supported me throughout this project.


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8.0 Appendix

8.1 NMR Calculation

As discussion in the experimental methodology, vanillin is used as an internal standard in the

calculation of compounds abundance in the extracts. The NMR peak for vanillin is found at

approximately 9.8 ppm, and can be seen below in Figure 27:

Figure 27: NMR graph showing peak for vanillin at 9.8 ppm.

To calculate our amount of a substance using this standard we:

Firstly integrate the vanillin peak, so that it is the reference peak.

Integration of the desirable peak range is then analysed (for example fatty acids).

Completing this action give us a parts per mole ratio between the vanillin and other

compound. Using this ratio as well as the moles of vanillin used, we can calculate the

moles and furthermore the mass of the desired product. (𝑀𝑜𝑙𝑒𝑠 = 𝑀𝑎𝑠𝑠

𝑅𝑀𝑀)


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8.2 NMR Results

Figure 28: Graph of NMR results using supercritical CO2 at 40oC and 75 bar.


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Figure 29: Graph of NMR results using supercritical CO2 at 35oC and 76 bar.

Figure 30: Graph showing NMR results of supercritical CO2 at 55oC and 75 bar.


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Figure 34: Graph showing NMR results for supercritical CO2 at 35oC and 76 bar.


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Figure 38: Graph showing NMR results for subcritical CO2 at 25oC and 75 bar.

Figure 39: Graph showing NMR results for exhaustive extraction on fresh Ascopyllum

nodosum


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Figure 40: Graph showing NMR results of exhaustive extractions on waste Ascophyllum

nodosum

8.3 Start-up of Supercritical Rig

Firstly, the systems cooler is turned on and set to -2oC. This ensures the carbon dioxide remains

liquid and can be pumped to the correct pressure.

The stainless steel extraction vessel containing the weighed out mass of Ascophyllum nodosum

is placed into the rig, with a pressure gauge fitted over it, to seal it into the system. For the

control of the rig, a computer programme called “Thar Process Suit” is used. It controls all

aspects of the rig. Pressure within the extraction vessel is set (up to 350 bar), and the flow of

carbon dioxide is turned on, by means of opening the flow from bottled carbon dioxide directly

attached to the rig. The Carbon Dioxide pump is then turned on and set to a flow rate of up to

15.0 g/min. The heat exchangers for heating the flow mixture are then turned on and set to the

desired temperature. The system is then left for a set amount of time (5 hours).


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8.4 Shut-down of Supercritical Rig

Once the desired extraction time is met, the rig then needs to be shut down. Firstly, the heat

exchangers are turned off, and the carbon dioxide pump turned off. Pressure within the system

is then slowly released from the extraction vessel until a pressure of 0 bar is met. This is done

my control of the manual BPR valve. The extraction vessel is then flooded with 100ml of

organic solvent and tapped off.

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