SEQUENTIAL EXTRACTION OF FUCOIDAN,

ALGINATE AND FUEL FROM BROWN MARINE ALGAE

BY

JOSEPH ATITSOGBUI BME0001223

PELUMI SAMUEL OMOWAIYE BME0008023

LAMIN KEBBEH BME0008523

PROPHET GABRIEL DADZIE BME0009223

SUPERVISOR

DR FELIX OFFEI

A PROJECT REPORT IN THE DEPARTMENT OF MARINE ENGINEERING,

FACULTY OF ENGINEERING SUBMITTED TO THE DEPARTMENT OF MARINE
ENGINEERING OF REGIONAL MARITIME UNIVERSITY, ACCRA-GHANA, IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A
BACHELOR OF SCIENCE DEGREE IN MARINE ENGINEERING

JUNE 2023
 DECLARATION
Candidates’ Declaration

“We, the undersigned, hereby declare that this project work (with the exception of quotations and
references which are duly acknowledged) is the result of our own original research and that no
part of it has been presented for another degree in this university or elsewhere.”

Name of student Signature Date

Joseph Atitsogbui ……………… ………/………/………

Pelumi Samuel Omowaiye ……………… ………/………/………

Lamin Kebbeh ……………… ………/………/………

Prophet Gabriel Dadzie ……………… ………/………/………

i
Supervisor’s Declaration
“I hereby declare that the preparation and presentation of the project work were supervised by me
in accordance with the guidelines on the supervision of projects laid down by the Regional
Maritime University, Accra, Ghana.”
Signature……………………… date………/…………. /…………..

Dr Felix Offei
(Lecturer)

Signature……………………… date………/…………. /…………..

Dr Isaac Animah
(Head Of Department, Marine Engineering)

ii
 ACKNOWLEDGEMENT

We would like to express our earnest gratitude to almighty God for the abundant grace he has
bestowed upon us through the course of our studies at the university and throughout our project.
We also want to acknowledge and appreciate our supervisor Dr Felix Offei for his professional
and academic guidance as well as insight for finding time out of their busy schedule to meet us
through various stages of our project work.

In addition, we would also like to express our profound gratitude to Mr. Gabriel for his time and
advice.

We also thank the BSc. Marine engineering class of 2023 for their love and support during our
BSc. Programme.

Finally, we are grateful to our parents and loved ones for their love, encouragement and support
in kind and cash throughout our studies.

i
 DEDICATION

We at this moment dedicate this project work to God almighty for His Grace and to our parents
and guardians for their support.

ii
 ABSTRACT

This study investigated how to sequentially extract the hydrocolloids, alginate and fucoidan, as
well as ethanol, from marine biomass in order to maximise the value of the biomass. It
particularly sought to optimise the extraction of the hydrocolloids, alginate and fucoidan from
brown seaweeds using a physicochemical method and to determine the potential ethanol yield
from the residual biomass obtained after hydrocolloid extraction. The motivation for this work is
to make good use of the sargassum that has invaded the beaches of Nzema in the western region
of Ghana and has become a threat to the livelihoods of local communities, fisheries, tourism, and
marine ecosystem. It also seeks to reduce the accumulation of sargassum seaweed from the coast
of Ghana in order to avoid its intoxicating health hazard because of foul odour. The study was
conducted through the sequential extraction of fucoidan and alginate under varying conditions of
time and temperature. It also included the production of ethanol from the residue obtained after
the extractions. The study established that the yield of fucoidan and alginate increases as
temperature and time increases and the potential ethanol yield from the substrate was 16.42%.

iii
 TABLE OF CONTENT
ACKNOWLEDGEMENT ............................................................................................................... i
DEDICATION ................................................................................................................................ ii
ABSTRACT................................................................................................................................... iii
CHAPTER ONE. INTRODUCTION ............................................................................................. 1
1.1 Background to the study ........................................................................................................ 1
1.1 Problem Statement ................................................................................................................ 3
1.2 Research Objectives .............................................................................................................. 3
1.2.1 Main objectives............................................................................................................... 3
1.2.2 Specific objectives .......................................................................................................... 3
1.3 Research Questions ............................................................................................................... 4
1.4 Limitations of Study .............................................................................................................. 4
CHAPTER TWO. LITERATURE REVIEW ................................................................................. 5
2.1 General overview of seaweed ............................................................................................... 5
2.2 Types of seaweed .................................................................................................................. 6
2.2.1 Brown seaweed (Phaeophyceae) overview .................................................................... 6
2.2.2 Red seaweed (Rhodophyceae) overview ........................................................................ 7
2.2.3 Green seaweed (Chlorophyceae) overview .................................................................... 8
2.3 Properties of seaweeds .......................................................................................................... 8
2.4 Hydrocolloid overview ........................................................................................................ 11
2.5 A general overview of alginate ........................................................................................... 12
2.5.1 Application Area of Alginate ....................................................................................... 13
2.5.2 Properties of Alginate ................................................................................................... 14
2.5.3 Side Effects of Alginate on Humans ............................................................................ 15
2.5.4 Limitation of Alginate .................................................................................................. 15
2.5.5 Extraction methods for alginate .................................................................................... 16
2.6 A general overview of fucoidan .......................................................................................... 18
2.6.1 Application Area of Fucoidan ...................................................................................... 18
2.6.2 Properties of Fucoidan .................................................................................................. 19
2.6.3 Extraction methods for fucoidan .................................................................................. 20
2.7 A general overview of ethanol ............................................................................................ 21

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 2.7.1 Production of ethanol from brown seaweed ................................................................. 21
2.7.2 Production methods of ethanol ..................................................................................... 22
2.7.3 Acid hydrolysis process of bioethanol production ....................................................... 22
2.7.4 Enzymatic hydrolysis process of bioethanol production .............................................. 23
CHAPTER THREE. METHODOLOGY ..................................................................................... 24
3.1 Harvesting and preparation of seaweed .............................................................................. 24
3.2 Preparation of standard solutions ........................................................................................ 25
3.3 Sequential extraction of fucoidan and alginate ................................................................... 26
3.3.1 Fucoidan extraction ...................................................................................................... 26
3.3.2 Alginate extraction ....................................................................................................... 26
3.4 Production of Ethanol.......................................................................................................... 27
3.5 Chemical Equations and Calculations ................................................................................. 28
3.6 Experimental Design ........................................................................................................... 29
3.7 Yield .................................................................................................................................... 30
CHAPTER FOUR. RESULTS AND DISCUSSIONS ................................................................. 31
4.1 Yield of Fucoidan ................................................................................................................ 31
4.2 Yield of Alginate ................................................................................................................. 32
4.3 Yield of Ethanol .................................................................................................................. 34
CHAPTER FIVE. CONCLUSIONS AND RECOMMENDATIONS ......................................... 35
Conclusions ............................................................................................................................... 35
REFERENCES ............................................................................................................................. 36

LIST OF FIGURES

Figure 1: Brown Seaweed in the ocean (Kåre, 2017; David, 2008) ............................................... 7
Figure 2: Red Seaweed on rocky platform (An Bollenessor, 2013; Akash 2022) .......................... 7
Figure 3: Green Seaweed on rocks at shore (Maggy, 2006; National education, 2008) ................ 8
Figure 4: Bioethanol production process from brown algae (Ok and Eun., 2016) ....................... 23
Figure 5: Harvesting and Washing of Seaweed ............................................................................ 24
Figure 6: Sun Dried and Milled Seaweed ..................................................................................... 25
Figure 7: Freshly extracted alginate. ............................................................................................. 27
Figure 8: Dried alginate (a) and dried fucoidan (b) extracted from sargassum. ........................... 27
Figure 9: Neutralised hydrolysate and bricks analysed using refractometer. ............................... 28

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Figure 10: Surface plot of fucoidan yield against temperature and time from Minitab. .............. 31
Figure 11: Regression analysis for fucoidan yield. ....................................................................... 32
Figure 12: Surface plot of alginate yield against temperature and time ....................................... 33
Figure 13: Regression analysis for alginate yield. ........................................................................ 34

LIST OF TABLES

Table 1: Compound extracts from seaweeds with bioactive properties. ........................................ 9

LIST OF EQUATIONS

Equation 1: Model equation of fucoidan yield ............................................................................. 32

Equation 2: Model equation of alginate yield ............................................................................... 33

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 CHAPTER ONE. INTRODUCTION

1.1 Background to the study

During the fall and winter months, the coastal floating brown macroalgae commonly called
Sargassum has been a regular occurrence on the Caribbean coastlines where the Sargassum species
exploded at a high extent and quantity popularly known as the 2013 invasion even crossing the
Atlantic Ocean to reach the west coast of Africa (Louime et al., 2017).

Seaweeds are marine plants that grow in water bodies, they are rich in nutrients such as fibre,
iodine, protein, and vitamin K. In some other parts of the world like Asian countries, seaweeds are
used for various foods unlike in the Western world where it is used for non-food applications.
Generally, the term seaweed invokes an image of smelly and rotten masses found on the beaches
of Western countries. They are categorised into three namely: red seaweed (Rhodophyceae), Green
Seaweed (Chlorophyceae), and Brown Seaweed (Phaeophyceae). The most abundant on the shores
of Western countries is Phaeophyceae (Tiwari et al., 2015). In 2015, the Mexican federal
government allocated USD 3.2 million to Sargassum extraction due to its environmental impact
and adverse effect on the tourism industry. The amount of seaweed exceeded the extraction
capacity and by late summer of 2019, Mexico spent USD 17 million on seaweed removal efforts
(Casas-Beltrán et al., 2020).

Scientific Research reveals that Sargassum blooming is tied to several factors. These factors
include climate change, and globalisation, changes in the ocean temperatures and circulation
patterns, high volume of Sargassum seaweed from the previous season serves as a seed source for
the next season, nutrient runoffs from industrialization, deforestation, and the use of excessive
nitrogen-based fertiliser.

To achieve global sustainability, society has to match the supply of food, feed, and fuel with the
demand of the world’s increasing population in the most appropriate way possible. Sargassum
mainly comprises two hydrocolloids namely Fucoidan and Alginate. Fucoidan is a sulfated
polysaccharide that is obtained from the cell wall of brown seaweed. The composition of Fucoidan
varies amongst species of brown seaweed. Fucoidan is a major component in pharmaceutical
products, it possesses biological functions such as Inflammatory, immune-modulatory, antitumor,

1
anti-bacterial, anti-viral, anticoagulant, antioxidant, neuro-protective, cardio-protection, and
growth-promoting effects amongst others (Saeed et al, 2021).

Present in the cell wall of the brown seaweed is an anionic polymer called Alginate. Alginate
provides mechanical strength to the cell walls of the seaweed, and it varies from 20% to 60% dry
matter in composition. Brown seaweed is not the only source of alginate (i.e., alginate can be
produced by bacteria). Alginate is widely applied in (food, cosmetic, paper, agricultural,
pharmaceutical, and medical) industries due to its gelation, viscosity, and stabilising properties.
The global seaweed market is worth more than USD 6 billion per annum of which 85% is in the
food area for human consumption. Seaweed-derived polysaccharides (carrageenan, Agar, and
alginate) make up almost 40% of the world’s hydrocolloid market. In 2016 the global alginate
market size value was USD 624 million, and the demand is bound to increase by high consumption
of frozen desserts, ice creams, beer, and yoghurt which can boost the market growth of alginate
value and use (Pereira et al, 2020).

Alginate has gained quite numerous applications in biomedical science and engineering because
of its properties which include biocompatibility and ease of gelation. Alginate is used in wound
healing, drug delivery, and tissue engineering because these gels have a similar structure to
extracellular matrices in tissues (Lee et al, 2012).

Furthermore, Sargassum comprises a significant quantity of sugar that can be converted to

bioethanol. Bioethanol can be produced from all plant materials due to the presence of cellulose,
hemicellulose, and lignin which are the three major construction compounds that are interwoven
to form the cell walls of all plants. As a result of the challenges involved in breaking down these
three major components into simple sugars, intensive research reviewed methods of obtaining
simple sugar from lignocellulosic plants. Bioethanol in current days is being produced
commercially from starch/sugar-based crops. Initially, with the production of ethanol in 1990, 4
billion gallons were made, and it increased slightly to 4.5 billion in 2000. Due to the spontaneous
increase in demand, it increased from 4.5 billion gallons to 23.3 billion gallons in 2010 (Guo et al,
2015).

The process to produce bioethanol begins with the conversion of carbohydrates by yeast through
fermentation followed by fractional distillation to purify the ethanol. Bioethanol can be extracted

2
from algae due to the presence of significant carbohydrates (20% of dry matter). Out of this 20%
half to two-thirds of the carbohydrate content can be converted to ethanol (Borowitzka et al, 2013).

1.2 Problem Statement

Sargassum invasion on the western coast of Ghana is a growing environmental and economic issue
that threatens the livelihoods of local communities, fisheries, tourism, and the marine ecosystem.
Sargassum seaweed accumulation on the western coast of Ghana is an intoxicating health hazard
because of foul odour, spawning a decline in fish catch and economic losses which requires an
effective management strategy to mitigate its negative impacts on the environment, local
communities, and economy.

1.3 Research Objectives

1.3.1 Main Objectives

The main objective of the study was to sequentially extract the hydrocolloids, alginate and
fucoidan, as well as the fuel, ethanol, from marine biomass in order to maximise the value of the
biomass.

1.3.2 Specific Objectives

The specific objectives of the study are:

1. To optimise the extraction of the hydrocolloid, alginate, from brown seaweeds using a

physicochemical methods.

2. To optimise the extraction of the hydrocolloid, fucoidan, from brown seaweeds using a

physicochemical methods.

3. To determine the potential ethanol yield from the residual biomass obtained after

hydrocolloid extraction.

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1.4 Research Questions

1. What are the optimal conditions for the extraction of alginate from brown seaweeds when
using physicochemical methods?
2. What are the optimal conditions for the extraction of alginate from brown seaweeds when
using physicochemical methods?
3. What would be the potential ethanol yield from the residual biomass obtained after
hydrocolloid extraction?

1.5 Limitations of Study

1. The yield of ethanol produced from seaweed is lower than that produced from maize, sugarcane,
and bamboo.

2. Machinery for efficient collection of sargassum from the shores

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 CHAPTER TWO. LITERATURE REVIEW

2.1 General overview of seaweed

Seaweed, also known as macroalgae, refers to a large number of macroscopic marine algae that
grows along rocky shorelines in the sea, rivers, lakes and other bodies of water. In simple terms,
seaweed in general does not have a formal definition, it is found in water bodies (ocean, river, sea,
etc.) and can be seen with the naked eye. Seaweeds are called macroalgae because they are
multicellular and visible, and this makes them different from microalgae. Seaweeds are widely
anchored to the bottom or solid structures of these water bodies by rootlike “clasp or holdfast”,
performing the function of attachment (Adam, 2020). According to Maine Seaweed Council,
seaweeds are mainly categorised into three namely: Brown seaweeds (Phaeophyceae) such as
focus and kelp; green seaweed (Chlorophyceae) such as sea lettuce; red seaweed (Rhodophyceae)
such as Irish moss and nori. They are separately differentiated by their colour which is controlled
by the pigment type and their chloroplasts (the subcellular structure accountable for
photosynthesis). In most cases, seaweeds are wrongly referred to as plants. Unlike land plants,
seaweeds do not have roots, leaves, and stems but rather have holdfast, stripes, and blades because
seaweeds do not have a vascular system (xylem and phloem) that circulates minerals and water
found in plants.

Seaweed species are edible, as many are likewise used for commercial purposes by humans
because they exhibit the most necessary extraordinary biochemical components such as protein,
carbohydrates, lipids, minerals (iron, iodine, and calcium), antioxidants, vitamins, essential fatty
acids, dietary fibre etc. amongst various marine biomasses utilised and traded around the world
(Birde et al., 2020; Edubirdie, 2022). These promote seaweeds to be easily incorporated in the
development and formulation of nutraceutical food products. Additionally, it is shown in a proven
study that the insertion of seaweed in daily illness is associated with low occurrences of numerous
diseases providing benefits to digestive health, bacterial, pathogenic virus and chronic diseases
such as congestive heart failure, diabetes, arthritis, heart attack, cancer, epilepsy (Silvia et al.,
2022). Seaweed is applied in various industries such as food, cosmetic, pharmaceuticals, medicine,
renewable energy etc. based on the valuable properties they exhibit including physicochemical
qualities, biologically active compounds, nutritional health benefits, biochemical components etc
(Birde et al, 2020).

5
According to the Maine Seaweed Council, seaweeds form an essential part of the marine
ecosystem by providing habitat and food for marine animals. Fish and invertebrates benefit from
low phosphorus and nitrogen concentrations since seaweed absorbs these excess nutrients because
of its complex structure. The excess nutrients have destructive effects on the marine ecosystem.
Seaweed also promotes ocean health by capturing carbon thereby reducing ocean acidification and
releasing oxygen for aquatic animals.

2.2 Types of seaweed

Different species of seaweed have been discovered around the world, but they have been grouped
into three types. These are brown seaweed (Phaeophyceae), red seaweed (Rhodophyceae) and
green seaweed (Chlorophyceae). Due to the importance of seaweeds, they are commercially
utilised as human food, fertiliser, drugs, fodder, etc (Mansilla, 2011). From marine ecosystems,
seaweeds are one of the most important natural resources and they are found on rocks at the bottom
of shallow coastal waters possessing biologically active compounds in abundance. (Sreenivasan,
2010); (Chojnacka K, 2012); (Munirasu, 2013); (Asaraja A, 2013).

2.2.1 Brown seaweed (Phaeophyceae) overview

Brown seaweed (Figure. 1) has about 1,500 to 2,000 species that are commonly found in cold
waters along continental coasts. They normally grow on rocky shores in cold areas of the world.
These seaweeds play a major role in the marine ecosystem by serving as food and potential habitat
for other species. Some common species of brown algae are Kelp, Sargassum, Rockweeds,
Fucales, Bladderwrack, etc. (Cock, 2011).

Brown seaweed has the largest and fastest-growing species of seaweed (Connor and Baxter, 1989).
All brown algae are multicellular because there are no species of brown algae discovered to exist
as a single cell or as colonies of cells (Bold et al., 1987). Most of the brown seaweed species are
edible and good for human health. The alginic acid (alginate) contained in the cell walls of brown
seaweeds is commercially extracted and used as a thickening agent in food and cosmetic products
(FAO, 2017).

6
Kelp (Laminariales), an example of brown seaweed utilises a great portion of carbon dioxide
through photosynthesis, and they can also store great amounts of that to contribute to the fight
against climate change (Vásquez et al., 2014).

Figure 1: Brown Seaweed in the ocean (Kåre, 2017; David, 2008)

2.2.2 Red seaweed (Rhodophyceae) overview

Red seaweeds (Figure. 2) are part of the oldest groups of eukaryotic algae (organisms whose cells
have a nucleus) (Lee, 2008). Red seaweeds are mostly found in marine habitats and are quite small
in freshwater bodies. (Dodds, 2019). Most of the few seaweed species that are found in freshwater
bodies are concentrated in warmer areas (Sheath and Robert, 1984). Red seaweeds are used as a
source of nutritional, functional food ingredients and pharmaceutical substances (Wang et al.,
2010). Red Algae are eaten raw in salads, soups and meals. (Wang et al., 2010)

Figure 2: Red Seaweed on rocky platform (An Bollenessor, 2013; Akash 2022)

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2.2.3 Green seaweed (Chlorophyceae) overview

Green seaweeds (Figure. 3) primarily contain chlorophyll A and B which reflects the green
colouration. They appear to be either bright green or dull green. Green seaweeds could either be
multicellular or unicellular and strive mostly in nutrient-rich salt water or fresh water with a high
concentration of phosphate and nitrate. They grow closest to the shore in shallow waters. Green
seaweeds contain a wealth of minerals and trace elements such as iron, iodine, and vitamin B12.
Green seaweeds provide the following benefits when consumed. These include improvement of
digestion and reduction in sugar absorption, balancing blood pH, antiviral against influenza,
soothing burns, toning, hydrating, and nourishing of the skin. The nutritional content of the
seaweed varies depending on the season and site of harvest (Pacific Harvest EST, 2002).

Figure 3: Green Seaweed on rocks at shore (Maggy, 2006; National education, 2008)

2.3 Properties of seaweeds

Apart from the fact that seaweeds are known as a rich source of minerals, they are also known as
a source of important bioactive compounds. Over the centuries, seaweeds have been used as
traditional medicines in several countries like China, Japan, the Philippines, and Korea (Kang et
al, 2016; Sanjeewa and Jeon, 2018). It has been shown in recent years that seaweeds contain high
antioxidative (Souza, 2012), anti-inflammatory, and antitumor polysaccharides (Yan, 2019).

Most of the bioactive compounds found in seaweeds were typically discovered from the aqueous
fraction of these seaweeds. This is a result of the traditional practices in most parts of the world
where seaweeds are boiled, prepared as a soup, or infused with hot water for the medicinal
properties of these seaweeds to be extracted and consumed by the patient (Anggadiredja, 2009;
Liu, 2012).

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Table 1: Compound extracts from seaweeds with bioactive properties.

Seaweed Extract/Compounds Bioactive References

Identified properties

Red Seaweeds

Kappaphyc Water extract Antioxidant, Dousip et al.

us alverazii Ethanol extract anticholesterol (2014)
Antiglycaemic Balasubrama
niam et al.
(2013)

Glacillaria Sulfated galactan Antioxidant Souza et al.

birdae (2012)

Glacillaria Acidic polysaccharide Antitumor Fan et al.

lemaneifor (2012)
mis

Gracilaria Chlorophyll proteins Anti- Shu et al.

changii inflammatory, (2013)
antiulcer

Gracilaria Methanol extract Anticancer Yeh et al.

tenuistipitat (2012)
a

Gracilaria Oligosaccharide-lysate Antiviral Wu et al.

sp. (2012)

Laurencia 5β-hydroxypalisadin B Anti- Wijesinghe et

snackeyi inflammatory al. (2014)

9
Green Seaweeds

Cauleurpa Caulerpin Antibacterial, Nagappan

racemosa anti- and
and inflammatory Vairappan
Caleurpa (2014)
lentilifera

Ulva Polysaccharide extract Anti- Sathivel et al.

Lactuca hepatoxicity, (2014)
antioxidant

Ulva rigida Fatty acids Antibacterial Ismail et al.

Ethanol extract Antibacterial, (2018)
antifungal Sirakov et al.
(2019)

Ulva Sulfated polysaccharide Immunomodula Tabarsa et al.

intestinalis tory (2018)

Brown Seaweeds

Sargassum Fucan Anticoagulant, Dore et al.

Vulgare antithrombotic, (2013)
Antioxidant,
and anti-
inflammatory

Sargassum Sulfoquinovosildiacylg Antiviral Plouguern_e

fusiforme lycerols et al. (2013)

Sargassum Phytosterols Anti-cholesterol Chen et al.

hemipphyll Oligofucoidan Antitumor (2014)
um Yan et al.
(2019)

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Dictyota Heterofucan Anti- Albuquerque
menstrualis inflammatory et al. (2013)

Dictyota Fucoidan Antiviral Rabanal et al.

dichotoma (2014)

Himanthalia Fucoxanthin Antioxidant, Rajauria et al.

elongata anticancer, (2017)
anti-
inflammatory

Laminaria Fucoidan Modulation of Kong et al.

japonica gut microbiota (2016)

Laminaria Β-glucan Modulation of Zhao and

digitata gut microbiota Cheung
(2011)

Undaria Essential oils Anti- Kang et al.

pinnatifida inflammatory (2016)

Ascophyllu Phlorotannins Immunomodula Corona et al.

m nodosum Low molecular weight tory (2016)
polysaccharide Modulation of Ramnani et
gut microbiota al. (2012)

2.4 Hydrocolloid overview

Hydrocolloids, also recognized as gels, gums, emulsions etc. are hydrophilic (strong affinity for
water) polymers derived from different sources including seaweed (e.g., alginate, fucoidan), plant
(e.g., locust bean gum, carrageenan, starch), modified or semi-synthetic (methylcellulose,
carboxymethylcellulose) animals (e.g., chitosan, egg white, gelatin) and microbial (e.g., xanthan
gum). Otherwise, defined as molecules of long chain, high–molecular–weight, water-soluble
polysaccharides. Hydrocolloids and other common gum are recognized as composite indigestible
polymers that contain microscopically dispersed insoluble particles mixed with water to give

11
viscous, emulsifying and thickening conditions. (Anderson and Andon, 1988; Bergenstahl et al.,
1988).

In recent times due to the hydrocolloid properties such as hydrophilic, gelation, thickening,
texturizing, stabilising, emulsifying etc., the use of hydrocolloids has broadly grown across food
industries, wound dressing, coating and adhesive agents, cosmetics, pharmaceuticals industries,
paper making etc. (Gino, 2020). In the food industry hydrocolloids are applied for improving gel
properties, food flavour, food consistency, microstructure control, food shelf life, food texture and
meat processing (Kunal and Jashbjai, 2017). In medicine specifically, dentistry, alginate and agar
hydrocolloids are good materials for making dental impressions. In pharmacy, research and
clinical laboratories hydrocolloids such as agar are used in the preparation of a solid or liquid
mixture of chemicals that can support microorganisms’ growth (T. Wüstenberg, 2014;
Medicosage, 2022). According to Byram Healthcare, hydrocolloids are used in wound dressings
such as bandages, body netting, composite dressings etc.

2.5 A general overview of alginate

Alginates are natural polysaccharides that are available in seaweeds and were first discovered by
E.C.C Stanford in 1881, during his search for useful products from kelp. He succeeded with the
process of alkali extraction of a viscous material, ‘align’, from the seaweed and he then precipitated
it using mineral acid.

They are anionic polysaccharide hydrocolloids naturally found on the cell walls of brown
(Phaeophyceae) seaweed tissues. Alginate from the cell walls of brown seaweed occurs as gels
containing calcium salt, sodium salt, potassium, magnesium, strontium, barium ions and acid
(Haug and Smidsrød 1967). Alginates exhibits poor mechanical strength, so combining them with
other polymers and composite provides the strength, flexibility, and mechanical resistance of the
tissues necessary to withstand the force of water in the marine environment where the seaweed
grows (Sudha, 2017; Leonel Pereira and João Cotas, 2020). Alginate is not found only in seaweeds
but is also produced by bacteria. Generally, alginate is commercially extracted from brown
seaweeds only (Draget et al, 2005).

The variation of alginate in the composition of brown seaweed (Phaeophyceae) is from 20 to 60%
dry matter. However, an average brown seaweed contains about 40% alginate. Alginates are

12
widely used in industries, bioengineering, biotechnology, etc. due to the suitable properties it
possesses such as biocompatibility, biodegradability, antibacterial, anticancer, probiotic, and lack
of toxicity. (Haug and Smidsrød 1967)

The applications of alginate in the pharmaceutical and food industry rely on the emulsifying,
viscous, thickening, stabilising, and gelation properties (Andriamanantoanina and Rinaudo, 2010;
Rhein-Knudsen et al, 2017); as food additives to stabilise and improve food consistency (e.g.,
jellies, mayonnaise, jams, baked food) (Peteiro, 2018), has influential potential in beverage
ingredient (e.g., beer from stabilisers, dairy products, drinks for lowering blood sugar level)
(Viswanathan and Nallamuthu, 2014), in the pharmaceutical industry (e.g., cosmetic, toothpaste)
(Mazumder et al, 2016), in textiles (e.g., textile prints additives) and medical textiles - threads as
a wound management material such as bandages, dental impression material (Hernandez et al,
2013), in agriculture alginate has the property of water retention thereby functions as a soil
conditioner (Fenoradosoa et al, 2010).

Further applications of alginate in biomedical engineering such as (wound healing, cell

encapsulation, cell scaffolding, stimulant immune agent, drug delivery, etc.) (Silva et al, 2012), in
tissue engineering alginate, acts as a tissue scaffold such as regeneration of cells in soft tissue
(Indrani and Budianto, 2013). The application of alginate in biotechnology relies on the covalent
bonding of alginate molecules and its variation with cations such as magnesium, calcium, or
sodium which permits a multitude of applications in several variations of the alginate molecule
configuration and structure. However, scientific studies on alginate structure and properties are in
trend for specialised knowledge for advanced biotechnology or a biomedical or pharmaceutical
ingredient and these investigations are prevailing to marks of scientific innovation associated with
practical knowledge which will eventually benefit traditional exploitation techniques of alginate
(Leonel and João, 2020).

2.5.1 Application Area of Alginate

The application of alginate across the food industry, biomedicine, pharmaceutical industry, textile
industry, bioengineering etc. is solely dependent on the properties alginate possesses such as non-
toxicity, biocompatibility, biodegradation, gelation, etc. Over the years, sodium alginate utility has
been productive in various food items such as milk products, drinkable yoghurts, fruit-flavoured

13
yoghurt, chocolate milk and eggnog to make a smoother mixture as a clarifying agent and as a
stabiliser. Also, due to the properties alginate possess such as emulsification, gelling, stabilisation,
texture functionality and thickening the polysaccharide hydrocolloid (alginate) is utilised on other
food product such as food packing, ice cream toppings, fruit jams, jelly, instant noodles etc.
(Leonel and João, 2020).

Alginate recognized for its substantial properties and non-toxicity is broadly used to bundle
moderate drugs quantity in alginate gels increasing the retention time of the drug to ooze in small
doses on a specific site of injury, muscle pains, and burns, occurring on the human body has a high
advantage over any ointments which could cause irritation or side effects. Alginate in wound
dressing materials such as foams, sponges, films, wafers, and nanofibers work readily to absorb
the wound fluid maintaining a physiologically moist atmosphere, to eventually protect the wound
site from bacteria. Alginates are utilised in the pharmaceutical industry because of their property
of non-toxicity within the interior part and exterior parts of the human body when consumed.
Unfortunately, some drugs and medicines produced in today’s medical field administered to
patients are causing various side effects. It is discussed to implement alginate on controlled,
sustained, and targeted drug delivery (Leonel and João, 2020).

2.5.2 Properties of Alginate

The subsequent list below converges on the relevance and uses of the properties of alginates.
Furthermore, the various species of brown seaweed vary with alginate properties. The most
commercially harvested species of brown seaweed are Sargassum, Laminaria, Turbinaria,
Ecklonia, Lessonia, Ascophyllum, Macrocystis and Durvillaea, in which Macrocystis,
Ascophyllum and Laminaria are the most important. However, the seaweed to be harvested is
based on the choice of its availability and the properties it possesses. (Dennis and McHugh, 1987)

Alginate biocompatibility simply refers to the responsive ability of alginate as a biomaterial to an

appropriate host. Alginate biocompatibility has been broadly analysed outside the cell walls and
within the cell walls of the brown seaweed to scientifically pronounce and conclude on the varying
level purity of alginate. Furthermore, alginic acid biocompatibility and strength are determined by
the quality and quantity of the acid (Giridhar, 2021). Calcium alginate was once injected into a
rat’s kidney as an experiment to study the biocompatible between the biomaterial (calcium

14
alginate) and the host (rat) with a conclusive promising result as well as an observation in
mammals’ inability to digest the biomaterial because of lack of ‘Alginase’ an enzyme responsible
for breaking down the chains of the anionic polysaccharide (alginate). (Becker et al., 2001;
Giridhar, 2021).

With the idea of alginate application to industry and biomedical sectors such as food, cosmetics,
tissue engineering, drug delivery etc. it is safe to say that sodium/calcium alginates are non-toxic
to cells primarily when used on mammals. (Dusel et al., 1986; Giridhar, 2021). Alginate viscous
property increases with the concentration of alginate and decreases with increasing temperature,
this occurrence is significant in high viscous alginates. With the increase in polyvalent metal ions
(Ca2+, Sr2+, Ba2+ Fe3+ Al3+) the flow property of alginate decreases as the viscosity increases.
(IRO, 1995).

In calcium alginate, the positive calcium ion from calcium chloride causes a rapid ion exchange
with alginate leading to an anti-reversibility gel property. The gel of varying rigidity is produced
with acid ratio control of mannuronic acid to guluronic acid such that gel formed by alginate with
high mannuronic acid possesses impoverished rigidity and effective flexibility while gel formed
by alginate with high guluronic acid mostly derived from glucose is frangible with high rigidity.
(IRO, 1995).

2.5.3 Side Effects of Alginate on Humans

1. Sodium alginates when consumed excessively cause diarrhoea, nausea and indigestion in
the gastrointestinal tract (stomach and intestine). (Jessica, 2018)
2. Alginate causes loss of appetite which eventually leads to weight loss. (Jessica, 2018)

2.5.4 Limitation of Alginate

1. Pure alginates possess weak mechanical strength. (Zhengyue et al., 2021)

2. Alginate is undegradable after implanting with salt but can be overcome by synthesising
partial oxidation. (Zhang et al., 2011).

15
2.5.5 Extraction methods for alginate

The extraction of alginate is generally composed of five stages which are: (a) acidification of the
seaweeds, (b) alkaline extraction with sodium carbonate (Na2CO3), (c) solid/liquid separation, (d)
precipitation, and (e) drying (Hernández et al., 1998). Some authors like (Fertah et al., 2017)
performed soaking of the brown seaweed in formaldehyde/formalin (for 1-12h) before the
extraction of alginates. This is done to soften the tissue of the seaweed, avoid alginate pigmentation
and bleach algal biomass (Hernandez-Carmona et al., 2013). However, innovative extraction
methods and techniques have gained recognition such as ultrasound-assisted extractions (UAE)
and microwave-assisted extraction. The parameters (e.g., temperature and time of extraction) have
a crucial impact on the hydrocolloid field of application and exhibit both mechanical and
physicochemical properties (Magdalena et al., 2019).

According to (Fenoradosoa et al., 2010), dried and milled sargassum turbinarioides from Nosy Be
in Madagascar were soaked in 2% of formaldehyde for 24 hrs at room temperature. Water and
0.2M HCl were added for 24hrs to wash the solids. Washing of solids and extraction with 2% of
Na2CO3 for 3hrs at 100°C. It was then centrifuged for the collection of a soluble fraction. Alginate
was precipitated with 95% of ethanol and washed with acetone. The extraction yield of alginate
was 10%. It was noticed that the extracted alginate was a polymer of uronic acids (no neutral
monosaccharide and sulphate were detected).

According to (Fertah et al., 2017), laminaria Digitata was taken from the Atlantic coast of Western
Morocco. This brown seaweed was dried and milled into two separate sizes (i.e.. <1mm and 1-
5mm). The dried algae were soaked in 2% of formaldehyde for one night. Washing of solids in
water and adding 0.2M of HCl for 24hrs. Washing of solids and extraction with 2% of Na2CO3 for
5 hrs at 25°C, 40°C, and 60°C. Alginate was centrifuged and precipitated with ethanol. The
alginate was purified with ethanol, methanol, and acetone. The extraction yield of alginate for the
<1mm size seaweed was 38.3% at 25°C, 51.8% at 40°C and 43.2% at 60°C; and for the 1-5mm
size seaweed was 35.3% at 25°C, 44.0% at 40°C and 40.2% at 60°C. From this experiment, it was
observed that there were higher extraction yields for the small-size algae and a maximum yield of
51.8% at 40°C, it was suggested that it can be used as a polyelectrolyte complex to produce drug
delivery micro and nanoparticles.

16
According to (Bertagnolli et al., 2014), sargassum filipendula was harvested from the Cigarras
beach in Brazil. The seaweeds were harvested in three seasons: Fall (May), Spring (November),
and Summer (February). Dried algae were soaked in 0.4% of formaldehyde for 0.5 hours and then
washed with distilled water and added to 0.1M of HCl for 24hrs. Bleached algae were washed with
distilled water and extracted with 2% of Na2CO3 for 5 hrs at 60°C. Alginate was separated from
the crude extract by ethanol precipitation. The extraction yield of alginate was 17.0±0.1% for the
fall season, 17.2±0.3% for the spring season and 15.1±0.1% for the summer season. It was
observed from this experiment that extraction yield depended on the season of seaweed harvest.

According to (Chee et al., 2011), turbinaria conoides, Sargassum baccularia, Sargassum

siliquosum, and Sargassum binderi, were all harvested from Port Dickson, Negeri Sembilan,
Malaysia. Using the cold method, air-dried brown seaweeds were soaked in 1% of CaCl2 at 27°C,
overnight for 18 h and washed with distilled water, then the seaweeds were stored in 5% HCl
solution for 1 h and washed with distilled water after which the algae was stored in 3% Na2CO3
solution for 1 h and adding water before it was left to stand overnight. Afterwards, separation of
the viscous mixture from its residue by centrifuging, then precipitation of sodium alginate with
ethanol/water mixture (1:1, v/v) was done through filtration, then washing with ethanol and drying
of the precipitate occurred. Using the Hot method, like the cold method, except for the first step:
the storing time was 3 h at 50°C in 1% CaCl2. The alginate yielded from the various species are
T. conoides: hot – 41.4%; cold – 40.5%; S. baccularia: hot method – 26.7%; cold – 23.9%; S.
siliquosum: hot – 49.9%; cold – 38.9%; S. binderi: hot – 38.9%; cold – 28.7%.

According to (Borazjani et al., 2017), sargassum angustifolium is harvested from Bushehr, Iran.
Milled brown seaweed was pre-treated to remove non-target compounds (pigments and lipids)
with 85% ethanol, overnight. At room temperature (25oC) the biomass was rinsed with acetone
and was dried at room temperature (25oC), the dried biomass was treated with distilled water at
65oC for 3 hours or HCl (0.1 M, pH 2, 65oC, 3 hours) or alcalase (5% w/w, pH 8, 50oC, 24 hours)
or cellulase (5% w/w, pH 4.5, 50oC, 24 hours). Then, extraction of alginates with 3% Na2CO3, pH
11, 65oC and 3 hours alongside precipitation with 75% ethanol occurred. Conclusively, the alginate
yielded from water treatment of brown seaweeds was 3.3%, for acid pre-treatment 3.4% and
alcalase and cellulase treatments 3.5%.

17
According to (Larsen et al., 2003), sargassum asperifolium, Cystoseira myrica Sargassum
dentifolium, Cystoseira trinode, and Sargassum latifolium were all harvested from Egypt. brown
seaweed was treated with formaldehyde before extraction with 0.2M HCl. The residue was
suspended in distilled water, neutralized (pH 7-8) and filtered. Then, extraction of the neutral
residue with 3% Na2CO3 was done to precipitate alginate with ethanol. The filtrate (neutral extract)
was also mixed with ethanol to precipitate alginate (without Na2CO3). The alginate yielded from
the various brown seaweed species is C. trinode – 3.3% C. myrica – 2.2% S. dentifolium – 3.3%
S. asperifolium – 12.1% S. latifolium – 4.3%.

2.6 A general overview of fucoidan

Fucoidan was first extracted and characterised by Kylin, a Swedish botanist in 1913, from various
species of ‘laminaria and fucus’. Fucoidan is a sulphated polysaccharide extracted from the cell
wall of brown seaweeds and serves to protect it from external stresses. It consists of fucus,
galactose, mannose, uronic acid, glucose, rhamnose, arabinose, and xylose. (Jing and Quanbin,
2017); (Luthuli, 2019).

High-purity organic fucoidan extract has been certified through clinical testing and confirmed to
be safe for human consumption. Hence, used in food and dietary supplements. (Fitton and Hellen,
2015). Fucose, one of the main constituents of fucoidan, varies in different species of brown
seaweed. The presence of fucose in various species of brown seaweeds is as follows; Sargassum
binderi - 34.5%, Cladosiphon okamuranus - 43.3% (Lim et al., 2016); Laminaria cichorioides -
72.0%, Laminaria Japonica - 54.0%, Fucus evanescens - 55.0% (Zvyagintseva et al., 2003); Fucus
distichus - 51.6% (Bilan et al., 2004); Saccharina latissimi - 30.8% (Bilan et al., 2010); Saccharina
longicruris - 12.8 - 21.5% (Rioux et al., 2009); Sargassum polycystum 18.6% (Bilan et al., 2013).
Fucoidan is contained in the cell walls of brown seaweeds such as Wakame Kombu and Mozuku
which promotes anti-ageing in humans when consumed. (Vishchuk et al., 2011).

2.6.1 Application Area of Fucoidan

Fucoidan is used as a medicinal nutritional supplement due to its anti-cancer and anti-inflammatory
characteristics. Fucoidan, when used in a dietary supplement, has benefits such as an improved

18
humane system, liver cleansing, stem cell repair – antiaging, hair growth, youthful look, decreased
fatigue, increased energy and weight loss. (Helen et al., 2016).

In the pharmaceutical industry, fucoidan is used in anticancer and anti-ageing drugs. Cancer has
been a huge burden on society and a leading cause of death in both developing and developed
countries. (Torre et al., 2015). The reserve has proven that fucoidan may reduce the toxicity of
certain anticancer drugs. (Alekseyenko et al., 2007).

2.6.2 Properties of Fucoidan

Fucoidans possess physicochemical properties such as crosslinking, solubility, non-toxicity,

rheological, biocompatibility and biodegradability aside from the physiochemical properties
fucoidans possess some strong biological properties which include immunomodulatory,
anticoagulant activity, antibacterial activity, anti-inflammatory, anti-oxidizing activity, antiviral
activity, and anti-tumour activity.

In rheological properties, fucoidan extracted from brown seaweed is rarely reported.

Biodegradability and biocompatibility facilitate fucoidan application in nutraceutical delivery
systems, it enhances the bioavailability and delivery of bio-active ingredients, however, the
polymerization and purification can improve biodegradability by increasing molecular weight.
(Alvarez-Vinas et al., 2019).

Furthermore, the anti-coagulant property in fucoidan inhibits platelet aggregation (high/low

viscosity of the blood) and therefore helps prevent blood clot which is a major factor in stroke and
heart attacks. (Perfect Nutrition, 2021). Immunomodulatory and anti-inflammatory properties in
fucoidan prevent detrimental effects on tissues and cells thereby helping in fighting infections,
restoring homeostasis, managing injuries and wound repair/healing. (Croasdell et al., 2015; Broggi
and Granucci, 2015). Thermotherapy on cancer cells has an adverse bad effect on neighbouring
normal cells but fucoidan thermotherapeutic drugs have been proven to be non-toxic and efficient
in suppressing cancer cells. (Vaikundamoorthy et al., 2018).

19
2.6.3 Extraction methods for fucoidan

Sargassum binderi was pre-treated with MeOH: CHCl3: H2O in a ratio of 4:2:1 respectively to
remove impurities. It was then sonicated in 2% CaCl2 solution and extracted at 85°C for 24hrs in
the same solution. This process was repeated six (6) times. It was precipitated with 10%
hexadecyltrimethylammonium bromide and iodide, dialysed and lyophilised. It was observed that
the extract was composed of fucose (contains fucoidan), galactose, glucuronic acid, mannose,
xylose, and glucose (Lim et al., 2016).

Adenocystis utricular seaweed was pre-treated with 80% EtOH (Ethanol) for 24hrs at 70°C. It was
then extracted with water (or 2% CaCl2; or HCl) for 7hrs at room temperature and then followed
by exhaustive extraction at 70°C. It was observed that the extract was composed of fucose
(contains fucoidan), rhamnose, glucose, galactose, xylose, mannose, uronic acid, and sulphate
(Ponce et al., 2003).

Focus evanescens was pre-treated with MeOH-CHCl3-H2O in a ratio of 4:2:1 respectively and
then extracted with 2% CaCl2at 85°C for 5hrs. It was precipitated and then the precipitate was
washed with water, stirred with 20% ethanolic solution and dissolved with water. From the
observation, the extract was composed of fucose (contains fucoidan), xylose, uronic acid, and
sulphate (Cumashi et al., 2007).

Focus serratus, Fucus Vesiculosus, and Ascophyllum nodosum was pre-treated with MeOH-
CHCl3-H2O in a ratio of 4:2:1 respectively and then extracted with2% of CaCl2 at 85°C for 5hrs.
The extracts were collected by centrifugation, combined, dialyzed and lyophilized. It was observed
that the extract was made up of fucose (contains fucoidan), xylose, mannose, glucose, galactose,
uronic acid and sulphate (Cumashi et al., 2007). The yield of fucose varies for different species of
brown seaweed with respect to the seasons. Fucus Serratus species in August is 46.6%, Fucus
Vesiculosus species in September is 26.1% and Ascophyllum nodosum in September is 33.0%
(Bilan et al., 2006; Medcalf et al., 1977)

20
2.7 A general overview of ethanol
The harmful effects of burning fossil fuels and their adverse effects on global warming have led
to the increasing demand for environmentally friendly and renewable sources of fuel like large
sustainable biofuels. Bioethanol has been regarded as a major substitute to replace liquid fossil
fuels. Bioethanol is mainly produced from sugarcane in commercial production, Bamboo, and
agricultural feedstocks (Saxena et al., 2009; Alvira et al., 2010).

Extraction of ethanol from seaweeds is a third-generation bioethanol production (Ge et al., 2011).
Seaweed has high productivity per unit area per year, and there is no competition with food crops
since it doesn’t require nutrients to cultivate and can grow on rocky seashores and sterile land,
unlike agricultural feedstock. It is also cheap since it doesn’t require expensive pre-treatment
compared to lignocellulosic biomass (Alvira et al., 2010). Brown seaweed can be grown rapidly
compared to land biomass.

The carbohydrate content of seaweed is in a range of 30-70% but depends on the species and
culture conditions. Due to the advanced mass production and cultivation method in Korea, brown
seaweed can be a good substrate for bioethanol production. The carbohydrates in brown seaweed
include fucoidan, alginate, laminarin, and mannitol (Dumitriu, 1998). A high concentration of salt
in the seaweed could act as an inhibitor in the production of ethanol as a result of low fermentation
yield. Bioethanol yield varies among seaweed species, as well as fermentation methods.

2.7.1 Production of ethanol from brown seaweed

Ethanol production from brown seaweed requires the conversion of the polysaccharides into
monosaccharides by hydrolysis (saccharification), fermentation of monosaccharides into ethanol,
and purification and concentration of ethanol (Figure. 4).

Bioethanol is made by microbial fermentation from the carbohydrates (alginate, fucoidan,

laminarin, and mannitol) present in brown seaweeds (Singh et al, 2014). The most abundant sugars
in brown algae are alginate, mannitol, and glucan (present as laminarin and trace cellulose). The
full potential of bioethanol production from brown seaweed has not yet been accomplished since
industrial sugar fermenting microbes are not available to metabolise alginate even though sugars
from mannitol and laminarin can be easily extracted from milled brown algae for bioconversion

21
(Pham et al., 2013; Horn et al., 2000). Despite these, the yield from sugar crops and lignocellulosic
biomass is 0.212 - 0.341kg ethanol/dry feedstock and that of mannitol and glucan is 0.152 -
0.196kg ethanol/dry brown algae. The catabolism of mannitol generates excess reducing
equivalents, causing an unbalanced redox environment under fermentative conditions compared
with glucose. There is a substantial loss in ethanol yield from brown seaweed because
ethanologenic microorganisms do not utilise alginate as a substrate and are unable to break down
alginate during fermentation (Horn et al., 2000). To effectively extract ethanol from brown algae,
co-fermentation of alginate with other fermentable sugars in an existing industrial microbe should
be presented. (Enquist-Newman et al., 2014). Hydrolysis of the pre-processed alginate is done to
release sugars which can be converted to ethanol through microbial fermentation.

2.7.2 Production methods of ethanol

Physicochemical and biological methods (acid, hydrothermal hydrolysis, and enzymatic) are
mainly used in converting polysaccharides into monosaccharides. The common method for the
pre-treatment of biomass is acid hydrolysis which is cheaper compared with enzyme cost. (Kishore
et al., 2020).

2.7.3 Acid hydrolysis process of bioethanol production

The pre-treatment is carried out by acid hydrolysis using either HCL, HNO3, or H2S04. The
treatment is done with the constituents: biomass concentration of 3-10% and acid concentration of
0-10%. The experiment was performed in 500-ml reaction bottles with 200ml of working volume.
The acid hydrolysis was carried out by keeping the flask at 100 degrees Celsius for 60 min keeping
the initial temperature constant. The residual seaweed biomass was removed after hydrolysis and
the hydrolysate was filtered through filter paper to remove the particles and the hydrolysate was
concentrated for further utilisation (Kumar et al., 2009).

Consequently, 20g/L of sugars derived from the dilute acid treatments is subjected to an anaerobic
fermentation process using commercially available yeast S.cerevisiae as an inoculum. The residual
sugars in the sample were determined after the fermentation process. After careful analysis, it was

22
concluded that H2SO4 hydrolysate yielded the highest amount of ethanol 9.31 ± 0.21g/L followed
by HCL hydrolysate 8.27 ± 0.19g/L and HNO3 hydrolysate 5.25 ± 0.13g/L.

An overall 95.16% of reducing sugars in the H2SO4 hydrolysate was converted to ethanol. The
least 51.08% conversion of sugars to ethanol was noticed in flasks containing HNO3 hydrolysate
(Kishore et al., 2020).

2.7.4 Enzymatic hydrolysis process of bioethanol production

Bioethanol production from different species and its yield using enzymatic hydrolysis; Padina
tetrastronica species through saccharification and fermentation of biodiesel-extracted residue by
saccharomyces cerevisiae yields 161mg/g of residue biomass (Ashokkumar et al., 2017). Ulva
rigida species through sugar extraction and fermentation using saccharomyces cerevisiae yields
65.3mg/g seaweed biomass (Korzen et al., 2015). Sargassum angustifolium species through
saccharification and fermentation of alginate-extracted residue using saccharomyces cerevisiae
yielded 44.5mg/g seaweed biomass (Ardalan et al., 2018). Kappaphycus alverazii species through
saccharification and fermentation using saccharomyces cerevisiae yields 206mg/g seaweed
biomass and glacilaria manilaensis through saccharification and fermentation using
saccharomyces cerevisiae yields 231mg/g seaweed biomass (Hssami et al., 2017).

Figure 4: Bioethanol production process from brown algae (Ok and Eun., 2016)

23
 CHAPTER THREE. METHODOLOGY

3.1 Harvesting and preparation of seaweed

Brown algae (Sargassum) was harvested from the shores of Regional Maritime University,
Nungua, Ghana at low tides on the 29th of October 2022 from 01:21 pm to 03:12 pm and washed
with fresh water to remove the salt content and debris collected during harvesting (Figure. 5). It
was sundried, chopped into smaller particles with scissors, debris sorted out, after which it was
milled using an industrial high-speed grinder (Figure. 6) with specifications (2500 amps, electric
voltage – 220 V, Motor Speed 32000 rev/min, working time 8 minutes, Work rate 4200 watt,
Crushing Degree ranging from 600 – 350 0C, time interval between 8 minutes), sieved using a 45-
micrometre mesh sieve and stored in a zip bag, then transferred to the lab and stored at room
temperature for further experiment (Kusumawati et al. 2018). Moisture Analysis was carried out
using the measuring balance and moisture analyser. 5g of seaweed was subjected to the analyser
for three separate experimental runs for which an average of 15.46% moisture was recorded at a
drying temperature of 105 degree Celsius for a drying time of 00:12:17.

Figure 5: Harvesting and Washing of Seaweed

24
Figure 6: Sun Dried and Milled Seaweed

3.2 Preparation of standard solutions

37% HCL solution was prepared by pipetting 4.1 ml of the stock solution into a 500 ml volumetric
flask which was partially filled with distilled water i.e., the solution was continuously swelled
while adding more distilled water until the solution reached the 500 ml mark on the volumetric
flask, the solution was gently transferred into an enclosed clean container and labelled
appropriately.

2.5% of Na2CO3 solution was prepared by measuring 12.5 g of Na2CO3 pellet and adding it to
distilled water in a 500 ml volumetric flask i.e., the solution was continuously swelled while adding
the distilled water to ensure uniform mixture. More distilled water was added until the solution
reached the 500 ml mark on the flask, the solution was gently transferred into an enclosed clean
container and labelled appropriately.

200 ml 2 M NaOH solution was prepared by measuring 16 g of NaOH pellet and adding it to
distilled water in a 200 ml volumetric flask i.e., the solution was continuously swelled while adding
the distilled water to ensure uniform mixture. More distilled water was added until the solution
reached the 200 ml mark on the flask, the solution was gently transferred into an enclosed clean
container and labelled appropriately.

25
500 ml 0.2 M H2SO4 solution from a stock solution of 97% purity was prepared by pipetting 5.49
ml of the stock solution into a 500 ml volumetric flask which was partially filled with distilled
with i.e., the solution was continuously swelled while adding more distilled water until the solution
reached the 500 ml mark on the volumetric flask, the solution was gently transferred into an
enclosed clean container and labelled appropriately.

70% Ethanol was obtained by diluting 707 ml of 99% Ethanol with 293 ml of distilled water.

3.3 Sequential extraction of fucoidan and alginate

3.3.1 Fucoidan extraction

150 ml of HCL was added to 8.66 g of the milled Sargassum and incubated at 25 0C, 35 0C and
45 °C for 50, 70 and 90 min respectively for each experimental run. The solution was filtered to
recover the filtrate and residue biomass. 70% ethanol was added to the filtrate (2:1 v/v) and left
overnight to precipitate. It was then centrifuged at 5000 rpm for 10 min and dried at 45°C for
48hrs to recover fucoidan (Figure. 8b) (Ale et al, 2012).

3.3.2 Alginate extraction

150 ml of Na2CO3 was added to residue biomass (obtained from the fucoidan extraction process)
and incubated at 70 °C for 2hrs, it was then filtered. The filtrate was mixed with 70% ethanol (1:2
v/v) and left for 2hrs for the alginate to precipitate. Alginate was filtered (Figure. 7) and dried at
45°C for 72hrs (Figure. 8a). The residue from the filtrate was labelled residue O (Sugiono et al,
2020).

26
Figure 7: Freshly extracted alginate.

(a) (b)

Figure 8: Dried alginate (a) and dried fucoidan (b) extracted from sargassum.

3.4 Production of Ethanol

The residue biomass (obtained from alginate extraction) was washed twice with water, 100 ml of
0.2 M H2SO4 added to the residue and the solution subjected to acid-hydrolysis at the temperature
of 121 °C in an autoclave for 15 min (Widyaningrum, 2016). The solution was filtered to recover
the hydrolysate and then neutralised by reacting it with 20 ml 2M NaOH. The sugar content

27
(bricks) was measured using a refractometer (Figure. 9). The potential ethanol yield was calculated
using stoichiometric ratio.

Figure 9: Neutralised hydrolysate and bricks analysed using refractometer.

3.5 Chemical Equations and Calculations

Neutralisation Reaction between NaOH and H2SO4

𝐻2 𝑆𝑂4 + 2 𝑁𝑎𝑂𝐻 → 𝐻2 𝑂 + 𝑁𝑎2 𝑆𝑂4

Volume of H2SO4 solution in the neutralisation reaction is 100 ml = 0.1 dm3

n = CV

𝑛 = 0.2 ∗ 0.1 = 0.02 𝑚𝑜𝑙

Mole ratio of H2SO4 to NaOH is 1:2.

Mole of NaOH required = 0.02 ∗ 2

= 0.04 mol

Volume of NaOH required = 𝑛(𝑁𝑎𝑂𝐻)/𝐶(𝑁𝑎𝑂𝐻)

= 0.04/2

28
 =0.02 dm3

=20 ml

∴Using mole ratios, the volume of 2 M NaOH required to neutralise 100 ml 0.2 M H2SO4 solution
is 20 ml.

Potential Ethanol Yield

Moisture content = 15.46%

Mass of wet seaweed = 8.66 g

Total Solid = 100 - 15.46% = 84.54%

Brix (Neutralised solution) - Brix (Hydrolysate) = 2.4 - 2.1 = 0.3

Dry Seaweed = 84.54/100 * 8.66 g = 7.32 g

7.32 g + 100 ml = 107.32 ml

0.3 g = 100 ml

x = 107.32 ml

x = 0.322 g

glucose conversion
𝑓𝑒𝑟𝑚𝑒𝑛𝑡𝑎𝑡𝑖𝑜𝑛
: 𝐶6 𝐻12 𝑂6 → 2𝐶𝐻3 𝐶𝐻2 𝑂𝐻 + 2𝐶𝑂2

The maximum (theoretical) ethanol yield under the above reaction can be deduced from its
stoichiometry to be 0.51g/g glucose.

Therefore, the potential ethanol yield = 0.322 * 0.51 = 0.164 = 16.42%

3.6 Experimental Design

Nine experiments were conducted under three different conditions of time (90 min,70 min and 50
min) and temperature (45 0C, 35 0C and 25 0C). Each experimental run was triplicated. The data
was analysed using Minitab software. The experimental design used was factorial design.

29
3.7 Yield

Fucoidan and alginate yield was calculated by dividing the mass of fucoidan or alginate recovered
by the total mass of the wet sample subjected to the experiment multiplied by 100% (Torres et
al.,2007).

30
 CHAPTER FOUR. RESULTS AND DISCUSSIONS

4.1 Yield of Fucoidan

In this study, the yield of the fucoidan of Sargassum was (0.8083140878 - 2.113163972%). The
fucoidan yield increases drastically with increasing time (50 - 90 minutes) and temperature (25 -
45 °C). The highest fucoidan yield extract of Sargassum (2.113%) was obtained at 70 °C for 35
minutes. In this study the yield obtained was higher than the yields for Sargassum reported by
Sugiono et al., (2020). It was discovered that an increase in time and temperature can enhance the
extractability of fucoidan. However, after reaching an optimal level, the extractability gradually
decreases (Figure. 11) also reported by Rodriguez et al., (2011). As a result of the multiple
regression data analyses carried out on Minitab software the results indicated that fucoidan yield
is more dependent on time i.e., time contributes more to fucoidan yield. The optimal setting
designed by Minitab to maximise the extraction of fucoidan was achieved at time 66.1616 min
with a predicted yield of 1.86410% (Figure. 11).

Figure 10: Surface plot of fucoidan yield against temperature and time from Minitab.

31
 Model Equation

𝐹𝑢𝑐𝑜𝑖𝑑𝑎𝑛 𝑌𝑖𝑒𝑙𝑑
= −2.65 + 0.1496𝑥1 − 0.044𝑥2 − 0.001147𝑥12 + 0.00076 𝑥22
+ 0.000072 𝑥1 ∗ 𝑥2

Equation 1: Model equation of fucoidan yield

Figure 11: Regression analysis for fucoidan yield.

4.2 Yield of Alginate

In this study, the yield of the alginate of Sargassum was (8.814472671 - 45%). The increase in the
extractability of alginate was obtained by increase in temperature and time as well as an exposure
to HCL acid also reported by Fertah et al., (2014). The highest alginate yield extract of Sargassum
32
(45%) was obtained at 45 °C for 90 minutes. As a result of the multiple regression data analyses
carried out on Minitab software the results indicated that alginate yield is more dependent on
temperature i.e., temperature contributes more to alginate yield. The optimal setting designed by
Minitab to maximise the extraction of alginate was achieved at temperature 45 °C with a predicted
yield of 43.0844% (Figure. 13).

Figure 12: Surface plot of alginate yield against temperature and time

Model Equation

Alginate Yield = 286.2 − 1.83x1 − 13.57x2 + 0.0130𝑥12 + 0.2087𝑥22 + 0.0020 𝑥1 ∗ 𝑥2

Equation 2: Model equation of alginate yield

33
Figure 13: Regression analysis for alginate yield.

4.3 Yield of Ethanol

In this study, the potential yield of ethanol produced from Sargassum is 16.42% at a temperature
of 121 °C for 15 min. Current data on the production of ethanol indicates that the yield is not much
as compared to the yield from other crops (maize, bamboo, and sugar cane) (Craggs et al., 2012).

Corn ethanol production is more complicated than sucrose-based feedstocks, but it produces about
5 times more ethanol per ton of feedstock (Manochio et al., 2017).

34
 CHAPTER FIVE. CONCLUSIONS AND RECOMMENDATIONS

Conclusions

Sargassum has overwhelmed the coastlines and poses a risk to the well-being of local populations,
fishing industries, tourism, and marine life. This study specifically sought to sequentially extract
fucoidan, alginate and produce ethanol from sargassum biomass. The study established that a
predicted yield (1.8456%) of fucoidan could be obtained at 66.166 min, a predicted yield of
alginate (43.0844%) at 45 °C and a potential ethanol yield (16.420%) from the residue. From these
findings it can be concluded that sargassum could be effectively utilised for the sequential
extraction of hydrocolloids (fucoidan and alginate) and production of fuel. This approach would
leave the western coastlines clean thereby allowing free and flexible fishing activities as well as
recreational activities.

Recommendations

Based on the results obtained from this study, it is recommended that:

1. Further studies should be conducted on the residual biomass obtained from ethanol production

2. The extraction process should be facilitated by using a modernized centrifuge machine to

increase the yield of fucoidan and alginate.

3. The refractive index detector of the HPLC unit should be repaired to validate the sargassum
extracts (alginate and fucoidan) and ethanol recovered.

35
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