Olatunji, 2020 Biopolymers

Springer Series on Polymer and Composite Materials
Ololade Olatunji
Aquatic
Biopolymers
Understanding their Industrial
Significance and Environmental
Implications
Springer Series on Polymer
and Composite Materials
Series Editor
Susheel Kalia, Army Cadet College Wing, Indian Military Academy Army Cadet
College Wing, Dehradun, India
The “Springer Series on Polymer and Composite Materials” publishes monographs
and edited works in the areas of Polymer Science and Composite Materials. These
compound classes form the basis for the development of many new materials for
various applications. The series covers biomaterials, nanomaterials, polymeric
nanofibers and electrospun materials, polymer hybrids, conducting polymers,
composite materials from macro- to nano-scale, and many more; from fundamen-
tals, over the synthesis and development of the new materials, to their applications.
The authored or edited books in this series address researchers and professionals,
academic and industrial chemists involved in the areas of Polymer Science and the
development of new Materials. They cover aspects such as the chemistry, physics,
characterization, and material science of Polymers, and Polymer and Composite
Materials. The books in this series can serve a growing demand for concise and
comprehensive treatments of specific topics in this rapidly growing field.
More information about this series at http://www.springer.com/series/13173

Ololade Olatunji
Aquatic Biopolymers
Understanding their Industrial Significance
and Environmental Implications
123
Ololade Olatunji
Chemical Engineering Department
University of Lagos
Lagos, Nigeria
ISSN 2364-1878 ISSN 2364-1886 (electronic)

Springer Series on Polymer and Composite Materials
ISBN 978-3-030-34708-6 ISBN 978-3-030-34709-3 (eBook)
https://doi.org/10.1007/978-3-030-34709-3
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Preface
The field of polymer science is a very broad one, due to the fact that polymers are
ubiquitous. Polymers are used in every industry, from food industries where they
are used as food additives or make up the bulk of the food itself, the biomedical
industry where polymers are used as scaffolds for tissue regeneration to the energy
industries where they are used as the starting feedstock for bioethanol production or
as substrates in the new generation of solar cells. For this reason, there have been
numerous texts which present polymers in various forms.
A review of the literature shows that there are currently no books which focus on
polymers from the aquatic environment as a whole. While there are texts which look
at different polymers which can be sourced from the aquatic environment, none
of these texts have considered the polymers with a focus on the aquatic source. This
is of much important in present time as the aquatic environment continues to be
faced with different challenges from pollution to increased acidification. It is
important that while considering the production, application and processing of these
polymers that the environment from which they are obtained and the impact of the
process of extraction are also taken into consideration. This ensures that the future
polymer engineers, producers, suppliers and consumers have a broader under-
standing of polymers which includes the global economy and environment.
Lagos, Nigeria Ololade Olatunji
v
Contents
1 Introduction to Aquatic Biopolymers . . . . . . . . . . . . . . . . . . . . . . . 1

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Overview of the Aquatic Ecosystem . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Types of Aquatic Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.1 Estuaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2.2 Springs and Aquifers . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2.3 Rivers and Streams . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.2.4 Lakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.2.5 Ponds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2.6 Lagoons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.7 Wetlands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2.8 The Coral Reefs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3 Aquatic Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3.1 Aquatic Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.3.2 Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.3.3 Aquatic Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.4 Carnivorous Aquatic Plants . . . . . . . . . . . . . . . . . . . . 23
2.3.5 Aquatic Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3.6 Aquatic Birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3.7 Aquatic Microorganisms . . . . . . . . . . . . . . . . . . . . . . 24
2.3.8 Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.4 Abiotic Components of the Aquatic Ecosystem . . . . . . . . . . . . 28
2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2 Occurrence in Nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
vii
viii Contents
3.3 Chemistry of Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.3.1 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3.2 Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.3.3 Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.3.4 Variation with Source . . . . . . . . . . . . . . . . . . . . . . . . 35
3.3.5 Deacetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3.6 Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3.7 Depolymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.4 Availability of Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . 37
3.5 Extraction of Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.5.1 Extraction from Crustacean Exoskeleton . . . . . . . . . . 40
3.5.2 Extraction from Mushrooms . . . . . . . . . . . . . . . . . . . 41
3.5.3 Extraction from Fish Scales . . . . . . . . . . . . . . . . . . . . 42
3.5.4 Extraction of Chitin from Insects . . . . . . . . . . . . . . . . 43
3.5.5 Microbial Extraction . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.5.6 Enzyme Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6 Environmental Implications . . . . . . . . . . . . . . . . . . . . . . . . . . 45
3.6.1 Resource Utilization . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.6.2 Water Consumption . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.6.3 Solid Waste/By-Products . . . . . . . . . . . . . . . . . . . . . . 47
3.6.4 Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . 47
3.6.5 Acid Usage and Disposal . . . . . . . . . . . . . . . . . . . . . 48
3.6.6 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.6.7 Energy and Electricity . . . . . . . . . . . . . . . . . . . . . . . 49
3.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.7.1 Packaging Films . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.7.2 Water Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.7.3 Antimicrobial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.7.4 Biomedical Application . . . . . . . . . . . . . . . . . . . . . . . 54
3.7.5 Gene Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.7.6 Anticancer Application . . . . . . . . . . . . . . . . . . . . . . . 55
3.7.7 Anti-inflammatory . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.7.8 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.7.9 Antimalaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.7.10 Papermaking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.7.11 Electronics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.7.12 Cosmetics and Toiletries . . . . . . . . . . . . . . . . . . . . . . 59
3.7.13 Agrochemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.7.14 Biodegradation of Chitin and Its Derivatives . . . . . . . 59
3.8 Commercial Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
3.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Contents ix
4 Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.3 Chemistry of Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3.1 Polymer Chain Structure . . . . . . . . . . . . . . . . . . . . . . 69
4.3.2 Rheological Properties . . . . . . . . . . . . . . . . . . . . . . . 71
4.3.3 Characterization of Alginate . . . . . . . . . . . . . . . . . . . 71
4.3.4 Decomposition of Alginate . . . . . . . . . . . . . . . . . . . . 72
4.4 Availability of Raw Material . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.5 Extraction of Alginates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.5.1 Extraction of Sodium Alginate . . . . . . . . . . . . . . . . . 74
4.5.2 Method 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.5.3 Method 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.5.4 Method 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.5.5 Extraction of Calcium Alginate and Other Salts of
Alginic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
4.5.6 Enzyme Extraction of Alginate . . . . . . . . . . . . . . . . . 77
4.6.1 Brown Algae Cultivation and the Environment . . . . . 78
4.6.2 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.6.3 Land Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.6.4 Energy Consumption . . . . . . . . . . . . . . . . . . . . . . . . 81
4.6.5 Water Consumption and Wastewater Generation . . . . 82
4.6.6 Carbon Dioxide Emission . . . . . . . . . . . . . . . . . . . . . 82
4.6.7 Solid Waste Generated . . . . . . . . . . . . . . . . . . . . . . . 82
4.6.8 Diversion of Resource for Alginate Production . . . . . . 83
4.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.7.1 Alginates in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
4.7.2 Textiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.7.3 Lowering Blood Sugar Level . . . . . . . . . . . . . . . . . . 85
4.7.4 Biomedical Application . . . . . . . . . . . . . . . . . . . . . . . 86
4.7.5 Alginate Oligomers and Monomers . . . . . . . . . . . . . . 87
4.7.6 Other Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5 Fucoidan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.3 Chemistry of Fucoidans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5.3.1 Degradation of Fucoidan . . . . . . . . . . . . . . . . . . . . . . 99
x Contents

5.5 Extraction of Fucoidans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.5.1 Acid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.5.3 Microwave Extraction . . . . . . . . . . . . . . . . . . . . . . . . 103
5.5.4 Subcritical Water Extraction . . . . . . . . . . . . . . . . . . . 104
5.6.1 Resource Utilization of Brown Algae
and Sea Cucumber . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.6.2 Use of Mineral Acids . . . . . . . . . . . . . . . . . . . . . . . . 106
5.7 Applications of Fucoidan . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.7.1 Biomaterials in Biomedicine and
Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.7.2 Anticancer Therapies . . . . . . . . . . . . . . . . . . . . . . . . 108
5.7.3 Drug Delivery Agent . . . . . . . . . . . . . . . . . . . . . . . . 109
5.7.4 Immune Modulation . . . . . . . . . . . . . . . . . . . . . . . . . 110
5.7.5 Antipathogenic Agent . . . . . . . . . . . . . . . . . . . . . . . . 111
5.7.6 Antiinflammatory . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
5.7.7 Renal and Hepatic Disease Treatment . . . . . . . . . . . . 112
5.7.8 Blood Anticoagulant . . . . . . . . . . . . . . . . . . . . . . . . . 113
5.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
6 Carrageenans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
6.3 Chemistry of Carrageenan . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
6.5 Extraction of Carrageenan . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.5.1 Chemical Extraction . . . . . . . . . . . . . . . . . . . . . . . . . 129
6.5.3 Semi-refined Carrageenan . . . . . . . . . . . . . . . . . . . . . 131
6.6.1 Cultivation and Harvest of Red Algae . . . . . . . . . . . . 133
6.6.3 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.6.4 Water Consumptions . . . . . . . . . . . . . . . . . . . . . . . . . 134
6.6.5 Use of Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.7.1 Milk Stabilizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
6.7.2 Gelling Agent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
Contents xi
6.7.3 Meat and Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

6.7.4 Toothpastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
6.7.5 Pet Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
6.7.6 Air Freshener Gels . . . . . . . . . . . . . . . . . . . . . . . . . . 138
6.7.7 Immobilized Cells and Biocatalysts . . . . . . . . . . . . . . 138
6.7.8 Antimicrobial Properties . . . . . . . . . . . . . . . . . . . . . . 138
6.7.9 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.7.10 Neuroprotective . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
6.7.11 Immunomodulatory Activity . . . . . . . . . . . . . . . . . . . 139
6.7.12 Antiviral Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
6.7.13 Polymeric Electrolytes . . . . . . . . . . . . . . . . . . . . . . . 141
6.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
7.3 Chemistry of Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
7.3.1 Polymeric Structure . . . . . . . . . . . . . . . . . . . . . . . . . 147
7.3.2 Gel Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
7.3.3 Viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
7.3.4 Interaction with Sugars . . . . . . . . . . . . . . . . . . . . . . . 149
7.3.5 Thermal Degradation of Agar . . . . . . . . . . . . . . . . . . 149
7.3.6 Biological Degradation of Agar . . . . . . . . . . . . . . . . . 150
7.5 Extraction of Agar from Red Seaweed . . . . . . . . . . . . . . . . . . 152
7.5.1 Alkali Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.5.2 Acid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
7.5.3 Hot Water Extraction . . . . . . . . . . . . . . . . . . . . . . . . 154
7.5.5 Ultrasound-Assisted Extraction . . . . . . . . . . . . . . . . . 155
7.6.1 Cultivation of Red Algae . . . . . . . . . . . . . . . . . . . . . 156
7.6.2 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
7.6.4 Use of Acid and Alkali . . . . . . . . . . . . . . . . . . . . . . . 158
7.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
7.7.1 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
7.7.2 Tissue Mimicry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
7.7.3 Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
7.7.4 Preservation of Post-Hatchery Chicks . . . . . . . . . . . . 161
7.7.5 Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
xii Contents
7.7.6 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

7.7.7 Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7.7.8 Preservation of Stone Structures . . . . . . . . . . . . . . . . 164
7.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
8 Ulvans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
8.3 Chemistry of Ulvans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
8.3.1 Biodegradation of Ulvans . . . . . . . . . . . . . . . . . . . . . 173
8.5 Extraction of Ulvan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
8.6.1 Cultivation and Harvest of Green Algae . . . . . . . . . . . 178
8.6.3 Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.6.4 Acetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
8.7.1 Immune Modulation . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.7.2 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.7.3 Anticancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
8.7.4 Anticoagulant Activity . . . . . . . . . . . . . . . . . . . . . . . 183
8.7.5 Antihyperlipidemic . . . . . . . . . . . . . . . . . . . . . . . . . . 184
8.7.6 Antiviral Property . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
8.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
9 Laminarins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
9.3 Chemistry of Laminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
9.3.1 Repeating Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
9.3.2 Molecular Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
9.3.3 Solubility Branching . . . . . . . . . . . . . . . . . . . . . . . . . 193
9.3.4 Methacrylated Laminarin . . . . . . . . . . . . . . . . . . . . . 193
9.3.5 Biodegradation of Laminarin . . . . . . . . . . . . . . . . . . . 195
9.3.6 Chrysolaminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Contents xiii
9.5 Extraction of Laminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

9.5.1 Acid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
9.5.2 Calcium Chloride Extraction . . . . . . . . . . . . . . . . . . . 198
9.5.4 Combined Extraction of Laminarin and Other Brown
Algae Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
9.6.1 Cultivation of Brown Algae . . . . . . . . . . . . . . . . . . . 200
9.6.4 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
9.6.5 Acid Consumption . . . . . . . . . . . . . . . . . . . . . . . . . . 201
9.6.6 Solid Waste Generation . . . . . . . . . . . . . . . . . . . . . . 201
9.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
9.7.1 Bioethanol Production . . . . . . . . . . . . . . . . . . . . . . . . 202
9.7.2 Gut Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
9.7.3 Anticancer Activity . . . . . . . . . . . . . . . . . . . . . . . . . . 205
9.7.4 Immunomodulatory Effect . . . . . . . . . . . . . . . . . . . . . 205
9.7.5 Plant Growth Agent . . . . . . . . . . . . . . . . . . . . . . . . . 206
9.7.6 Food Processing and Preservation . . . . . . . . . . . . . . . 207
9.7.7 Antimicrobial Activity . . . . . . . . . . . . . . . . . . . . . . . 207
9.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
10 Aquatic Plants and Algae Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 211
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
10.3 Chemistry of Aquatic Proteins . . . . . . . . . . . . . . . . . . . . . . . . 213
10.5 Extraction of Proteins from Algae and Aquatic Plants . . . . . . . 216
10.5.1 Physical Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 216
10.5.2 Chemical Extraction . . . . . . . . . . . . . . . . . . . . . . . . . 217
10.5.4 Ultrafiltration and Diafiltration . . . . . . . . . . . . . . . . . . 218
10.5.5 Assisted Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . 218
10.6.1 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
10.6.2 Solid Waste Generation . . . . . . . . . . . . . . . . . . . . . . 221
10.6.4 Acids and Alkali . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
xiv Contents
10.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

10.7.1 HIV Microbicides . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
10.7.2 Phycobiliproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
10.7.3 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
10.7.4 Animal Feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
10.7.5 Antihypertensive . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
10.7.6 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
10.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
11 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
11.2.1 Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
11.2.2 Symbiotic Microorganisms . . . . . . . . . . . . . . . . . . . . 236
11.2.3 Fish Viscera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
11.2.4 Carnivorous Plants . . . . . . . . . . . . . . . . . . . . . . . . . . 238
11.2.5 Extremophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
11.3 Chemistry of Some Aquatic Enzymes . . . . . . . . . . . . . . . . . . 239
11.3.1 Enzyme Stability in the Deep Sea . . . . . . . . . . . . . . . 241
11.5 Extraction of Enzymes from Aquatic Organisms . . . . . . . . . . . 243
11.5.1 Enzyme Extraction from Fish Viscera . . . . . . . . . . . . 243
11.5.2 Extraction of Enzyme from Algae . . . . . . . . . . . . . . . 244
11.5.3 Enzyme from Marine Microorganisms . . . . . . . . . . . . 245
11.6 Immobilization of Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 248
11.6.1 Chemical Immobilization . . . . . . . . . . . . . . . . . . . . . 248
11.6.2 Physical Immobilization . . . . . . . . . . . . . . . . . . . . . . 249
11.7.1 Cultivation of Algae . . . . . . . . . . . . . . . . . . . . . . . . . 250
11.7.2 Replacement of Alkali or Acid Catalyst
in Biodiesel Production . . . . . . . . . . . . . . . . . . . . . . . 250
11.7.4 Use of Salts and Buffers . . . . . . . . . . . . . . . . . . . . . . 251
11.8 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
11.8.1 Biological Cleaning Agents . . . . . . . . . . . . . . . . . . . . 252
11.8.2 Biodiesel Production . . . . . . . . . . . . . . . . . . . . . . . . . 253
11.8.3 Antifungal Agents and Pesticides . . . . . . . . . . . . . . . 253
11.8.4 Bioactive Oligomers . . . . . . . . . . . . . . . . . . . . . . . . . 253
Contents xv
11.8.5 Fish Processing: Skin and Scale Removal . . . . . . . . . 254

11.8.6 Fish Sauce and Fish Protein Hydrolysate
Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
11.8.7 Caviar Production . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
11.8.8 Biomarkers for Environmental Pollution
Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
11.8.9 Pharmaceutical Application . . . . . . . . . . . . . . . . . . . . 256
11.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
12 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
12.3 Chemistry of Collagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
12.5 Extraction of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
12.5.1 Extraction of Collagen from Combined
Fish Waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
12.5.2 Hydrothermal-Based Extract of Hydrolyzed
Collagen from Fish Scales . . . . . . . . . . . . . . . . . . . . 269
12.5.3 Extraction of Collagen from Jellyfish . . . . . . . . . . . . . 270
12.6 Environmental Implication . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
12.6.1 Jellyfish Blooms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
12.6.2 Fisheries Waste Management . . . . . . . . . . . . . . . . . . 271
12.6.5 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
12.6.6 Salts, Acids and Alkali . . . . . . . . . . . . . . . . . . . . . . . 273
12.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
12.7.1 Skin Mimicking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
12.7.2 Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
12.7.3 Bone Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . 276
12.7.4 Biomedical Implants . . . . . . . . . . . . . . . . . . . . . . . . . 277
12.7.5 Microneedles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
12.7.6 Cosmetics and Skin Care . . . . . . . . . . . . . . . . . . . . . 279
12.7.7 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
12.7.8 Antioxidant Activity . . . . . . . . . . . . . . . . . . . . . . . . . 280
12.7.9 Anti-inflammatory Activity . . . . . . . . . . . . . . . . . . . . 281
12.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
xvi Contents
13 Starch ......................... . .. . . . . . .. . . . . . . . . . . . . 287

13.1 Introduction . . . . . . . . . . . . . . .
. .. . . . . . .. . . . . . . . . . . . . 287
13.2 Occurrence in Nature . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . 288
13.3 Availability of Raw Material . . . . .. . . . . . .. . . . . . . . . . . . . 290
13.4 Chemistry of Aquatic Starch . . . . .. . . . . . .. . . . . . . . . . . . . 292
13.4.1 Biodegradation of Starch . .. . . . . . .. . . . . . . . . . . . . 295
13.5 Floridean Starch . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . 296
13.6 Extraction Process . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . 297
13.6.1 Extraction of Starch from Microalgae . . . . . . . . . . . . 297
13.6.2 Extraction of Starch from Duckweed . . . . . . . . . . . . . 299
13.6.3 Extraction of Starch from Macroalgae . . . . . . . . . . . . 299
13.6.4 Extraction of Floridean Starch from Red Algae . . . . . 300
13.7 Environmental Impact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
13.7.1 Waste Utilization . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
13.7.2 Cultivation of Aquatic Plants and Algae
for Starch Production . . . . . . . . . . . . . . . . . . . . . . . . 301
13.7.4 CO2 Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
13.7.5 Water Remediation . . . . . . . . . . . . . . . . . . . . . . . . . . 302
13.8 Applications of Aquatic-Sourced Starch . . . . . . . . . . . . . . . . . 303
13.8.1 Third-Generation Biofuel Production . . . . . . . . . . . . . 303
13.8.2 Bioplastic Production . . . . . . . . . . . . . . . . . . . . . . . . 303
13.8.3 Waxy Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
13.8.4 Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
13.8.5 Textile and Paper Industry . . . . . . . . . . . . . . . . . . . . 305
13.8.6 Pharmaceutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
13.9 Commercial Production and Applications . . . . . . . . . . . . . . . . 306
13.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
14 Cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
14.3 Chemistry of Aquatic Cellulose . . . . . . . . . . . . . . . . . . . . . . . 314
14.3.1 Decomposition of Cellulose . . . . . . . . . . . . . . . . . . . 316
14.5 Extraction of Aquatic Cellulose . . . . . . . . . . . . . . . . . . . . . . . 317
14.5.1 Extraction of Cellulose from Aquatic Plants . . . . . . . . 318
14.5.2 Extraction of Cellulose from Algae . . . . . . . . . . . . . . 318
14.6 Environmental Implications . . . . . . . ............ . . . . . . . 320
14.6.1 Energy for Drying . . . . . . . ............ . . . . . . . 320
14.6.2 Chemical Consumption . . . . ............ . . . . . . . 321
14.6.3 Methane Emission . . . . . . . ............ . . . . . . . 321
14.6.4 Land Space Occupied . . . . . ............ . . . . . . . 322
Contents xvii
14.6.5 Aquatic Plants and Algae Bloom . . . . . . . . . . . . . . . . 322

14.6.6 Wastewater Treatment . . . . . . . . . . . . . . . . . . . . . . . . 323
14.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
14.7.1 Textile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
14.7.2 Bioethanol Production . . . . . . . . . . . . . . . . . . . . . . . . 325
14.7.3 Cellulose Filler in Composites . . . . . . . . . . . . . . . . . . 326
14.7.4 Cellulose Nanofilters . . . . . . . . . . . . . . . . . . . . . . . . . 326
14.7.5 Drug Carrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
14.7.6 Papermaking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
14.7.7 Production of Cellulose Derivatives . . . . . . . . . . . . . . 327
14.7.8 Conductive Cellulose Paper . . . . . . . . . . . . . . . . . . . . 328
14.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
15 Polyesters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
15.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
15.3 Chemistry of Cutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
15.3.1 Biodegradation of Cutin . . . . . . . . . . . . . . . . . . . . . . 337
15.5 Extraction of Cutin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
15.6 Environmental Implication . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
15.6.1 Use of Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . 341
15.6.2 Use of Hydrochloric Acid . . . . . . . . . . . . . . . . . . . . . 341
15.6.3 Wastewater Generated . . . . . . . . . . . . . . . . . . . . . . . 342
15.6.5 Aquatic Plant Harvesting . . . . . . . . . . . . . . . . . . . . . 342
15.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
15.7.1 Sunscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
15.7.2 Food Packaging and Additives . . . . . . . . . . . . . . . . . 343
15.7.3 Cosmetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
15.7.4 Pharmaceutical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
15.7.5 Biomimicry of Cutin Matrix for Wastewater
Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
15.7.6 Biodiesel Production . . . . . . . . . . . . . . . . . . . . . . . . . 345
15.7.7 Biopolyester Production . . . . . . . . . . . . . . . . . . . . . . 346
15.9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
16 Others Aquatic Biopolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
16.1 Phlorotannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
16.2 Bioluminescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
xviii Contents
16.3 Biofluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

16.4 Xyloglucan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
16.5 Pectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
16.6 Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
16.7 Peptides from Frog Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
16.8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
17 Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Chapter 1
Introduction to Aquatic Biopolymers
Polymers refer to macromolecules made up of repeating units of smaller molecules

covalently bound together to form a bigger molecule. For example, proteins are
polymers made up of amino acids, cellulose is made up of repeating units of glucose
and polyethylene is made up of repeating units of ethylene. The units which join
together to form polymers are referred to as monomers, and the process through which
this is achieved is termed polymerization. Biopolymers refer to polymers which are
produced by living organisms. The term aquatic biopolymers refers to biopolymers
which are produced by living organisms that inhabit the aquatic ecosystem.
Since the beginning of the ages, polymers have played a significant role in human
life in providing the most basic need, food, in the form of carbohydrates and pro-
teins. As life advances, polymers have come to be of great importance to modern
civilization, from the food we eat, the portable plastic bottles that give us convenient
access to clean drinking water, the medicines we take, basic hygiene, tools and the
fuels that run our engines. Polymers are fundamental to our existence as modern
humans. Considering that the polynucleotides which form our DNA that carry the
instructions for every life function are also polymers, it can therefore be said that
polymers indeed make life. The aquatic environment is a rich source of a wide range
of natural resources, and among those resources are polymers. They are found in
the cell walls, tissues, exoskeletons, secretions and anatomical regions of aquatic
organisms across the five kingdoms.
A large variation of factors exists in the aquatic environment. These variable
factors include salinity, depth, light intensity, temperature, pressure, density, pH,
prey/predator presence, nutrients, dissolved gases, water flow rate, flora and fauna.
These factors determine the types of biochemicals produced by the different organ-
isms to survive their environment, even more so where they are in such direct contact
with the continuous medium (Christophe et al. 2015). Such variation in the envi-
ronmental factors also translates to a diverse range of polymers. Over the course
of evolution of life, living organisms both plants and animals have devised means
to produce, absorb, store and process different forms of biopolymers. This ranges
from polysaccharides used for energy and structure, proteins for metabolism and
cell renewal and polyesters for water repelling among others. These are stored in
© Springer International Publishing 2020 1
O. Olatunji, Aquatic Biopolymers, Springer Series on Polymer and Composite Materials,
https://doi.org/10.1007/978-3-030-34709-3_1
2 1 Introduction to Aquatic Biopolymers
different anatomical regions of the plant. Carbohydrates, for example, are stored
in chloroplasts, cytoplasm, periplastic compartment, peri-plastid membranes, in the
cytoplasm, and vacuole localized in the cytoplasm (Prabhu et al. 2019).
The diverse chemistry and bioactivity that can be derived from naturally sourced
polymers add to their significance. This is of particular importance as increasing
occurrence of cancer and other degenerative disease and resistance to existing antimi-
crobial drugs calls for more diverse and complex compounds existing in nature. The
rain forests are often described as the medicine chest of the world. This is attributed
to the presence of a diverse range of bioactive compounds which can be obtained
from the diversity of organisms in the rain forest. The aquatic ecosystem consists of
even more diverse range of organisms, and its existence and subsistence are crucial
to that of the rain forest and life on earth. This book provides the reader with a rich
understanding of the wide range of polymers which can be sourced from the aquatic
environment, some of which have bioactivities such as anticancer and antimicrobial
activities.
Due to limited access compared to land where humans are better adapted to inhabit,
the aquatic world is relatively underexplored. As technology advances, various tools
have been developed to better explore the world that exists below sea level. Today,
submersibles have been developed which can reach the deepest part of the ocean
which have previously been inaccessible. High-resolution specialized cameras which
can capture the faintest light underwater have also been developed alongside more
sophisticated analytical tools and techniques (Linley et al. 2016). This improved
access to the world below sea level has significantly improved knowledge and access
to the aquatic world in recent years. Within this book, we also explore some of the
polymers which have been discovered in recent years from deep-sea organisms.
When evaluating the commercial implications of production of biopolymers from
aquatic sources, it should be noted that apart from the cost of raw materials, the
downstream processing constitutes around 60% of the production cost. Downstream
processing is where the bulk of the value addition to the final product occurs. This
includes solid–liquid separation, purification, solvent recovery and product recovery.
Therefore within this book, some of these stages are included when the extraction
process for each polymer is described. This is with the aim to understand the pro-
cesses involved in the production of the biopolymers and how these impact on the
environment.
As the aquatic ecosystems of the world are increasingly facing threat from pol-
lution as a result of human activity and poor waste management (Hitchcock and
Mitrovic 2019), it is important, therefore, to understand what is at stake as far as the
aquatic environment is concerned. This book aims to provide an understanding of
the biopolymers existing within the aquatic ecosystem, how they are obtained, their
role in ecosystem as well as their existing and potential economic value.
The benefits of aquatic biopolymer production can be summarized as follows:
• Utilize aquatic waste from fisheries and aquaculture especially as aquatic activities
have shown an increase in recent times due to increased demand for aquatic food.
1 Introduction to Aquatic Biopolymers 3
• Source biopolymers with more diverse chemistry to address complex health issues,
meet the demand for novel materials and address bacteria resistance.
• Replace non-biodegradable plastics which presently are having deteriorating
impact on aquatic life and water resource by augmenting the feedstock for bioplas-
tic production with aquatic biopolymers which have faster biomass accumulation
rate.
• Alternatives to depleting fossil resources which do not require use of land or
freshwater to cultivate.
• Value addition to aquatic resources to serve as a means of improved income for
developing countries and emerging economies where a lot of the fishing activities
occur.
• Development of biorefinery through increased feedstock from aquatic waste and
excess abundant resources.
This book takes a new approach to developing understanding of polymers by
focusing on polymers derived from specifically the aquatic ecosystem. In so doing,
it presents a much broader view of polymers, the opportunities, impact and key issues
in the polymer and aquatic industry. Each chapter introduces the reader to the different
chemical structures of polymers and how these could vary from simple linear to quite
complex non-uniform structures. In exploring the production processes, the reader
learns about different techniques which are used in the polymer industry and where
possible some case studies of specific companies are included.
In the process of discussing the sources of polymers, a variety of species of organ-
isms are explored, showing how polymers exist across the five kingdoms and all over
the food web. The ubiquity of polymers is further established through reviewing the
numerous applications of each aquatic biopolymer covered. The current issues facing
the aquatic environment and the environmental impact of the extraction processes are
also discussed. This gives the reader a rich understanding of the biopolymer resource
of the aquatic ecosystem, their economic and environmental significance.
References
Christophe M, Rachid A, Mario L (2015) Fish as a reference species in different water masses.
Aquat Ecotoxicol, 309–331
Hitchcock JN, Mitrovic M (2019) Microplastic pollution in estuaries across a gradient of human
impact. Environ Pollut 247:457–466
Linley TD, Gerringer ME, Yancey PH, Drazen JC, Weinstock CL, Jamieson AJ (2016) Deep sea
research part I: oceanographic research papers, 114:99–110
Prabhu M, Chemodanov A, Gottlieb R, Kazir M, Goldberg A (2019) Starch from the sea: the
green macroalga Ulva ohnoi as a potential source for sustainable starch production in the marine
biorefinery. Algal Res 37:215–227
Chapter 2
Overview of the Aquatic Ecosystem
Abstract The chapter gives an overview of the aquatic ecosystem as a whole. The
aquatic ecosystem is classified based on different criteria: depth, water flow, salinity
and features. The organisms within the aquatic environment are also described; these
include algae, aquatic plants and animals. In the process, some current issues facing
the aquatic environment are also discussed. Some of the terms described here are
used in other chapters of the book; therefore, this chapter also serves as a reference
point for the rest of the book. The chapter also includes illustrations and images of
some aquatic ecosystems from different parts of the world.
Keywords Biopolymers · Marine · Freshwater · Littoral · Pelagic · Estuaries
2.1 Introduction
Evidence suggests that water was brought to earth by water carrying meteorites and
asteroids colliding with the planet during its formation. This water formed the oceans
of the earth which then spill into other bodies of water. Therefore, every drop of water
on earth is billions of years old (Takir et al. 2018). This water is recycled through the
water cycle and changes state between gas, liquid and solid through the processes of
evaporation, condensation and freezing. The oceans absorb a significant amount of
energy from earth’s revolution which would otherwise cause extremely high wind
speeds which could have destructive effects on life on land. The aquatic ecosystem is
made up of the living and non-living components that exist within a body of water. It
consists of such diverse life-forms that there exists more diversity of life in the water
than on land. The survival of an aquatic ecosystem depends on maintaining a balance
between the existence and activities of the producers, consumers, decomposers as
well as the abiotic components. This is same in terrestrial ecosystem except that the
aquatic ecosystem has an abundance of moisture, continuity of the medium (water),
limitation of air and variation in temperature and light.
To say that water plays an important role in sustaining life is almost an under-
statement. Water is indeed life. The aquatic world is full of diverse life-forms, and
it makes up 70% of the earth. Water has served as the main evidence of life on
other planets; to ascertain the existence of life, one must first confirm the presence

6 2 Overview of the Aquatic Ecosystem
of water. Humans, plants, animals, prokaryotes and protists all depend on water to
exist. More than just hydration, many biochemical reactions and processes depend
on water. Dissolving of nutrients for uptake, the maintenance of erect upright form
in plants and much more require water, such is the extent of the relevance of water
for life on earth.
Aquatic ecosystems refer to the complex web of relationships between living and
non-living organisms which exist around a body of water. In order to understand the
industrial and environmental significance of aquatic biopolymers, it is also important
to understand the ecosystem within which these biopolymers exist. The aquatic envi-
ronment holds a world of resources which play important roles in both the present
and future industries. In addition, it is a vital food source and means of generating
income for many communities. Biopolymers form a significant part of the aquatic
environment, and various aquatic sourced biopolymers are explored in the different
chapters of this book.
The United Nations Convention on climate change sets the global goal of ensuring
that the global temperature rise does not go 2 °C above the preindustrial temperature.
The global temperature rise is due to many factors, part of which is hypothesized to
be the continuous dependence on fossil fuel which results in the release of gaseous
compounds which result in global warming. To this end, this book seeks to explore the
range of available resources in the aquatic environment, their production process as
well as their economic value both existing and potential. It explores how the aquatic
environment could indeed offer alternatives to the biopolymers which we require in
various aspects of life.
Optimum utilization of the vast resources of the aquatic world requires an under-
standing of the different resources and the role they play in the environment and the
economy. The sustainable millennium goal SDG 14 states: “Conserve and sustain-
ably use the oceans, seas and marine resources for sustainable development” (FAO
2018). Even where one is interested in only a single resource, it is important to have
a general overview of the range of biopolymers and how they are connected. This
helps to explore alternatives, co-production and the connection of that particular
biopolymer to the rest of the aquatic ecosystem.
While still at its infancy, the marine biorefinery could be built around developing
a synergy with the already-existing offshore facilities. This will enable a more seam-
less transition from mostly fossil-based polymer products to more diverse polymer
industry that includes the use of aquatic biopolymers sourced from aquatic waste
and by-products in a sustainable manner. Furthermore, deep-sea mining, oil and gas
drilling and intensive unsustainable fishing methods are reported as the major threats
to the aquatic ecosystem. For example, a recent study revealed that the increase
in the copper concentration in the water during deep-sea mining results in reduced
metabolic activities and immune response in B. azoricus, a mussel which inhabits the
deep sea (Martins et al. 2017). By utilizing the biopolymers from aquatic wastes and
by-products, the demand for these unsustainable sources of polymers is reduced, and
through value addition to the aquatic resource, there is less pressure on fishermen to
adopt unsustainable fishing methods.
2.1 Introduction 7
This predicted rise in population is necessitating the expansion of the food industry
in general. Aquatic food serves as a key source of protein and essential nutrients in
which many developing and advanced economies rely on. This will mean a rise
in the amount of aquatic waste and by-products being generated—thus indicating
long-term availability of feedstock such as shrimp and lobster shells, fish scales and
internal organs to power the marine biorefinery. Going further, we shall explore the
availability trends of the respective aquatic feedstocks for biopolymer production.
This chapter gives the reader an overview of the aquatic environment and in doing
so provides an insight into the diversity of the aquatic ecosystem which serves as a
source of a wide variety of biopolymers which will be discussed in the subsequent
chapters.
2.2 Types of Aquatic Ecosystems
Through exploring the various aquatic biosystems, we can then understand the var-
ious roles played by these biopolymers within the living organisms and within the
aquatic environment as well as how to extract and utilize them. Aquatic ecosystem
can refer to either inland or marine waters. Inland waters refer to aquatic ecosys-
tem within the land borders, while the marine waters refer to generally the seas
and oceans. These coastal lands/waters refer to the boundary between the aquatic
ecosystem and terrestrial ecosystem. These are generally referred to with terms such
as beach, shore, coastlines and waterside.
Based on source and salinity, aquatic ecosystem can be classified as marine and
freshwater. Brackish water is formed where the saltwater from the marine mixes with
freshwater. There are five oceans on earth: the Atlantic, Pacific, Indian, Arctic and
Antarctic oceans. These feed into the numerous seas and inland waters (Table 2.1).
Aquatic habitat can further be characterized by other factors, an example is char-
acterization by flow rate of water. Based on flow rate aquatic habitat can be either
flowing or standing waters. Organisms living in either of these have features which
allow them to cope at the respective flow conditions (Table 2.2).
Table 2.1 Classification of

Aquatic ecosystem
aquatic ecosystem based on
water salinity Marine Freshwater
Examples: ocean, sea, Examples: ponds, lakes,
estuaries, lagoons, salt rivers, springs streams,
marshes wetlands
Table 2.2 Classification of

Aquatic ecosystem
aquatic ecosystem based on
water flow Lotic-flowing waters Lentic-still waters
Rivers, streams, springs Ocean, lakes, ponds, swamps
Table 2.3 Different Lake zones

Lake zones Subdivision Depth range Organisms and features
Littoral zone Epilimnetic Surface to 6 m Aquatic plants
Hypolimnetic 6–10 m
Limnetic zone From 10 to depth of effective Planktons
light penetration
Profundal zone From point beyond light Heterotrophs
penetration to bottom
The aquatic environment can also be classified based on depth. Table 2.3 shows
the classification of lake zones (Peters and Lodge 2009), and Table 2.4 shows the
classification of the ocean depths (Honjo 2009; Linley et al. 2016; Staby and Salvanes
2019). The lake zones are similar for rivers; however, in ponds, the profundal zones
do not exist since they are relatively shallow and light can penetrate to the bottom.
Different aquatic organisms have adapted to live in waters at different depths, tur-
bulence, salinity, temperature, light and pressure. Other adaptations include defense
mechanisms and competition for food. For example, organisms living in the deepest
region of the water where no light penetrates have adapted features which enable
them to create light and/or sense prey in the dark.
Other factors by which aquatic ecosystem can be characterized include pH,
turbidity, dissolved gases, temperature, light penetration and areal distribution.
Types of aquatic ecosystem include:
• Aquifers
• Springs
• Rivers
• Lagoons
• Streams
• Lakes
• Seas
• Ponds
• Wetlands
• Beaches
• Bays
• Estuaries
• Oceans
• Coral Reefs
All of these aquatic systems play a significant role in the survival of different
life-forms and the accumulation of a wide range of biopolymers and other resources
on earth. The following sections describe each type of aquatic ecosystem and the
different life-forms which exist within the respective ecosystems.
2.2 Types of Aquatic Ecosystems 9
Table 2.4 Different zones of the ocean

Ocean zones Depth range Organisms and features
Epipelagic zone (Sunlight Surface to 200 m Abundant sunlight, minimal
zone) pressure, higher
temperatures. Coral reefs and
photosynthetic organisms,
more ocean life and human
activities
Mesopelagic zone (Twilight 200–1000 m Lower light intensity (mostly
zone) blue light). Zooplankton and
rarer sea life such as the
Barreleyes, ribbonfish and
snipe eels. Vertical migration
of organisms and gradient of
oxygen level, light intensity,
temperature and salinity
Bathypelagic zone (Midnight 1000–2000 m Dark, pressure up to
zone) 5.858 lbs/square inch,
bioluminescent organisms.
Nematodes, bivalve molluscs,
polychaetes, crustaceans,
sipunculid worms and
gastropods
Abyssopelagic zone (Abyssal 2000–6000 m Near freezing point, high
zone) pressure, rare species and
invertebrates such as the giant
squid. Includes some parts of
the ocean floor
Hadalpelagic zone (The 6000–11,000 m (valleys Deepest known is the
trenches) below the ocean floors) Mariana Trench in Japan.
temperature and pressure,
few rare species recently
discovered include the
Mariana Snailfish and
Ethereal snailfish
2.2.1 Estuaries
Estuaries are very important in the maintenance of the aquatic biodiversity. They are
water bodies which are enclosed by structures such as vegetation and the organisms;
they have easy access to the sea (Dame 2008). The water in the estuaries is brackish as
a result of ocean water mixing with freshwater. There is also variation in turbulence
depending on the contact with the sea. These estuaries serve as nurseries for young
species. The underwater plant roots protect them from bigger prey simply by serving
as a hiding place that only allows entry of organisms below a certain size. This allows
them a better chance of survival and growing to a safer size after which they can then
easily access the sea and begin their journey to their final home in the deeper waters.
In recent times, some estuaries are under threat from environmental pollution,
disease outbreak and overfishing (Dame 2008). Some of these are due to the release
of toxins from factories and homes. This in turn distorts the balance in the ecosystem.
Losing estuaries means losing several species of aquatic plants and animals.
One of the recent threats studied in the estuaries of the Clyde, Bega and Hunter
estuaries in east coast of Australia revealed that increased human activities resulted
in higher microplastics in the estuaries (Hitchcock and Mitrovic 2019). In the three
estuaries studied, the microplastic pollution was as high as 1032 parts per m3 in
the coast with the most populated Hunter estuary, to 98 part per m3 in the Clyde
estuary which the coastal land is least populated by humans while Bega estuary had
a microplastic pollution of 246 parts per m3 . Most of the microparticles had a particle
diameter below 200 µm.
2.2.2 Springs and Aquifers
In some parts of the earth, water accumulates under layers of rocks and sediments.
These are often artificially obtained by digging wells deep into the earth’s core and
either drawing out or pumping. Such water is relatively high quality as it is naturally
filtered through layers of rock and sand on the way out of the aquifer. They are used
for human consumption, agriculture and other activities important for life. In order
to conserve water and ensure the availability of aquifer-sourced water for future use,
it is important that the rate at which water is returned to the aquifer is balanced
with the rate at which it is being removed. Aquifer forms a relatively low energy
requiring source of clean water. Recharge zones are points in which rainwater seeps
back into the aquifer beneath and this makes up for the water removed either through
natural springs or by human activity. Recharge zones can be natural or man-made,
and these recharge zones experience dry and wet periods. While some aquifers show
rapid response to climate change, others show slow response. The response of an
aquifer to change in climate depends on the type of aquifer. An example is the
Northern Sudan Platform subbasin where average annual precipitation of 85 mm
was recorded between 2002 and 2012, and between 2013 and 2016, average annual
precipitation recorded was 107 mm (Abdelmohsen et al. 2019).
The underground water trapped within aquifers does not always need to be pumped
or drained by human activities. Due to the topology of the ground, the water escapes
from aquifers into the surface due to pressure difference and gravitational force. The
path through which the water flows is known as springs. This serves as a source of
natural spring water. These could either be slow moving or generate enough pressure
to form bubbling springs. The water from springs is usually of high quality due to
the fact that the rainwater flowing into the aquifers is filtered through different layers
of soil and rocks. Water flowing from springs forms a large part of waters in streams
and rivers in addition to annual rainfall.
2.2.3 Rivers and Streams
Rivers serve as a means of transportation, food, recreation and biodiversity. Large

bodies of freshwater which flow are referred to as rivers. Generally, a flowing body
of water that spans a relatively large area can be classified as a river. The Nile River
is the longest river in Africa and possibly in the world; it is measured 6825 km in
length and runs through 11 countries including Rwanda, Ethiopia, Kenya, Uganda,
Tanzania, Sudan and others (Abd-Elbaky and Jin 2019). Other examples are the
Volga River, which is the largest river in Europe; the Danube River, which is the
second largest river in Europe and runs through the heart of Europe; the Amazon
River, which is crucial water flow for the Amazon rain forest and contends with the
Nile River in length as the Amazon River also measures over 6000 km in length.
Figure 2.1 shows Mbagathi River in Kajiado County in Kenya. The image also shows
the terrestrial plants and trees on the land through which the river flows.
The constant flow of a river is due to the force of gravity as a river flows over a
sloped floor. It is important for a river to flow rather than be stagnant as flowing water
allows air to mix into the water to support life-forms which require a level of dissolved
Fig. 2.1 Mbagathi River, Kajiado county, Kenya. August 2019. Photograph courtesy of Ross van
Horn
oxygen to survive. The constant flow also prevents the settling of pathogenic larvae
of organisms such as mosquitoes on the surface of the water. Several aquatic and
terrestrial life-forms depend on the water cycle in the river. For example, some life-
forms such as the tadpole stage of frogs and the dragonflies begin life in the river
until they reach adulthood and transform to live on land or fly. Even in adulthood,
some of these organisms still depend on the water as a source of food and refuge.
Rivers are linked to streams, pools, water channels and floodplains. The life-forms
and structure of the abiotic and biotic components around a river vary at different
parts of the river. Figure 2.2 is an example of a stream flowing through the town
of Stellenbosch, South Africa. The water from this stream also serves as irrigation
water for the farms in the area.
2.2.4 Lakes
A large standing body of water surrounded by land is referred to as a lake. Over a

thousand lakes exist in the world. A lake is formed by the deposition of water from
rainfall, ice formation, river water and other factors which lead to water flow and
formation ending up in the lake. Since the lake is formed from these variable factors,
the level of water and conditions in a lake therefore varies at different periods as
water evaporates and is deposited. Lakes form a habitat for diverse life-forms such
as water plants, snails and fish. Waterbirds such as swans, ducks and geese are also
a regular feature in lakes, and these in turn serve as food for land animals as well as
aesthetics and recreation for humans. Example of lakes are the Lake Chad which is
surrounded by the seven Chad basin countries: Nigeria, Cameroon, Central African
Republic, Niger, Chad, Algeria and Sudan (FAO 1997), Lake Victoria in Tanzania
and numerous other lakes across the world.
The life in the lake is held in a sensitive balance between the photosynthesizing
life-forms and the decomposition of the organic matter. A lake is comprised of the
oxygen-dissolved zone, the light-penetrating zone and the dark oxygen-depleted
layer. These lake zones are classified as littoral, limnetic and profundal zones,
respectively (Table 2.3).
Some of the lakes in the world are drying up due to climate change, pollution and
some due to the natural cycle of a lake. Lake Victoria, the second largest freshwater
lake in the world, is possibly an example of such, which is located in Tanzania and
covering an area of over 69,000 km2 as of 2018. It was reported to have shrunk
by 203 km2 from 1984 to 2018 with 0.3% shrinkage in area (Awange et al. 2019).
Similarly, the Lake Chad has been reported to have shrunk from a surface area of
25,000 to 2000 km2 (Mahmood and Jia 2019). This shrinkage of the lakes is attributed
to a combination of climate change and human activities.
Fig. 2.2 Three different parts of a stream from shallower low turbulence to slightly deeper slow-
moving to the steeper higher turbulence. Stellenbosch Town, South Africa, June 2019 (Winter).
Photograph by Ololade Olatunji
2.2.5 Ponds
Ponds refer to water-filled depressions on the earth’s surface. Ponds, like lakes, are
formed from water deposited from rain, snow, river runoffs or other water forma-
tions. However, ponds are much smaller than lakes. While lakes could extend for up
to hundreds of acres, ponds only cover a few acres of area. Due to the relative smaller
Fig. 2.3 A partly dried pond on the hill in Stellenbosch, South Africa June, 2019. Photograph by
Ololade Olatunji
size, ponds can become dry at different times, depending on the environmental con-
ditions. Ponds are usually shallow and therefore have stronger light penetrations and
dissolved oxygen along its depths such that ponds support more aquatic plant and
algae growth. Ponds are susceptible to drying up, especially during periods of lower
rainfalls. Figure 2.3 shows an example of a pond with a decreased water level in June
in the Town of Stellenbosch, South Africa.
2.2.6 Lagoons
Lagoons are bodies of water separated from the ocean by barriers such as reefs. They
can be natural or man-made and generally saline, low tide and relatively shallow
compared to the sea with minimal mixing with freshwater. Unlike estuaries, the
ocean water in lagoons is trapped behind coastal dune systems, sandpits or island
barriers and therefore does not provide the aquatic organisms therein the same ease
of access as the estuaries (Harris 2008). An example is the Lagos Lagoon in Nigeria
(Fig. 2.4), the Vermelha Lagoon in Brazil, the Nichupte Lagoon of the Mexican
Caribbean, Venice Lagoon in Italy, Manzala Lagoon in Egypt, Santos Andre Lagoon
in Portugal, Tunis Lagoon in Tunisia, Vistula Lagoon of the Baltic Sea and the La
Pletera salt marsh Lagoon in northeast Catalonia. The lagoon serves as host to some
aquatic life such as fish, amphibians, crustaceans, aquatic plants and waterbirds.
Species such as the Aphanius iberus fish are endemic to lagoons like the La Pletera
salt marsh Lagoon (Casamitjana et al. 2019). Lagoon serves as a source of fish,
recreation and transportation and forms a major attraction for tourism.
Fig. 2.4 A view of Lagos Lagoon from the University of Lagos. The third mainland bridge is built
over the Lagoon; some aquatic plants are also visible in the image. Photograph by Ololade Olatunji
2.2.7 Wetlands
The way in which the land and water meet varies. In some parts, the sea meets the land
at rocky shores or sandy beaches, while in some parts the meeting point is demarcated
by wetlands connecting aquatic and terrestrial environments. Flood plains and rain
forests are watered by the cycle of the river flowing through them. One of much
global importance is the Amazon rain forest in Brazil, through which the Amazon
River flows and is one of the most diverse wildlife. Wetlands are important to the
environment as they support a diverse range of life-forms such as frogs, birds, turtles
and aquatic plants. They are wet soil regions close to a water body. Wetlands play a
significant role in abating floods by absorbing rainwater. Examples of wetlands are
marshes, swamps, floodplains and bogs.
The amount of water retained in wetlands varies with rainfall, riverflow, season and
climate change. The water from the river which flows into wetlands is important for
agriculture; for example, growing of floating rice species in rice-growing regions of
countries such as Vietnam and Japan is carried out in sync with the wet and dry cycle
of flow of water from the river into the wetlands on which the rice is grown (Iizumi
and Ramankutty 2015). The amount of water retained by the land from the aquatic
environment is measured as the terrestrial water storage (TWS). For example, the
average TWS measured for the Nile River basins between September and November
was 42.66 and −23.34 mm between March and May. These values are based on
average between 2003 and 2016 (Abd-Elbaky and Jin 2019). The TWS at different
parts of the year therefore varies with the season depending on rainfall.
Currently, the Amazon rain forest is threatened by deforestation. The felling of
trees at a rate faster than the forest can grow new trees denies several species their
habitat, cover and a source of food, consequently leading to a decline in population.
This results in the loss of plant species which depend on the larger trees for cover and
temperature control and species such as woodpecker which depends on the trees for
shelter and protection from prey. Likewise as the population of the organisms which
are a source of food for the larger prey decline, the larger organisms which prey on
them also begin to die out.
2.2.8 The Coral Reefs
The coral reef is commonly referred to as an underwater city as it is made up of

living structure which houses a very diverse range of aquatic species with complex
relationships. The reef itself is made up of organisms referred to as the corals which
build a protective housing made up of limestone within which they permanently live.
The corals have a symbiotic relationship with algae where they make use of the energy
produced by algae from photosynthesis to build their limestone structure. Coral reefs
exist within the epipelagic zone (sunlight zone), usually within the 35 m depth of the
sea fringe. Although it is commonly referred to as the coral reef, the term biological
reef is also used as a way to better highlight its significance and also provides a term
that also credits the significant role of another organism which plays a very important
role in creating these reefs, the algae. Algae provide the energy which is required
by the corals to build these reefs. The energy produced from photosynthesizing
organisms such as the sea grass and algae serves as a power source for the coral reef,
such that these underwater habitats are essentially solar powered. Collectively, corals
can build the underwater structure known as coral reefs extending up to thousands
of miles. The largest coral reef on earth is that which exists in the northern coast
of Australia, referred to as the Great Barrier Reef. Organisms which can be found
within and around the coral reef include a range of finfish species, sea horses, starfish,
anemones, sea cucumbers, clown fishes, mantis shrimp, sea turtles and much more.
The algae, the shells of crustaceans, the skin and even scales of the fish all comprise
a range of biopolymers which will be explored in different sections of this book. The
coral reef serves an important role in maintaining the aquatic biodiversity which in
turn is significant toward the availability of diverse range of aquatic biopolymers.
Recent studies reveal that the Great Barrier Reef is a source of net release of
CO2 to the environment (Lonborg et al. 2019). The coral reefs therefore potentially
contribute to global warming based on the theory that CO2 results in the overall
increase in the earth’s temperature. On the other hand, the Great Barrier Reef is valued
at AU$ 56 billion (Pendleton et al. 2019). This is value derived from recreational
activities, tourism and fisheries. The coral reefs are also quite fragile and are at risk
from factors such as ocean acidification (Pendleton et al. 2019) and nutrient runoff
from sugarcane plantations (Deane et al. 2018). The coral reefs therefore serve as an
example of how the aquatic life and activities impact on terrestrial life and activities
and vice versa.
2.3 Aquatic Organisms
Life inhabiting the aquatic environment includes the five kingdoms: plants, animals,
prokaryotes, protista and fungi. These could be large organisms such as the whales
and giant squids or they could be microorganisms such as bacteria and microalgae.
2.3.1 Aquatic Plants
One of the differentiating features between aquatic and terrestrial plants is that aquatic
plants growing in flowing water possess larger and narrower leaves while land plants
or plants grown in poorly oxygenated water had smaller and broader leaves. However,
the size or distribution (number of stomata per unit cm2 of leaf) of the stomata does
not vary (Penfound and Earle 1948).
Aquatic plants are also referred to as hydrophytes. Some aquatic plants are invasive
while others are extractive primary producers which photosynthesize using nutrients
such as phosphorus, manganese, zinc, iron and nitrogen and carbon dioxide from the
aquatic environment and releasing oxygen in exchange (Oyedeji and Abowei 2012).
In some aquatic environment where oxygen supply from the atmosphere is low,
aquatic plants serve as the primary oxygen source for the other aquatic organisms
within. Aquatic plants also add some aesthetics to the environment if well controlled.
The water lily, for example, blooms with colorful flowers and some accounts of it
being grown for its ease of growth and blooming beautifully in the United States
after the First World War.
Some aquatic plants can be beneficial to the aquaculture or wild aquatic environ-
ment serving as nutritional base to shell and finfish, zooplankton and other inver-
tebrates as well as controlling the growth of invasive phytoplankton and moss by
removing the metabolites required by these organisms. Others may pose a nuisance
by disrupting water transportation and blocking navigational channels or serve as
hosts to pathogens and pests. Reported cases of water hyacinth disrupting water
transportation exist in the Niger Delta region of Nigeria (Oyedeji and Abowei 2012).
Water hyacinth which was once said to be an admirable plant grown for aesthetic
appeal has since then gone from an admirable water plant to one of the biggest
pests in the world. Water hyacinth pest problems have resulted in millions of dollars
worth of loss in terms of destruction of aquatic life and human activity related to
aquatic environment. This includes blocking of drainage, destruction of aquatic food
source, impediment of runoff leading to flooding, hindering of water transportation
and loss of income from water-based recreational activities. Water hyacinth is a self-
pollinating, mat-forming vegetative water plant. Its structure comprises a flowering
leaves, inflorescences supported by a stolon and rhizomes with fibrous unbranched
roots reaching down to the water. They can grow up to 3 feet high.
The fact that aquatic plants are adapted to cope in the aquatic environment indi-
cates some significant differences between their structure and that of terrestrial plants.
This abundance of water means they do not require cutin or suberin in their leaves
and roots for the prevention of water loss. They instead have other features which
address the need for buoyancy to float on water as well as survive the tides. Other
features of aquatic plants include lacunae, which are holes in the root cortex which
aid in moderating gas exchange (North and Peterson 2005). Aquatic plants can be of
three types based on their biological structure; these are:
• Phytoplanktons
• Periphytons and
• Multicellular Macrophytes
Phytoplanktons are photosynthetic microscopic plants (and animals) which drift in
water. They require light for synthesis; therefore, they are located in the littoral zones
of water. They serve as primary producers as they can convert nutrients from the water
such as zinc, nitrogen and magnesium into carbon and oxygen. Most of the oxygen
productions from photosynthesis in the oceans, lakes and rivers are carried out by
phytoplankton. Planktons which do not photosynthesize are referred to as zooplank-
tons. Phytoplankton also comprises algae and bacteria as well as plant phytoplank-
tons. There are about 43 species of phytoplankton. They serve as a nutrient source
to other aquatic organisms such as finfish and shellfish and therefore are required for
a healthy aquatic ecosystem. Their environmental and economic significance lies in
the sustenance of the other aquatic organisms of economic importance.
Periphytons are aquatic organisms which require a substrate to grow on. They could
be heterotrophic or autotrophic (Petters and Lodge 2009). Rocks and plants could
serve as such substrate as well as other surfaces. These surfaces provide the structural
support for them to survive aquatic environment. They obtain nutrients from the
water, and they serve as a food source for herbivores.
Multicellular macrophytes are more independently growing and anchored multi-
cellular aquatic plants. They can be emergent, submerged or free-floating. Many
submerged aquatic plants do not possess cuticle as they are surrounded by abundant
water. Likewise, they partially or completely lack xylem and stomata remains open.
Roots may be present in submerged plants to anchor them to the surface or it could
be absent. While floating aquatic plants are adapted with air spaces trapped within
their structure to enable them to remain afloat, submerged aquatic plants are adapted
to survive in low light due to their distance from the surface of the water. The leaves
of the floating aquatic plants are flat to aid flotation while their roots are specially
adapted to aid oxygen uptake. Emergent aquatic plants grow in the shallowest end
of the water. Their roots are buried in the soil at the bottom of the water while the
rest of the plant grows above water. Figure 2.5 illustrates these three different types
of aquatic plants.
2.3 Aquatic Organisms 19
Emergent
Floating
Submerged
Fig. 2.5 An illustration of floating, submerged and emergent aquatic plant
In some cases, some common features may exist between aquatic plants and
terrestrial plants which serve different purposes in aquatic plants. An example of
such is the presence of cuticles in some aquatic plants such as duckweed for the
purpose of protection against ultraviolet radiation and pathogens rather than for
water retention as it is used in terrestrial plants (Borisjuk et al. 2018). There are also
plants that are classified as amphibious and plants that are both aquatic and terrestrial
such as the marshland and shore weeds (Littorella uniflora) (Braendle and Crowford
1999).
Effects aquatic plants can have on aquatic system include reduction in temperature,
and decayed plants can cause high biochemical oxygen demand and reduce dissolved
oxygen. They can also result in buildup of toxins and bad odor and generally destroy
the aesthetics of the environment. Therefore, in order for aquatic plants to serve their
beneficial role, there is a need for the aquatic system to be well maintained with
controlled plant growth. Some common examples of aquatic plants are wild rice,
wasabi, watercress, water caltrop, chinese water chestnut and water mimosa among
others. Duckweed and water hyacinth are often used as animal feed. Non-edible
aquatic plants include water lilies and water lettuce. The edible water plants include
rice and water chestnuts.
While aquatic plants play an important role in the aquatic ecosystem, uncontrolled
population growth of any species could cause an imbalance in the ecosystem and
eventual destruction of other life-forms in the ecosystem. Water hyacinths are one of
such. These invasive plants mostly exist in slow-moving or still waters such as lakes
and ponds. It survives in temperature between 12 and 35 °C with optimal at around
25–30 °C. It can also survive in slightly acidic to neutral water of pH between 5 and
7. Water hyacinths do not survive in saltwater; hence, they do not exist on the sea.
They can barely survive in brackish water and can be found in some lagoons.
At a low controlled level, aquatic plants add aesthetics to the aquatic environment
as some have colorful flowers. Aquatic plants also absorb the water waves and abate
the impact of flowing water which may lead to erosion. However, at uncontrolled
levels they tend to starve essential aquatic life of light and oxygen which are essential
to their growth. The death of the aquatic flora, the process of decay of these dead
plants commences which then results in the decomposing bacteria taking up all the
dissolved oxygen in the water, this then leads to the death of the fish and other aquatic
lives which require dissolved oxygen to survive. The humans who live around these
water bodies also depend on the fish as food and trade, once this depletes this also
affects the survival of the humans living around the hyacinth-infested waters.
2.3.2 Algae
Algae are polyphyletic; they are a group of photosynthesizing organisms which

belong to different kingdoms; some are plants while some are not; some are micro-
scopic in nature while others are meters in length; and some are unicellular and
others multicellular. What they have in common is that they possess chlorophyll,
they photosynthesize, and however, they grow on water and do not have roots, stems
or leaves as terrestrial plants do (Cavalier-Smith 2007).
Although there are thousands of species of algae, they can be classified into
three main types: green, red and brown (Chlorophyta, Rhodophyta and Phaeophyta,
respectively) (Kadam et al. 2015; Bleakley and Hayes 2017). This classification is
mainly based on their domination pigments which give them the visible color. The
green algae include species such as Ulva lactuca, the red algae include species such
as Kappaphycus alvarezii, and the brown algae include species such as Laminaria
Japonica. Most brown and red algae are found in marine waters while green algae
can be found in freshwater. Some freshwater red algae exist albeit rare and brown
algae are almost exclusively found in marine water. Fucoxanthin is the pigment
responsible for the brown coloration of the phaeophyta, that of red algae are caused
by phycobilins while that of green algae is due to the presence of only chlorophyll.
The brown algae are all multicellular and could grow to tens of meters in length. The
red algae are also mostly multicellular while the green algae comprise unicellular
and multicellular species (Bleakley and Hayes 2017).
One of the direct uses of algae, particularly marine macroalgae, is as food for
humans and other animals (Fougere and Bernard 2019). Currently, algae have been
having both detrimental and potentially revamping effect to food security. Harmful
algal blooms threaten aquatic life, so does the potential for algae serving as a more
sustainable source of food (FAO, IFAD, UNICEF, WFP and WHO 2018).
Marine algae are usually found in the photic zone of the sea as they require
sufficient sunlight for photosynthesis. The importance of light for algae growth is
further emphasized in studies where in the absence of sufficient light, with surplus
nutrient supply, very little growth occurs even in the presence of abundant nutrients
(Prabhu et al. 2019). In the absence of light, the organisms are unable to convert
the nutrients for growth and metabolism. Light is therefore as important for aquatic
algae life as it is for terrestrial plants.
Algae can either be macroalgae or microalgae. There a thousands of species of
both microalgae and macroalgae. Microalgae are photosynthetic, mostly unicellu-
lar, microscopic organisms, while macroalgae also called seaweeds are multicellu-
lar, photosynthesizing organisms. Algae can either be heterotrophic, autotrophic or
mixotrophic. Autotrophic algae can fix inorganic CO2 from the atmosphere through
photosynthesis and produce storage carbohydrates while heterotrophic algae can
fix small organic molecules into lipids and proteins while mixotrophic algae can
do both. This makes algae potentially suitable for the production of both biodiesel
and bioethanol, since the lipids can be taking through transesterification and carbo-
hydrates can be saccharified and fermented. Algae have been explored for several
decades for potential commercial production of third-generation biofuel (Nigam and
Singh 2010)
Algae are of particular environmental and economic importance for several rea-
sons. They are able to accumulate carbohydrate at a much faster rate than land plants
due to much higher photosynthetic efficiency. They can grow in wastewater with
high nitrogen content unlike terrestrial plants, and they take up CO2 at a much faster
rate. Algae can also grow in a variety of environments from saline to fresh, from high
turbulence ocean water to slow flowing municipal wastewater (John et al. 2010).
Microalgae are more compliant to genetic engineering for the optimization of
desired output than higher plants being microscopic in nature. They are more sus-
ceptible to control of metabolic pathways and DNA alteration (Rosenberg et al.
2008). Microalgae make for good third-generation biofuel as they contain both lipid
and starch, for the production of biodiesel and bioethanol, respectively. Furthermore,
microalgae does not contain lignin and hemicellulose (Mahapatra and Ramachan-
dra 2013), which makes the process of extraction of the fermentable carbohydrates
less expensive and rigorous as these biopolymers are more complex to saccharify.
Like macroalgae. The microalgae also has the ability to remediate wastewater by
removing sulfur and nitrates (Lv et al. 2017). Microalgae can be found in fresh and
saltwater. Example of freshwater microalgae is the Chlorococcum sp.
Green, brown and red algae are distinguished from one another based on their
cellular structures and composition. While the cell walls of brown algae contain
alginates and fucoidan with laminarin as reserve photosynthetic pigment, red algae
contain agar and carrageenan as their cell wall polysaccharide alongside cellulose,
those of green contain ulvan in their cell wall and starch within their chloroplast.
Diatoms are eukaryotic microalgae that belong to the group of plankton and benthic
algae. Their skeleton is made of amorphous silica, and some diatoms are photosyn-
thetic while others are not. They can be found in both marine and freshwater (Chetia
et al. 2017).
Algae can either be sourced from natural stocks; some fishermen simply dive into
the sea to pick algae by hand. Algae can also be cultivated in open sea or aquaculture.
There are three main methods of cultivating algae as illustrated in Fig. 2.6. These are
broadcast, off button, and longline methods.
(a) Water Surface

Algae
Sea bottom
(b)
Water Surface
Hanging pole
String Algae
Sea bottom
(c)
Water Surface
String Hanging pole
Algae
Sea bottom
Fig. 2.6 An illustration of the three methods of cultivation of red algae. a Broadcast, b off-bottom
and c longline methods
In the broadcast method, algae seedlings are cultivated close to the bottom of the
water. This is usually in slow-moving shallow waters in ponds or lakes or artificial
water systems. In the off-bottom method, the algae seedlings grow on lines tied to
poles such that the algae extend about 20 cm from the bottom of the water, midway
in the shallow water depth. In the longline method, the algae seedlings grow closer
to the surface hanging from ropes attached to poles. Different algae species have
preferences for particular growth method.
2.3.3 Aquatic Animals
These include the vertebrates: amphibians, mammals, birds, fish and reptiles as
well as invertebrates such as crustaceans, mollusks, insects, squids, echinoderms.
Although some mammals, amphibians and birds spend some, most or all of their
time outside of water, they are classified as aquatic if they mostly depend on water
for their survival and feeding. Examples are polar bears who mainly feed on seals
and the kingfisher birds that feed on small fish from rivers. Some of the animals of
particular interests which are further discussed in other chapters are the frogs whose
skin serves as a source of some bioactive polymers and other aquatic animals with
special features which aid them in surviving in the deepest part of the ocean.
The decline or surge in the population of fish in aquatic environment has long been
used as an indication of the presence or absence of contaminants within such aquatic
environment. In industrial quality checks, the product safety for human consumption
is confirmed by testing on fish in controlled aquatic systems such as aquarium or
ponds (Christophe et al. 2015). The aquatic animals serve as a source of food and
livelihood for various life-forms including humans. There is need to maintain a bal-
ance between the population of different aquatic animals in order to ensure constant
supply of aquatic resources.
One of the aquatic invertebrates which recently pose a nuisance to the fisheries
industry is the jellyfish. In recent years, giant jellyfish population explosion in the
ocean has been reported (ref). The cause of this bloom has been attributed to the
decline in the population of the larger aquatic organisms which prey on these jellyfish
due to overfishing, eutrophication of coastal waters, global warming, changes to the
aquatic habitat and translocation (Dong 2019). The jellyfish bloom has resulted in a
decline in the population of smaller finfish as they are poisoned by these jellyfish. This
results in devastating loss to fishermen, aqua tourism industry and also destruction
of cooling systems of power plants located on the coast. Measures taken to address
this include the development of jellyfish bloom detection tools (Azmar et al. 2017),
consuming the jellyfish for food and use as feedstock in biopolymer industry as
reviewed in the subsequent chapter in this book.
2.3.4 Carnivorous Aquatic Plants
Whether on land or in the water, carnivorous plants are very rare. Globally, there are
over 700 species of carnivorous plants from over 5 orders and 10 genera (Lima et al.
2018). Examples of species of aquatic carnivorous plants are Aldrovanda vesiculosa,
Utricularia vulgaris, U. reflexa Oliver, U. stygia Thor and U. intermedia Hayne
(Adamec 2010). Two common aquatic carnivorous plants are the water wheels and
the pitcher plants. The evolution of plants to feed on insects is thought to originate
from the natural defense which plants have against fungi and bacteria (Schulze et al.
2012). Since fungi and insects have the chitin-based structure in common, similar
chitin degrading mechanism is involved in breaking down the chitinous shells of
insects. Carnivory in plants is thought to be an adaptation to the limitation of nutrients
in the growth medium. While to other plants, the roots serve as the main organ for
the uptake of nutrients. In carnivorous plants, the leaves have adapted a feature for
taking up nutrients from the trapped animal (mostly insects and larvae).
Some carnivorous plants adopt passive techniques for attracting prey, while oth-
ers adopt active techniques for attracting prey (Forterre et al. 2005). The passive
technique involves insect being lured toward a sticky surface where they feed on the
plant nectar; however, by the architecture of the plants they are unable to leave due
to the adhesive surface and are eventually entrapped within the hairs and tentacles
and are eventually digested by the plant enzymes (Schulze et al. 2012). In the active
capture system, the plant traps prey via a mechano-electrical sensor and trap system
where the movement of the prey in contact with a biological sensor, usually a hairlike
sensor, stimulates the closure of a trap which seals and crushes the insect. This is
then followed by digestion and the nutrients extracted from the insect. The enzymes
produced by the aquatic carnivorous plants are a form of aquatic biopolymers as well
as polymeric compounds which these enzymes act on.
2.3.5 Aquatic Insects
Insects form an important part of the aquatic system. Some depend on the aquatic
plants for food and in some cases shelter. They also depend on the aquatic environ-
ment for nursery where they lay their eggs and larvae. They in turn serve as food and
a source of nutrition for other aquatic animals and carnivorous plants. Aquatic insects
generally inhabit rocky shores, remain buried in sand or settle on aquatic plants and
algae (Merritt and Wallace 2009). They therefore require the aid of other structures
or aquatic life-form, in order to inhabit the aquatic environment. Examples of insect
which inhabit the aquatic environment include Gerridae, Diptera, Coleoptera and
Collembola (Merritt and Wallace 2009).
2.3.6 Aquatic Birds
Some flying and flightless birds inhabit the aquatic environment. These include those
which mostly stay afloat on water for most of the time and mainly depend on the
aquatic environment for food. They feed mainly on fish and other aquatic organisms.
Some are able to fly away from the water and however depend solely on fish for food.
Examples are the penguins, ducks, swans, geese and the kingfisher bird.
Some of the challenges faced by these birds include the release of toxins into the
aquatic environment. They face risk of toxins present in the water and in the organisms
they feed on from the polluted water. One of such is lead toxin, toxins from algal
blooms of blue-green cyanobacterium, oil spills, Clostridium botulinum, zinc and
lead from industrial waste. These toxins could lead to conditions such as hypochromic
anemia, pneumonia, secondary aspergillosis, neuromuscular malfunction and even
unexplained death while body is still in good condition (Backues 2015).
2.3.7 Aquatic Microorganisms
Microorganisms exist both on land and in water. They are as important to the aquatic
environment as the larger organisms. The microorganisms present in water include
bacteria, viruses, protozoa and fungi. They can be of various classification such as
filamentous, rod-shaped or helical, aerobic, anaerobic or facilitators, heterotrophs,
autotrophs or mixotrophs (Little et al. 2001). Some exist within other organisms
where they have a symbiotic relationship with the host organisms. Examples of
such are the microbes which produce light within bioluminescent aquatic organisms.
These are discussed in later chapters of this book under bioluminescent proteins.
Other examples of symbiotic aquatic bacteria include those in the internal organs
of fish and the digestive bacteria in the utricle and stolon of the carnivorous aquatic
plant Utricularia breviscapa (Lima et al. 2018). Such bacteria aid in the digestion of
food and organic matter.
Aquatic microorganisms can either drift with the current suspended in the water.
These are generally classified as plankton (Meaning vagabond in Greek). Other
microorganisms are attached to substrates or within sediments in water. These are
known as the benthos (Greek word meaning depth) (Callieri et al. 2019). Planktons
are generally stationary; however, some possess some features sufficient to enable
them to maintain buoyancy. This allows them to migrate and aggregate as the nutrient
content and conditions vary, generally to move toward regions with nutrients and
favorable living conditions. Planktons serve as a food source for various species
which in turn are also a source of food to other organisms.
Benthos depend on the organic matter settled on the substrate they are attached to.
These types of microorganisms form biofilms which are the polymeric extracellular
matrix of the aggregates of microorganisms. The biofilms formed by these microbes
are crucial to their survival as it serves as a barrier through which nutrients and other
compounds are exchanged with the immediate environment (Kostakioti et al. 2013).
The biofilm enables the microorganism to adhere to the substrate in water; it also
serves a protective purpose to these benthic microorganisms. Outside of the natural
aquatic ecosystem, biofilm-producing bacteria also form on wet surfaces in everyday
life such as bathrooms, labs and hospitals. Microbes present in the water could be true
aquatic microorganisms which only exist in water while others are introduced from
land. The introduction of land microorganisms into water could be from seepage
from land or deliberate or accidental introduction of fecal waste into the water.
2.3.8 Humans
Since water serves as a source of food, means of transportation, source of energy,

recreation and aesthetics, for a very long time in human history, people have settled
along aquatic ecosystem. There are also communities in different parts of the world
who have settled on water. An example is the Makoko community in Lagos, Nigeria,
which is made up of humans who have constructed floating wooden houses on water
and use canoe and other floating objects as the primary means of transportation
within the water community (Riise and Adeyemi 2015). Similarly, the Satoyama
community in the ancient city of Kyoto in Japan have for centuries established a
harmonious relationship with the aquatic life which promotes biodiversity and also
benefits human health. The humans living in these water-rich community cultivate
the aquatic resources such as fish, rice and aquatic plants in a sustainable manner
that benefits both humans and aquatic life. This harmonious relationship existing in
Satoyama community has recently been reported to be on the decline (Takeuchi et al.
2016). It is therefore important to develop a wider understanding of the importance
of the aquatic environment to human life.
Humans use water as a means to move people and goods across continents, coun-
tries, cities and communities over the oceans, seas, lakes and rivers. Such means
include ships, boats and submarines. Permanent structures such as bridges and under-
water tunnels are build to allow land-based transportation systems over water. Water
also serves for recreation and major sporting events such as kayaking and water ski-
ing. Deep-sea oil exploration, fishing and mining are some of the major activities
which take place in the aquatic environment. An example of human activity is shown
in Fig. 2.7, an image of men fishing and a motorized boat moving across the Lagos
Lagoon.
Beyond transportation on the surface of water, humans have explored the deepest
depth of the ocean and beyond. The location referred to as the Challenger Deep in
the Mariana Trench, a depth of 10,994 km below sea level (Kobayashi et al. 2012)
has been confirmed as the deepest part of the ocean. Although the conditions are
so extreme that no human has physically been at that depth, recently submersibles
equipped with high-resolution cameras have discovered life-forms that have adapted
to live in what was previously considered to be a barren depth in the ocean. This
discovery has been of benefit to humans in ways such as discovering sources of new
enzymes these organisms use to digest food and produce light in the dark depths of the
sea (Kobayashi et al. 2012; Altun et al. 2008). Such discoveries lead to development
Fig. 2.7 Fishing activity on the Lagoon in Lagos, Nigeria. August 2019. Photograph by Ololade
Olatunji
of new ways to produce better environmentally sustainable fuels and new compounds
for detecting and treating diseases such as cancers.
Living aquatic resources become available through two main routes: capture from
the waters or through fish farming, also referred to as aquaculture. Another form
which is a hybrid between these two methods is mariculture, a process where aquatic
organisms are cultivated on the sea within enclosed areas. Algae, shellfish and sea
cucumber are some of the organisms which are cultivated in mariculture. This is done
to obtain either whole biomass or parts of these organisms for use as food, medicine,
jewelry or other applications (Engle 2009). The mode of sourcing a particular aquatic
resource is important as it affects the logistics involved in sourcing the feedstock
for the production process. Some existing manufacturing plants which processing
aquatic waste and by-products into other products are located close to the sea or river
from where the aquatic resource is sourced or farmed for the reason of nearness to
raw material which serves as the feedstock for the production plant. Where there is
not such plants situated close by the transport and storage of the raw materials (e.g.
shells, scales, viscera and bones) further adds to the cost of production.
The population of aquatic organisms is significantly affected by the level of fishing
by humans. According to the FAO, an estimated 94.6 million tonnes of fish were
sourced through capture in 2014 (FAO 2016). This includes both marine and inland
waters. Aesthetically valued shells and pearls which are used as ornaments for art,
decorative, jewelry or other aesthetic application are also sourced by humans from
the aquatic environment (FAO 2018).
The human activities have played key roles in the decline of the aquatic ecosys-
tems. This includes overfishing, introduction of species to waters they would not
naturally exist in, discharge of polluted water into the waters, agriculture leading
to run off of fertilizers into the water, building of hydroelectricity dams, dredging
and diversion of the waters for activities such as irrigation, constructions near, on,
in water bodies and oil spillage. Pollution of the seas and oceans by dumping of
municipal and industrial waste has been shown to lead to diseases and illnesses such
as hepatitis, typhoid fever and gastroenteritis. These occur as a result of consuming
shellfish such as oysters and mussels which have fed on the contaminants (Kwaasi
2003).
Human activity in and around aquatic environment has also been responsible for
the eutrophication of lakes. This refers to the process whereby the nutrient level in
the water rises due to run off of nutrients from surrounding land resulting in the
increase in the population of aquatic plants which eventually leads to the death of
other aquatic life and depletion of the water. In nature, eutrophication occurs as a
slow process over long periods of time. At the natural pace, eutrophication does
not have such a pronounced negative impact on the environment. When this process
is accelerated, it distorts the balance in the ecosystem most often with devastating
effects.
The aquatic industry cuts across all three economic sectors: primary, secondary
and tertiary. Activities such as fishing, deep-sea mining and trawling belong to the
primary sector; the secondary sector comprises fish processing which includes activ-
ities such as commercial finfish canning and extraction of biopolymers from aquatic
wastes. The tertiary sector of the aquatic industry relates to activities such as boat
rental and aqua tourism. Biopolymer resources of the aquatic environment play
numerous roles in the life of humans; these range from polymers used in water
purification, those with medicinal applications to those used to produce biofuel.
These will be discussed in more detail in subsequent chapters of this book.
2.4 Abiotic Components of the Aquatic Ecosystem
The abiotic components of an ecosystem include the physical and chemical parts
such as the organic matter such as from the dead organisms, inorganic matter such
as the silica of diatoms and the calcium carbonate deposited by the shedding of zoo-
planktons and exoskeleton of crustaceans (Wilson 2013). The temperature, salinity,
pH, water flow rate, size of rocks and soil at the bottom of the water, minerals, light
intensity, dissolved oxygen and water level, all contribute to the abiotic factors.
2.5 Conclusion
In this chapter, we have taken an overview of the aquatic ecosystems, the living
and non-living components therein, their impact on the environment and economy.
The aquatic ecosystems serve as habitat to diverse life-forms and play a significant
role even to life on land. The aquatic environment also serves as a source of a
variety of natural resources, and of particular interest in this book are the polymer
resources of the aquatic environment. In the subsequent chapters, we shall look at the
biopolymers which exist within the aquatic environment, their sources, chemistry,
industrial processing and applications and the environmental significance of such.
References
Abd-Elbaky M, Jin S (2019) Hydrological mass variations in the Nile River Basin from GRACE
and hydrological models. Geodesy and geodynamics (in press)
Abdelmohsen K, Sultan M, Ahmes M, Save H, El Kaliouby B, Emil M, Yan E, Abotalib AZ,
Krishnamurthy RV, Abdelmalik K (2019) Response of deep aquifers to climate variability. Sci
Total Environ 677:530–544
Adamec L (2010) Mineral cost of carnivory in aquatic carnivorous plants. Flora 205:618–621
Altun T, Celi F, Danabas D (2008) Bioluminescence in aquatic organisms. J Ani Vet Adv. 7(7):841–
846
Awange JL, Saleem A, Sukhadiya RM, Ouma YO, Kexiang H (2019) Physical dynamics of Lake
Victoria over the past 34 years (1984–2018): is the lake dying? Sci Total Environ 658:199–218
Azmar F, Pujol M, Rizo R (2017) A swarm behaviour for jellyfish bloom detection. Ocean Eng
134:24–34
Backues KA (2015) Anseriformes. Fowler’s Zoo and wild animal medicine, 8, pp 116–126
References 29
Bleakley S, Hayes M (2017) Algal proteins: extraction, application and challenges concerning
production. Foods 6:33 (1–34)
Borisjuk N, Peterson AA, Lv J, Qu G, Luo Q, Shi L, Chen G, Kishchenko O, Zhuo Y, Shi J (2018)
Structural and biochemical properties of duckweed surface cuticle. Front Chem. https://doi.org/
10.3389/fchem.2018.00317
Braebdle R, Crowford RMM (1999) Plants as amphibians. Perspect Plant Ecol Evol System 2(1):56–
78
Callieri C, Eckert EM, Di Cesare A, Bertoni F (2019) Microbial communities. Encycl Ecol 1:126–
134
Casamitjana X, Mencio A, Quintana XD, Soler D, Comptee J, Martinoy M, Pascual J (2019)
Modeling the salinity fluctuations in salt marsh lagoons. J Hydrol 575:1178–1187
Cavalier-Smith T (2007) Evolution and relationships. In: Brodie J (ed) Unravelling the algae: the
past, present, and future of algal systematics. CRC Press, Boca Raton, pp 21
Chetia L, Kalita D, Ahmed GA (2017) Synthesis of Ag nanoparticles using diatom cells for ammonia
sensing. Sens Bio-Sens Res 16:55–61
Christophe M, Rachid A, Mario L (2015) Fish as a reference species in different water masses.
Aquat Ecotoxicol, 309–331
Dame RF (2008) Estuaries. In: Encyclopedia of ecology, pp 1407–1413
Deane F, Wilson C, Rowlings D, Webb J, Mitchell E, Hammam E, Sheppard E, Grace P (2018)
Sugarcane farming and the Great Barrier Reef: the role of a principal approach to change. Land
Use Policy 78:691–698
Dong Z (2019) Blooms of the moon jellyfish Aurelia: causes, consequences and control. World
seas: an environmental evaluation, 2nd edn., vol III, pp 163–171
Engle CR (2009) Mariculture, economic and social impacts. In: Encyclopedia of ocean sciences,
2nd edn., pp 545–551
FAO (1997) Irrigation potential in Africa: a basin approach. Food land and water bulletin 4. FAO
land and water development division. ISBN 02-5-103966-6
FAO (2016). The State of World Fisheries and Aquaculture 2016. Contributing to food security and
nutrition for all. Rome. pp 200. ISBN 978-92-5-109185-2
FAO (2018). The State of World Fisheries and Aquaculture (2018). Meeting the sustainable
development goals. Rome. Licence: CC BY-NC-SA 3.0 IGO ISBN 978-92-5-130562
FAO, IFAD, UNICEF, WFP, and WHO (2018) The state of food security and nutrition in the world.
Building climate resilience for food security and nutrition. Rome, FAO. Licence: CC BY-NC-SA
3.0 IGO. ISBN 978-92-5-130571-3
Forterre Y, Skotheim JM, Dumais J, Mahadevan L (2005) How the venus flytrap snaps. Nature
433:421–425
Fougere H, Bernard L (2019) Effects of diets supplemented with starch and corn oil, marine algae, or
hydrogenated palm oil on mammary lipogenic gene expression in cows and goats: A comparative
study. J Dairy Sci 102(1):768–779
Harris G (2008) Lagoons. In: Encyclopedia of ecology, 2nd edn., vol. 2, pp 539–545
Hitchcock JN, Mitrovic M (2019) Microplastic pollution in estuaries across a gradient of human
impact. Environ Pollut 247:457–466
Honjo S (2009) Biological pump and particle fluxes. Encyclopedia of ocean sciences, 2nd edn., pp
371–375
Iizumi T, Ramankutty N (2015) How do weather and climate influence cropping area and intensity?
Global Food Secur 4:46–50
John RP, Anisha GS, Nampoothiri KM, Pandey A (2010) Micro and macroalgal biomass: a
renewable source for bioethanol. Biores Technol 102:186–193
Kadam SU, Alvarez C, Tiwari BK, O’Donnell CP (2015) Extraction of Biomolecules from
seaweeds. In: Tiwari BK and Troy DJ (eds) Seaweed Sustainability. Academic press. pp 243–269
Kobayashi H, Hatada Y, Tsubouchi T, Nagahama T, Takami H (2012) The hadal amphipod Hiron-
dellea gigas possessing a unique cellulase for digesting wooden debris buried in the deepest
seafloor. PLoS One 7(8):e42727, 1–8
Kostakioti M, Hadjifrangiskou M, Hultgren SJ (2013) Bacteria biofilms: development, dispersal

and therapeutic strategies in the Dawn of the postantlantibiotic era. Cold Spring Harb Perspect
Med 3(4):a01306
Kwaasi AAA (2003) Microbiology: Classification of Microorganisms. In: Caballero B (ed)
Encyclopedia of food sciences and nutrition, 2nd edn. Academic Press, USA, pp 3877–3885
Lima FR, Ferreira AJ, Menezes G, Miranda VFO, Dourado MN, Araujo WL (2018) Cultivated
bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae)
from floodplains in Brazil. Braz J Microbiol 49:714–722
Linley TD, Gerringer ME, Yancey PH, Drazen JC, Weinstock CL, Jamieson AJ (2016) Deep sea
research part I: oceanographic research papers, 114:99–110
Little BJ, Ray RI, Pope RK (2001) Bioactive environments: corrosion. In: Encyclopedia of materials:
science and technology, 2nd edn., pp 533–537
Lonborg C, Calleja ML, Fabricius KE, Smith JN, Achterberg EP (2019) The great barrier reef: a
source of CO2 to the atmosphere. Mar Chem 210:24–33
Lv J, Guo J, Feng J, Liu G, Xie S (2017) Effect of sulfate ions on growth and pollutants removal of
self-flocculating microalga Chlorococcum sp. GD in synthetic municipal wastewater. Bioresour
Technol 234:289–296
Mahapatra DM, Ramachandra TV (2013) Algal biofuel: bountiful lipid from Chlorococcum sp.
proliferating in municipal wastewater. Curr Sci 105:47–55
Mahmood R, Jia S (2019) Observed and simulated hydro-climatic data for the Lake Chad basin,
Africa. Data in Brief, 25(1014043):11–15
Martins I, Goulart J, Martins E, Morales-Roman R, Marin S, Riou V, Colaco A, Bettencourt R
(2017) Aquat Toxicol, 40–49
Merritt RW, Wallace JB (2009) Aquatic habitats. In: Encyclopedia of insects, 2 edn., pp 38–48
Nigam PS, Singh A (2010) Production of liquid biofuels from renewable resources. Prog Energy
Combust Sci https://doi.org/10.1016/j.pecs.2010.01.003
North GB, Peterson CA (2005) Water flow in roots: Structural and regulatory features. Vasc Transp
Plants, pp 131–156
Oyedeji AA, Abowei JFN (2012) The classification, distribution, control and economic importance
of aquatic plants. Int J Fish Aquat Sci 1(2):118–128
Pendleton L, Guldberg OH, Albright R, Kaup A, Marshall P, Marshall N, Fletcher S, Haraldson
G, Hansson L (2019) The great barrier reef: vulnerabilities and solutions in the face of ocean
acidification. Reg Stud Mar Sci 31:100729
Penfound WT, Earle IT (1948) The biology of the water hyacinth. Ecol Monogr 18:447–472
Peters JA, Lodge DM (2009) Littoral zone. Encyclopedia of inland waters, pp 79–87
biorefinery. Algal Res. 37:215–227
Riise J, Adeyemi K (2015) Case study: Makoko floating school. Curr Opin Environ Sustain 13:58–60
Rosenberg JN, Oyler GA, Wilkinson L, Betenbaugh MJ (2008) A green light for engineered algae:
redirecting metabolism to fuel a biotechnology revolution. Biotechnology 19:430–436
Scherzer S, Sanggaard KW, Kreuzer I, Knudsen AD, Bemm F, Thogersen IB, Brautigam A, Thomsen
LR, Schliesky S, Dyrlund TF (2012) The protein composition of the digestive fluid from the venus
flytrap sheds light on prey digestion mechanisms. Mol Cell Proteomics 11:1306–1319
Staby A, Salvanes AGV (2019) Marine life: mesopelagic fish. Encyclopedia of ocean sciences, 3rd
edn., pp 283–289
Takeuchi K, Ichikawa K, Elmqvist T (2016) Satoyama landscape as social-ecological system:
historical changes and future perspective. Curr Opin Environ Sustain 19:30–39
Takir D, Howard K, Yabuta H, McAdam M, Hibbitts C, Emery J (2018) Linking water-rich asteroids
and meteorites: implications for asteroid space missions. In: Primitive meteorites and asteroids,
pp 371–408
Wilson B (2013) Benthic shelf and slope habitats. In: The biogeography of the Australian North
West shelf, pp 259–265
Chapter 3
Chitin
Abstract Chitin occurs in a variety of organisms in aquatic ecosystems. It is a poly-

mer of acetyl glucosamine monomer. It has wider commercial applications in its
deacetylated form as chitosan. Extraction process requires high-temperature treat-
ment under alkaline as well as acidic conditions, depending on the source. Chitin is
one of the most explored aquatic biopolymers with existing commercial applications
as well as a wide range of applications in research. This chapter covers extraction pro-
cesses from different sources, the chemistry, conventional and emerging applications,
availability of aquatic feedstock for chitin production as well as the environmental
and economic impact of chitin.
Keywords Chitin · Polymer · Chitosan · Crustaceans · Shrimps
3.1 Introduction
Chitin is the second most abundant polymer on earth (after cellulose). It is the most
abundant aquatic biopolymer and so far has proven itself to be the most ubiquitous
of all the biopolymers as it finds application in almost every industry. Chitin is one
of the polymers which have a relatively diverse source. It can be found in organisms
which are completely restricted to living in water such as shrimps as well as those
found in wetlands such as crabs, those in moist environment such as mushrooms
and even in insects. Biodegradability is a most desirable feature of polymers, and
there is an increasing demand for biodegradable polymers which will degrade into
non-toxic compounds once they have completed their service life. Chitin is one of
such polymers.
In this chapter, we present the chemistry of chitin, its occurrence in nature, role
within the environment and organisms from which they are sourced and the various
methods employed in the extraction and processing into other forms, particularly
its deacetylation into chitosan, the most commercially relevant form. The chapter
also explores the global demand for chitin and its numerous conventional, novel
and potential applications as well as the environmental impact of its production.
The chapter also identifies some of the companies and countries involved in chitin
production from different sources.

32 3 Chitin
3.2 Occurrence in Nature
Chitin is a naturally occurring amino polysaccharide. It is a structural feature in the

exoskeletons of arthropods, shells of mollusks and the cell walls of fungi, forming an
integral part of the outer shells of crustaceans such as shrimps, crabs and lobsters and
mollusks such as snails and slugs. Some research studies have reported the presence
of chitin in the scales of certain species of fish (Rumengan et al. 2017). However,
the yield of chitin from fish scale is much lower than other sources. Yield of about
20% has been reported for Nile tilapia (Boarin-Alcalde and Graciano-Fonseca 2016).
Figure 3.1 illustrates the presence of chitin in the cell wall of mushroom.
Chitin is a non-toxic biopolymer. Crustacean shells generally consist of 20–
30% chitin, 30–40% protein and 30–50% calcium carbonate and calcium phosphate
(Majekodunmi 2016). Much of the commercial production of chitin is from crabs
and shrimp shells as these are more available and more extensive research has been
carried out on the extraction of chitin from these sources.
As a lesser explored source, fish scales are a particularly attractive source of chitin
as the scales can be collected before the fish is sold or taken for further processing
unlike in crustaceans where in some cases such as lobsters and crabs, the shells are
served with the food and can only be collected after cooking and serving. How-
ever, not all fish scales contain chitin. Fishes which have been reported to contain
a detectable amount of chitin include parrotfish (Chlorurus sordidus), red snapper
(Lutjanus argentimaculatus) and Nile tilapia (Oreochromis nIloticus) (Rumengan
et al. 2017; Boarin-Alcalde and Graciano-Fonseca 2016). However, fish scales have
a much lower chitin content than other sources. In contrast, scales of other fish
have no chitin present in them. These scales are generally composed of collagen and
hydroxyapatite in an orthogonal plywood structure (Gil-Duran et al. 2016). Such fish
with no chitin present in their scales include Atlantic tarpon (Megalops atlanticus).
The composition of fish scales, the presence or absence of chitin, depends on the
requirement of the fish in adapting to its environment, and this varies for different
Beta -1,3/1,6 Water

soluble glucans
Alpha - 1,3 Alkali

soluble glucans
Chitin nanofibers
Beta - 1,3 Alkali

soluble glucans
Cell membrane
Fig. 3.1 Occurrence of chitin within the mushroom structure

3.2 Occurrence in Nature 33
species of fish. Similarly, the composition of chitin in other aquatic organisms varies
for the different species.
The major issue which is presently limiting a larger commercial exploitation of
the available chitosan resource is the environmental, technical and economics of
scale surrounding its production process. The chitin market is expected to be more
than triple in the next 9 years. The major drivers for this growth are expected to
be the numerous emerging applications of chitin derivatives, especially chitosan, in
the healthcare industry (Future Market Insights 2019). Researchers have presented
a plethora of ways to use chitin; however, the major challenge is the cost of chitin
production both financially and environmentally. There needs to be further research
into more economic yet environmentally friendly means of extracting chitin and
converting it into useful products, thus making the chitin industry an exciting one
in which new opportunities for researchers and businesses lie in the development of
new chitin-based products and discovering new sources of the second most abundant
polymer on the planet.
3.3 Chemistry of Chitin
3.3.1 Structure
The repeat unit of chitin is an acetylglucosamine structure linked by a beta 1-4

linkage between the monomer units. Chitin is a linear long-chain polymer with a
high molecular weight which varies largely depending on factors such as source,
extraction method and extraction conditions and from batch to batch. It is quite
similar to cellulose, except that one of the hydroxyl (–OH) groups of glucose as
seen in cellulose is replaced by an amine group (–NH2 ) (Kaya et al. 2017). Chitin is
rather unreactive in its native form. However in its derivative forms, it becomes more
reactive and much more useful. One of the most important reactions that is often
required of chitin is the deacetylation reaction. Chitin in its pure form is about 90%
acetylated, and when it is deacetylated to over 60%, it can be classified as chitosan
(Kalut 2008). Thus although deacetylation of chitin results in chitosan, chitin also
has a level of acetylation. A chitosan polymer chain that has up to 10,000 repeating
units will have at least 6000 of these being glucosamine while the other 4000 will be
acetyl glucosamine. The level of acetylation affects the properties of the chitosan. At a
degree of deacetylation of over 98%, we begin to have a chitosan with good solubility
and minimal aggregation. Figure 3.2 shows the structure of a chitin repeating unit.
34 3 Chitin
Fig. 3.2 Chitin structure showing the acetylated glucosamine unit
3.3.2 Solubility
For broader applicability, it is important for a polymer to be moldable in order to be

reformed into useful products such as films, sheets and other shapes. This is usually
achieved in polymers either by melting as in the case of thermoplastics or by dis-
solving in a suitable solvent. For most natural polymers, the latter is often the case.
The polymer needs to readily dissolve in a non-toxic, non-mutagenic and relatively
low-cost solvent to be suitable for industrial application. In the solution form, the
polymer can then be processed using any of the various polymer processing tech-
niques such as solvent casting, dip coating, spin coating and micromolding (Olatunji
and Olsson 2015).
Chitin is insoluble in all the common organic and inorganic solvents; however
in its deacetylated state as chitosan, it becomes soluble in acetic acid and in acidic
solution below a pH of 6.5 (Roy et al. 2017). Like other polymers, chitosan increases
the viscosity of the solvent when dissolved within it. For example when chitosan is
added to acetic acid, it forms a viscous sticky solution which gets more viscous as the
concentration of chitosan increases. This is a unique property of polymers in general,
and it is one of the simple tests used in the laboratory to determine the presence of chi-
tosan following extraction and deacetylation. Chitin has a stronger crystalline form
than chitosan and is relatively more hydrophobic. Upon deacetylation, the structure
becomes more hydrophilic due to the amino group becoming available to form hydro-
gen bond with solvents. Chitin will not dissolve in water, acidic, alkaline or other
organic solvents. It will however dissolve in rarer solvents such as dimethylacetamide
mixed with lithium chloride and hexafluoroisopropyl alcohol and hexafluoroacetone
(Agboh and Qin 1997; Rinaudo 2006). However, these solvents are hazardous and
not suitable for industrial applications.
3.3 Chemistry of Chitin 35
While the insolubility of chitin poses a difficulty in processing it into other prod-
ucts, this characteristic of chitin however comes in use during the extraction process.
Extraction of chitin from crustacean shells such as crabs involves dissolving the
collagenous component of the shell in a strong alkali followed by dissolving the
minerals (mostly calcium carbonate) in acid. What’s left behind is the only compo-
nent of these shells that does not dissolve in either acidic or alkali solvents, chitin.
Solubility of chitosan increases as the chain length decreases. Chitosan oligomers
are chitosan chain with short chain lengths. Degree of polymerization on chitosan
could be as low as 5. We could also have chitosan dimers and trimers. This shorter
chain allows for improved solubility which aids certain applications (Azuma et al.
2015).
The secondary and tertiary structures of polymers are dependent on the presence
of functional groups within the chain and the interactions between the functional
groups within the chain and surrounding chains. Chitin has within its chain acetyl,
hydroxyl and amino groups. Intermolecular bonding and intramolecular hydrogen
bonding between these groups cause chitin to fold in on itself and aggregate, making
it unready to form hydrogen bonds with the solvents, hence the insoluble nature of
chitin.
3.3.3 Isomers
Chitin exhibits polymorphisms in three different forms, α, β and γ. The α and β

forms are the more crystalline forms and have received more attention (Kalut 2008).
The chitin tertiary structure comprises of six strands of chitin polymer chain wound
together in a protein-like helical structure to form crystalline chitin microfibrils (Roy
et al. 2017). The α conformation is the most abundant in nature. In the α conformation,
the chitin polymer chains are arranged in alternating manner. In the β chitin, the
polymers are arranged in the same direction while in the γ conformation they are
randomly arranged.
3.3.4 Variation with Source
The distribution of the acetyl group within the chitin and chitosan structure is com-
pletely random. This poses a challenge in achieving a precise standard for solubility
and solution properties. Results from one research group may depend highly on the
batch being used. There is yet to be a standard correlation between parameters such
as degree of acetylation and species such that chitin derived from a particular species
would correlate with, for example, molecular weight, solubility and degree of acety-
lation. The solubility of chitin and chitosan is dependent on the primary, secondary,
tertiary and quaternary structure of the polymer; these are affected by the degree
of deacetylation which in turn varied from species to species and batch to batch.
36 3 Chitin
For example, the degree of deacetylation of mollusks is different from that extracted
from crustaceans. Chitin properties vary even within the same species. For example,
different types of mushrooms produce chitin with varying fiber length (Ifuku et al.
2011).
3.3.5 Deacetylation
This is where some or all of the acetyl (COOCH3 ) functional groups attached to the
N-acetylglucosamine units (2-acetamino-2 deoxy-β-D-glucopyranose) are replaced
with a hydrogen leaving behind an amine (–NH2 ) end group of glucosamine (2-
amino-2-deoxy-β-d-glucopyranose). This occurs when chitin is reacted with 40–
50% w/v of sodium hydroxide (Kalut 2008). The extent to which this occurs within
the polymer chain is referred to as the degree of deacetylation (DDA).
3.3.6 Molecular Weight
Chitin from the same source can vary significantly in properties depending on the
batch. For example, a study reported molecular weight of chitin extracted from
chemical method as 33,400 g/mol while that from lactic acid fermentation was
967,000 g/mol (Castro et al. 2018). Although through methods such as gel per-
meation chromatography, chitosan can be separated into different molecular weights
such that high and low molecular weight chitosans are available in the market. Chi-
tosan of high and low viscosity is also available since molecular weight is related to
viscosity.
The molecular weight of chitin plays a very important role in its applicability.
Molecular weight affects properties such as solubility, viscosity of the solution,
mechanical properties of the film or other products produced and its interaction
and reaction with other polymers and materials in either composite forms or reacted
forms and other forms. The higher the molecular weight of chitin and chitosan, the
lower the solubility. However, this ceases to apply at low molecular weight below
2.43 kDa when the short chain does not aid the formation of hydrogen bonding with
the solvent (Roy et al. 2017). Depolymerization of chitin to obtain lower molecular
weight oligomers can be achieved by acid or enzyme hydrolysis.
3.3.7 Depolymerization
Another important reaction of chitin is the depolymerization into monomers. Chitin

can be completely depolymerized into its monomeric form, acetyl glucosamine, or
deacetylated and depolymerized into glucosamine. Glucosamine has become another
3.3 Chemistry of Chitin 37
derivative of chitin which has gained demand over the years. Glucosamine is the
monomer sugar unit obtained from depolymerization of chitosan. It has the chemical
structure shown below (Hulsey 2018). Glucosamine and acetyl glucosamine have
been proven to have some bioactive properties such as anticancer and treatment
of osteoarthritis when administered intravenously or orally as a food supplement
(Azuma et al. 2015).
3.4 Availability of Raw Materials
The Food and Agriculture Organization of the United Nations reported global pro-
duction of fish, crustacean, mollusk and other aquatic animals (excluding aquatic
mammals and reptiles) of 171 million tonnes in 2016. The highest ever recorded
since 1961. This rise in aquatic food production and capture can be attributed to var-
ious factors both ecological and economic. There are increased global efforts toward
sustainable development of fisheries and aquaculture.
The consumption of fish-based food has also doubled in recent years. This increase
in consumption of aquatic sourced food also implies an increase in generation of
aquatic waste. These aquatic wastes are a rich source of biopolymers of much envi-
ronmental and economic significance. With the majority of fish being exported from
developing countries as at 2017, further value addition to aquatic resources would
significantly boost these economies as non-food products; for example, biopolymers
are sourced from the aquatic resources. On the other hand, high-value fish such as
lobsters and shrimps are being imported into developing countries as the middle-
class demand for higher-quality products as spending capacity increases. The shells
for chitin production are usually sourced from post-consumption as the aquatic food
such as lobsters is usually served or sold to the consumer with the shell in order to
preserve freshness. This implies an increase in the availability of the chitin resource
in these regions or at best a wider spread availability in multiple regions.
Up to 100 billion tonnes of chitin can be produced by a species of crustacean annu-
ally. The Prince Edward Island located in eastern Canada, for example, is reported to
produce around 15 million pounds in weight of shells. This includes around 4 mil-
lion pounds of lobster shells and 8 million pounds of crab shells. Taken that two-thirds
of the mass would be water and based on the reported composition of chitin in crus-
tacean shells, a quarter of this is estimated to be chitin. About 50% would be mostly
calcium carbonate, while about 25% would be protein. Additionally, a valuable dye
can also be extracted from the shells.
The main sources of chitin are from crabs and shrimp shell. Although research
output has shown availability from other sources, these are presently the sources
being most explored on a commercial scale. For chitin production for biopolymer to
be practical, sustainable and economically viable, there first needs to be a reliable
and sustainable source of the aquatic resources which serve as the source of the
feedstocks. Here, we review the availability of the main sources of chitin.
Shrimps, lobsters, crabs and gastropods are among the high-value captured
seafood species, and the rate of catch has been reported to have shown a record
38 3 Chitin
increase from 1998 to 2016. These high-value sea animals are valued between USD
3800 and USD 8800 per tonne. Reduction in shrimp production has been reported
in Thailand in recent years due to disease; however, many other countries reported
a rise in production of shrimp among other seafood by 2014. A steady catch of 3.5
million tonnes have been recorded for shrimp since 2012 (FAO 2016); this rose to a
record high in 2016 with capture of shrimp rising from just over 1 million tonnes in
1970 to 3.4 million tonnes in 2016. Vietnam, for example, exports an estimated USD
7.3 billion worth of aquatic products in 2016 most of which is catfish and shrimp.
The availability of these aquatic resources depends on a number of factors which
could be economic or ecological. For instance, increase in per capita income could
mean more demand for higher-value seafood, and ecological reasons could be reduc-
tion in particular population of sea animal or a particular aquatic disease causing
reduction in a particular population. The most abundant species of shrimp, the
Argentine red shrimp, has shown the record high in 2016 and an annual increase
of 22% since 2011. While a record high of Argentine red shrimp (Pleoticus muel-
leri) of 144,000 tonnes was recorded in 2015 and this species was classified as being
fished within biological limit presently, shrimp stock in the southern Indian Ocean is
being overexploited. China is presently the highest producer of crustaceans, marine
mollusks and aquaculture in general.
The availability status of shrimp varies from region to region; for instance in
the Gulf of Mexico, shrimp is maximally fished within sustainable limit while in
the Caribbean and Guianas and the Southwest Indian Ocean penaeid shrimps are
presently overfished. Deepwater shrimps are maximally sustainably fished, however
tending toward n = being overfished. Aquaculture has been on the rise in recent
years as a means to meet the demand for fish and make certain species of aquatic
food available in regions where they are otherwise scarce and to meet the high cost
of deep-sea fishing. The aquatic organisms commonly farmed using aquaculture are
salmon, shrimp and bivalves. Aquaculture contributes 7,862,000 tonnes to production
of crustaceans globally in 2018. In the same year, 17,139,000 tonnes of mollusks
were also produced through aquaculture. Although aquaculture has seen tremen-
dous growth in the past few years, captured species still contribute significantly to
availability of crustacean and mollusks. Captured shrimp, crab, lobsters and gas-
tropods recorded for 2016 were 3,400,000, 1,700,000, 315,000 and 170,000 tonnes,
respectively (FAO 2016).
Based on the available data, we can generally say that there is sufficient sustainable
supply of a variety of crustaceans and mollusks to produce chitin. For example if
an estimated two-thirds of the crustacean shells from aquaculture alone is water
and 40% of the dry mass comprises of chitin, then an estimate tonne of over 2
million tonnes of chitin annually from crustaceans sourced from aquaculture alone.
Other sources report a global production rate of chitin from natural source of up
to 100 billion tonnes annually (Elieh-Ali-Komi and Hamblin 2016). However, the
present production quantity of 10,000 tonnes reported in 2000 is quite far from
the potential production rate. Table 3.1 shows some yields of chitin obtained from
different studies.
3.4 Availability of Raw Materials 39
Table 3.1 Chitin yield reported from different sources

Chitin source Chitin yield Extraction method References
Shrimp shell 13.4% Ammonium-based ionic Tolesa et al. (2019)
liquids
Sea snail 21.65% Alkali and acid Mohan et al. (2019)
Crab shell 6.9% Fermentation Castro et al. (2018)
7.5% Alkali and acid Castro et al. (2018)
34.4% Fermentation Flores-Albino et al.
(2012)
Nile tilapia 20% Alkali and acid Boarin-Alcalde and
Graciano-Fonseca
(2016)
Beetle (Holotrichia 15% Alkali and acid Liu et al. (2012)
parallela)
Penicillium camemberti 18% Alkali and acid Aili et al. (2019)
Mushrooms
Stipes 7.4% Alkali and acid Hassainia et al. (2018)
Pileus 6.4%
Gills 5.9%
Other than their use for chitin production, the shells of crustaceans also find use
in agriculture for use as soil nutrient and organic fertilizers. While chitosan can
act as an adsorbent to remove impurities from water, the shells in crude form just
grounded and dried show superior adsorbent property compared to pure chitin. At a
concentration of 2.1 mg/mL, the non-treated shrimp shells are capable of removing
textile dyes from wastewater (Massimilian and Ludovico 2016). Therefore, the chitin
and chitosan manufacturers need to consider the competition from other applications
of the aquatic resource.
3.5 Extraction of Chitin
Chitin in nature always exists as a composite or in a complex embedded alongside

other compounds such as proteins, glucans, minerals and other compounds within the
organism. Therefore, obtaining pure chitin requires treatment stages to isolate it from
the other components; some of these could also be useful components for other appli-
cations. Extraction processes of chitin from the shells of crustaceans generally involve
the same process of demineralization, deproteinization and decolorization followed
by further separation and purification. Several concentrations and conditions have
been reported from different research groups.
A summary of the processes involved in the extraction of chitin is given in Fig. 3.3.
The exact process involved in achieving each stage varies based on biomass used
and method being employed.
40 3 Chitin
Shell
waste
Pretreatment Demineralization Deproteinization
Chitin
Drying
Purification
Deacetylation
Chitosan
Fig. 3.3 Processes in extraction of chitin
In the following sections, typical example processes are given for extraction of
chitin from crustacean shells: mushrooms, insects and fish scales. The difference in
extraction processes from one source to another is due to the variation in the structural
composition of the shell or other parts of the organisms from which chitin is to be
extracted. The yield from a particular source also varies with extraction methods and
the efficiency of the process. Chitin could be lost during separation and purification
processes.
3.5.1 Extraction from Crustacean Exoskeleton
Crustaceans are characterized by the presence of a hard shell forming an exoskeleton

which serves mainly structural roles and some role in calcium regulation in the
organism (Ahearn et al. 2004). These hard shells consist of chitin fibers within a
protein matrix calcified with calcium carbonate and calcium phosphate (Gadgey and
Bahekar 2017). These shells have sites for muscle attachments such that the wastes
from their processing may also contain some lipids and other proteins and compounds
such as carotenoids.
Extraction of chitin from exoskeletons of crustaceans generally involves three
main stages. One stage is removal of the protein in a process referred to as the depro-
teinization, and this is achieved by immersing the shells in alkali solution. Sodium
hydroxide is most commonly used for this. The next stage is then to remove the cal-
cium content which is mainly acid-soluble calcium carbonate. This is referred to as
the demineralization step. Since calcium carbonate and most minerals are soluble in
acidic solutions, the demineralization process is carried out by immersing the depro-
teinized residue in acid solution. While hydrochloric acid is most widely used, other
acids such as nitric acid (HNO3 ), sulfuric acid (H2 SO4 ), acetic acid (CH3 COOH) and
oxalic acid (HCOOH) can also be used for demineralization. The type of acid used
3.5 Extraction of Chitin 41
will determine the extraction time and required condition. The order of demineraliza-
tion and deproteinization can also be reversed according to the dominant component
in the shells. If the protein content is predominant, then the deproteinization can be
carried out before the demineralization; however if the mineral content is higher,
then the demineralization process can proceed the deproteinization for improved
efficiency of chitin extraction (Kalut 2008). The efficiency of the extraction process
is also affected by the level of contacting between the solid mass and the chemicals
being used for extraction. This can be improved by particle size reduction through
pulverization.
The quality of chitin is significantly affected by the type of acid used, temperature
and the pH. Isolation of the chitin from the dissolved minerals and proteins is achieved
by physical separation by either filtering or centrifugation. It is therefore important
that the chitin does not dissolve in the alkali or acid solution. Chitin is insoluble;
however, it becomes soluble when deacetylated into chitosan such that some of the
chitin may be lost during extraction, if not all. High temperature and high pH may
also result in the depolymerization and hydrolysis of chitin which diminishes the
physical properties, and this is undesirable. Care must therefore be taken to use the
right operating parameters that retain the chemical structure and integrity of the chitin
(Gadgey and Bahekar 2017).
3.5.2 Extraction from Mushrooms
Mushrooms are a species of fungus which form fleshy fruity bodies. They are used in
food and for medicinal applications. They grow in soil, on standing or fallen trees or in
their food source and comprise a stem and a cap with gills. Although not exclusively
aquatic in nature, mushrooms grow naturally near moisture-rich areas such as rain
forests (Ficket et al. 2017). Chitin is present in the cell wall of mushrooms where it
is embedded within the alkali-soluble beta-1,3 glucans. It plays a structural role in
cell wall of fungi.
The morphology of mushrooms varies from that of fish scales and crustacean
shells due to the fact that mushrooms contain glucans alongside proteins and chitin,
thus requiring a slightly different extraction method. The process begins with the
fresh mushrooms being crushed to reduce the particle sizes. This can be done in a
domestic blender or food processor. This is then followed by filtering and washing
in distilled water. The process of washing in water is the first extraction step which
removes the water-soluble parts of the mushroom cell wall, and these are the glucans
and the minerals. This also shows how some of the nutrients in the food are lost
during washing, although in this case it is desirable. The next step is treating with
2% w/v sodium hydroxide for 24 h at a temperature of 100 °C. This deproteinization
stage is similar to that used for crustaceans and fish scales. It is used here not just to
remove proteins but also to remove alkali-soluble glucans. The residue left behind
after separation is then repeatedly washed with distilled water until it is neutral.
What now remains after removal of the glucans and proteins is the residual minerals.
42 3 Chitin
These are removed by treatment with 2M hydrochloric acid for 48 h. The sample
which by now consists mainly of chitin is then further treated to remove the pigments
followed by further treatment with sodium hydroxide to remove residual proteins and
alkali-soluble glucans. Further processing could then follow to obtain chitin fibers
or powder.
This method of extraction of chitin from mushrooms has proven effective in five
different species of edible mushrooms (Ifuku et al. 2011). These include the com-
mon mushroom (Agaricus bisporus), king trumpet mushroom (Pleurotus eryngii),
maitake (Grifola frondosa), shiitake (Lentinula edodes) and buna-shimeji mushroom
(Hypsizygus marmoreus). This method yields between 1.3 and 3.5% chitin depend-
ing on the variety of mushrooms. Chitin production from edible mushrooms faces
the challenge of competing with food, especially in vegan diets where mushrooms
are used as a substitute for meat as a source of nutrients and desired meaty texture.
3.5.3 Extraction from Fish Scales
Relatively, fewer studies have gone into extraction of chitin from fish scales than
from crustacean shells. This is largely due to the lower yield of chitin in fish scales
compared to crustaceans and mollusks. Nonetheless, the extraction of chitin from
fish scales is well worth further exploring as the demand for more sources of chitin-
based biopolymers rises. Furthermore unlike crustaceans such as crabs and lobsters,
fish scales are always removed before the fish is served as food. Therefore, sourcing
of fish scales for chitin production requires more sustainable logistics. Increasing
worldwide farming of tilapia, a scaly fish would also mean increasing demand for
ways to utilize the fish scales generated as waste.
The process of chitin extraction from fish scales begins with separating the scales
from the fish. This is usually done at the point of sale at the markets or prior to
processing in fish factories or cooking at restaurants. The fish scales are then collected
by the processor and washed with water at room temperature to remove fins, skins
and other residues.
The main extraction process then commences at demineralization where the min-
erals present within the structure of the fish scale are dissolved in a 0.5M acid solu-
tion, usually hydrochloric acid. The ratio of dried scales to acid solution is generally
between 1:10 and 1:20 ratio of grams of dry fish scales to volume of acid solution in
ml. Continuous stirring is also applied to ensure even distribution as the demineral-
ization process progresses. A time period of 90 min to 2 h is allowed for this process.
This is then followed by washing to remove the acid filter and drying.
The next stage is then to remove the proteins, deproteinization. This is achieved
by treating the demineralized residue with 1% solution of sodium hydroxide at 50 °C
for 3 h under low stirring (~250 rpm). What remains at this point is mostly chitin with
some pigments and impurities (Boarin-Alcalde and Graciano-Fonseca 2016). Further
treatment is then carried out to remove the pigments and odor causing impurities.
This can be achieved with the addition of sodium hypochlorite or ethanol.
3.5.4 Extraction of Chitin from Insects
Although insects are not exclusively aquatic organisms, they can be found in aquatic
areas either surrounding stagnant water or releasing their lava on the water. Some
aquatic insects such as the dragonfly start life as aquatic larvae form for many years
before they move out of the water and fly. These tend to spend most of their life in
water, and their life as adults is relatively short (Berg 2009). Making use of the cuticle
of insects for chitin production is a potential solution to sustainable pest control or
simply making use of an easily cultivated abundant source of chitin.
Chitin extraction from insect is a relatively recent development. A few insect
species have been found to contain chitin; these include desert short-horned grasshop-
pers, green bugs, German cockroach, vespid wasp and yellow jacket wasp (Badawy
and Mohamed 2015). To extract chitin from insects, the first step is to kill the insects
by freezing or dead insects could be collected. This is then followed by deproteiniza-
tion with potassium hydroxide at 40 °C for 48 h. The deproteinized mass is then
washed repeatedly with distilled water until neutral. The next stage is then to remove
the mineral content using acid for this, and 5% acetic acid is used. The use of acetic
acid is favorable as it allows use of acid from a sustainable and non-fossil-based
source as acetic acid can be produced through fermentation. Dehydration is then
carried out with ethanol by series.
Other studies have extracted chitin from bumble bee (Majtan et al. 2007) and bee-
tles (Liu et al. 2012) using the conventional sodium hydroxide deproteinization and
hydrochloric acid demineralization. The structure of chitin in terms of demineraliza-
tion, crystallinity and molecular weight varies for different species of insects. Some
studies nonetheless report similarities between the chitins extracted from insects to
the commercial chitin (Liu et al. 2012). There is yet to be a defined taxonomic clas-
sification of chitin from various species; however, it is concluded in the different
studies that the chitin obtained from insect has similar characteristics to those from
aquatic sources.
3.5.5 Microbial Extraction
Certain microbes are able to metabolize the proteinous parts of the chitin sources,
while the acidic condition generated by the acids created by the microbes dissolves
the minerals leaving behind a solid crude chitin which can then be further processed
and purified to obtain chitin. The obtained chitin can then be further deacetylated
to obtain chitosan. The fermentation process achieves partial deproteinization and
demineralization. This is then followed by further chemical treatment to obtain purer
chitin. Although some acids and alkali are still used in this process, it is at a much
lower concentration since most of the demineralization and deproteinization are
achieved during the fermentation process. Up to 99.6% and 95.3% of deproteinization
and demineralization have been achieved, respectively, from lactic acid fermentation
44 3 Chitin
of crab shells to obtain chitin for example (Castro et al. 2018). The fermentation
process therefore reduces the environmental impact of chemical chitin extraction.
The process could take up to 80 h as reported by different research studies (Cira
et al. 2002; Castro et al. 2018). Another advantage of the fermentation process is the
ability to recover the proteins for use as animal feed. Unlike the chemical process
where the deproteinization is done with a high concentration of alkali, the protein
extracted cannot be recovered and is usually discarded hence generating more waste.
Lactic acid bacteria (Lactobacillus sp.) fermentation of crustacean waste, for
example, involves the inclusion of a carbon source and in some cases mild acid such
as acetic acid to provide the right pH for the growth of the bacteria in the lag phase.
The lactic acid bacteria ferment the carbon source which could be whey, sugarcane
or other as well as the carbon present on the biowaste. Lactic acid is released as
a by-product of the fermentation process. The lactic acid reacts with the calcium
carbonate producing calcium lactate which precipitates and can then be separated by
washing. Proteolytic enzymes are produced from either the gut bacteria present on
the shrimp or strains added to the fermenter or the biowaste. These break down the
protein, leaving behind crude chitin. The low pH caused by the presence of lactic acid
and other by-products of the fermentation such as acetone also prevent the growth
of spoilage bacteria.
Lactococcus lactis, Terendinobacter turnarae, Lactobacillus plantarum, Lacto-
bacillus pentosus and Lactobacillus salivarius are some examples of bacteria that
have been used in lactic acid fermentation of shell waste to produce chitin. Other
than the limited rate of demineralization and deproteinization achieved in chitin pro-
duced by fermentation, another concern is the microbial contamination of the chitin
produced (Gortari and Hours 2013). Any possible contamination makes this form of
chitin not suitable for human or animal consumption. For this reason, despite envi-
ronmental and economic advantage posed by microbial extraction of chitin, many
of the applications of this method of chitin extraction have been limited to research
and laboratory experiments.
Other strains of organisms have been explored for production of chitin by fer-
mentation. Filamentous fungi have also been used in the biological production of
chitin (Gortari and Hours 2013). The proteolytic enzymes are released by the fungi
which results in the deproteinization and demineralization of the shrimp shells. The
consequent release of amino acids in the process of deproteinization acts as the nitro-
gen source which the fungi require for growth and multiplication. This results in the
lowering of the pH of the system, thus aiding the demineralization of the shells.
3.5.6 Enzyme Extraction
Enzyme extraction involves the deproteinization of the shell waste through the action
of proteolytic enzymes. This has the advantage of not including microbes, thereby
minimizing the risk of microbial contamination of the product. However, the pro-
teolytic enzymes only act to hydrolyze the protein; therefore, a pretreatment stage
is required to chemically demineralize the shell waste to get rid of the calcium
carbonate.
These enzymes are commercially available and can be isolated from a variety
of organisms. Examples of such enzymes include Alcalase, Pancreatin, Delvolase,
Cytolase, Econase, Maxazime and Cellupulin. Enzymatic deproteinization can
achieve between 54 and 97% protein removal from shell waste and minimizes the
need for alkali deproteinization (Gortari and Hours 2013).
While enzyme and lactic acid fermentation-based extractions of chitin from shell
waste show potential for more environmental and economical alternative to chemical-
based extraction using acid and alkali, more research needs to be carried out toward
a safe, eco-friendly and reproducible method for commercial extraction of chitin of
high quality.
3.6 Environmental Implications
In order to assess the environmental impact of chitin and chitosan production, here
we make use of the life cycle assessment report by Munoz et al. (2018). The life cycle
assessment was carried out using data from two chitosan production companies: one
of which is Mahtani Chitosan and the other an anonymous European company herein
referred to as Company X. Table 3.2 summarizes typical consumption in the process
of extraction of chitin from shrimp shells.
Mahtani Chitosan produces general-purpose chitosan, while Company X produces
chitosan for medical use. They both use the chemical extraction method; however, a
number of differences in their operations exist. (1) Mahtani Chitosan is close to the
source of the starting material (shrimp shell) while Company X outsources its chitin
production to China where it ships the dried crab shells from Canada to china. The
chitin produced is then shipped to Europe where the chitosan production takes place.
Table 3.2 Typical

Consumption Quantity
consumptions in chitin
extraction from shrimp shells Water consumption 167 L/kg chitin
Sodium hydroxide 1.3 kg/kg chitin
5.18 kg/kg chitosan
Energy consumption 1.4 L diesel per tonne of
(transportation and shrimp
electricity) 0.02 L per kg of chitin
1.3 kWh per kg chitin
CO2 emission 0.7 kg/kg chitin
Shell waste/resource 33 kg shrimp shell/kg chitin
utilization
Solid waste generated 1.5 kg calcium salts/kg chitin
4 kg protein/kg chitin
46 3 Chitin
(2) Mahtani Chitosan uses shrimp shell, while Company X uses crab shells as feed
material. (3) Mahtani Chitosan uses its protein sludge by-product as fertilizer, while
Company X uses theirs as animal feed.
However, it should be considered that the information presented here is based on
data from the company’s operation and therefore may vary for other companies as
such is by no means exhaustive. The goal here is to give an overview of the resource
requirements and environmental impact of the chitin production process.
3.6.1 Resource Utilization
The amount of raw material required depends on the type of feedstock being used
and the efficiency of the process. Crab and shrimp are most commonly used for
commercial chitin production due to their high chitin content. Table 3.1 shows chitin
yield/content for different sources of chitin based on reports from the literature.
Company X reports using 10 kg dry crab shell per kg of chitin produced, while
Mahtani Chitosan reportedly uses 33 kg of shrimp shells per kg of chitin produced.
Use of the waste aquatic materials for chitin and chitosan production diverts the
use of these materials as animal feed and as organic fertilizers, the main alternative
uses of these materials. The direct environmental impact therefore lies in the pressure;
this diversion puts on other resources used for animal feed and fertilizers.
3.6.2 Water Consumption
A lot of water is used in the process of washing the shells, washing after deminer-
alization and deproteinization to get rid of the acids and alkali used to return the
solids to neutral. The chemical process generates more wastewater for this reason.
The water is either treated and recycled in-house or sent to external water treatment.
An alternative to the use of shell waste in the production of chitosan is to use
the shell waste as animal feed. Comparing the quantity of water used in chitosan
production to that use in preparation for crab shells as animal feed, less water is
actually consumed in the production of chitosan compared to using the crab shells
for animal feed. Further consideration must be made to the fact that the diversion of
the shell waste from animal feed to chitin production means increased demand for
other sources of animal feed such as soybeans and barley.
Mahtani Chitosan reportedly uses 167 L of freshwater per kg of chitin it produces
from shrimp shell. A further 250 L of water is then used for each kg of chitosan
produced per 1.4 kg of chitin. Company X in Europe reportedly uses 300 L of water
per Kg of chitin produced from crab shells. The data for water consumed during
chitosan production from chitin by Company X was not provided for confidentiality
reasons. At Mahtani Chitosan, the wastewater from the production process is treated
3.6 Environmental Implications 47
on site. This involves neutralization, settling, biological treatment followed by sand

filtration before it is then released into the sea.
3.6.3 Solid Waste/By-Products
Protein extracted is recycled and reused as fertilizers or animal feed. The calcium
salts produced are used either deposited in landfill sites or spread on the roads as
road-filling material. The manner in which the by-products are processed, discarded
or reused depends on the regulations in the region of operation and company choice.
This will therefore vary from company to company. For every kg of chitin produced,
1.5 kg of calcium salt and 4 kg of protein are produced. The European company is
estimated to produce 2.84 kg protein per kg of chitin produced. These solid wastes
generated from the extraction of chitin find application as fertilizers and animal feed
in the case of the protein and as road fillers in the case of the calcium salt. Shrimp
shells are also often used as animal feed, and the diversion of shrimp shells for chitin
production which in turn results in additional pressure on other animal feed sources
such as barley and soybeans is partly compensated for by the use of the protein from
the extraction as animal feed. Similarly, crab shells are often sent to composting sites
where they are used as organic fertilizers. The use of the protein sludge as fertilizers
also partly compensates for the crab shells diverted from agricultural use to chitin
production.
3.6.4 Sodium Hydroxide
From the chemical-based extraction processes discussed, it is obvious that there is

a relatively large amount of acids and alkalis used from deproteinization to dem-
ineralization and even much more sodium hydroxide (up to 50%) is required for the
deacetylation of chitin to its more industrially relevant form, chitosan. This has a
lot of environmental impact, and the cost of the chemicals and the cost of disposing
them need to also be considered.
A life cycle assessment of sodium hydroxide which evaluated the environmental
impact of sodium hydroxide from the production to its use and disposal shows that for
every kg of sodium hydroxide used, 3.5 MJ of fossil energy is consumed, 0.6329 kg
of carbon dioxide is emitted which contributes to global warming, 1.298 g of 1,4
dichlorobenzene equivalents are released into the aquatic environment, 0.706 of sul-
fur dioxide equivalents are released into the atmosphere which further contributes to
acid rain and 0.4927 g of carcinogens are released to human exposure (Thannimalay
et al. 2013).
Mahtani Chitosan reports 1.3 kg sodium hydroxide used per kg of chitin and a
further 5.18 kg used per kg of chitosan produced from the chitin. Company X reports
48 3 Chitin
8 kg of 4% vol of sodium hydroxide used per kg of chitin produced; however, no

data was provided for the amount used for chitosan production.
Use of proteolytic enzymes in the deproteinization process is an alternative; how-
ever, much of this is still in the research stage and is yet to be commercialized. The
use of microbial extraction could potentially eliminate the need for sodium hydroxide
in the production of chitin. However, the higher concentration is used in the deacety-
lation process for production of chitosan. An alternative or accompanying process
could be enzymatic deacetylation using the enzyme deacetylase. This is presently
under research, and the mechanism of action and specificity of this enzyme is still
under study. Biotechnology-based deacetylation of chitosan would be a great leap in
a truly green process for chitosan production.
3.6.5 Acid Usage and Disposal
In the chemical process, the acid is mainly used in the demineralization stage. Accord-
ing to data produced by Mahtani Chitosan company (Munoz et al. 2018), to produce
1 kg of chitin 8 kg of hydrochloric acid at 32% concentration is used. In the lactic acid
fermentation process, lactic acid is produced by the bacteria and poses less environ-
ment adverse effect. Since the acid unlike the alkali actually reacts with the calcium
carbonate, it is not recovered, and it is converted into calcium chloride, water and
carbon dioxide, the products of the reaction between hydrochloric acid and calcium
carbonate. The impact of the hydrochloric acid used is therefore the carbon dioxide
emission resulting from the process.
3.6.6 CO2 Emission
In the demineralization stage, carbon dioxide is emitted as the calcium carbonate

reacts with hydrochloric acid to form calcium chloride water and carbon dioxide as
shown in Eq. (3.1).
2HCl + CaCO3 → CaCl2 + H2 O + CO2 (3.1)
CO2 emitted from organic degradation of the shrimp and crab shell waste is rated
as zero, while CO2 emission from clearing of land for building of chitosan factory
and the increased pressure on clearing of land for growing of plant-based animal
feed such as soybean and barley is rated as GWP-100 (global warming potential) of
1. This is because with organic degradation the carbon being emitted is that which
was removed recently during formation to the shells, while that from clearing of
long-standing plants at a much larger scale had more impact on CO2 emission.
Approximately, 0.7 kg of CO2 is released per 1 kg of chitin produced during the
acid mineralization process. This means the production process is resulting in CO2
emission of 70% of its own weight. Further CO2 emission occurs as a result of the
mineralization of the protein when it is used as fertilizer. Other emissions from this
process include ammonia, dinitrogen monoxide and nitrogen oxides.
For chitin production from crab shells, the CO2 emission during the acid deminer-
alization was estimated at 0.9 kg/kg of chitin produced. For both sources assessed,
crabs and shrimp, the chitin production process had more impact on acidification of
the environment and climate change than any other process in the supply chain from
transportation to chitosan production. However in terms of water use, the shrimp
shell supply had more impact on water use than chitin or chitosan production while
in crab shell supply chain, chitin production had more impact on water use. Likewise
in crab shell production, the chitosan production led to more toxins being released
into the environment while in shrimp shell production chitin production and chitosan
production had similar level of impact on Fr. Ecotox.
3.6.7 Energy and Electricity
The energy consumption starts from the fuel used in the transportation of raw mate-
rials to the factory. For Mahtani Chitosan, a reported 1.4 L of diesel is consumed
per tonne of shrimp transported using a tractor. A further 0.02 L of diesel is used
up per kg of chitin produced in bulldozer operations. The process is also reported
to consume 1.3 KWh of electricity per kg of chitin produced. Conversion of chitin
to chitosan through deacetylation consumes a further 1.06 KWh of electricity and
31 MJ of burning wood as fuel per kg of chitosan produced from shrimp shell.
For chitin production from crab waste, Company x reports an electricity consump-
tion of 1.2 KWh and 6 kg of coal fuel for heating per kg of chitin produced. The
chitin is then transported to Europe from China, an estimated distance of 22,874 km
by sea, adding to the energy consumption.
In developing a greener supply chain for chitin and chitosan production, trans-
portation systems such as bicycles could be considered to transport the aquatic waste
serving as the raw materials from the point of generation to the factory. Such is
being adopted in some recycle models in countries like Nigeria and India where
bicycles, rickshaws and pushcarts are used to transport used plastics from homes and
businesses where they are generated to recycle factories.
Therefore although chitin is a biopolymer, the process of extraction may not nec-
essarily be biologically friendly. For this reason, researchers have explored green
chemistry for production processes which require the use of less resources and less
harmful chemicals in the production of chitin, chitosan and the chemicals used for
extraction. Other alternatives for extraction includes the use of enzymes and fermen-
tation by microbes. Although these have been explored by researchers, the chem-
ical process is still preferred in commercial production due to the high cost of the
biological extraction methods.
Chitin in its crude form without isolating it from the minerals and collagen has
also shown potential in applications such as agriculture and pharmaceuticals. Other
50 3 Chitin
than the chemicals used in chitin production, the process of removing chitin from the
environment should also be considered. Chitin deposited by molting arthropods, dead
diatoms, insects and mushrooms is otherwise used up by microorganisms including
bacteria and fungi for metabolism. Removal of these chitinous materials makes them
unavailable to these microorganisms within the aquatic environment. This is likely
to impact on the biodiversity of microbes and other organisms within the environ-
ment, and this would have an effect on the ecosystem. The ecosystem involves every
organism playing a role in the carbon and nitrogen cycle as well as the food chain.
The reduction or increase in one population will affect that of another and the ecosys-
tem as a whole. We must therefore weigh the benefits of manufacturing chitin and
chitin-based products against the environmental cost of removing them from their
originating environment.
From the environmental impacts outlined thus far, it becomes apparent that the
energy and resources consumed and emissions from chitin and chitosan production
vary significantly from company to company. Other than the source of raw material,
other factors such as transportation mode, distance, choice of fuel and method of
extraction have an effect on the impact the production of chitin will have on the
environment. Here, we have compared two processes from two sources (shrimp and
crab), from two different countries.
3.7 Applications
The demand for biodegradable polymers lies chiefly in their demand for using alter-
natives to non-biodegradable, petroleum/fossil fuel-based plastics. Despite the rise
in the production of natural derived biodegradable plastics, presently of the 300 mil-
lion tonnes of plastic being produced annually, only about 1% are from natural source
(Brostow and Datashvili 2016).
Presently at 80.413 billion USD and predicted to rise to 105,549 billion USD by
2027, the global demand for chitin-based product is on the rise. The largest demand
for chitin derivatives is for chitosan. Glucosamine gained the majority of the market
share for chitin derivatives, while chitosan came second. Up to 64.9% of chitin was
converted to glucosamine (Future Market Insights 2019).
The global chitin market is as of 2017 is worth 900 million USD with Asia Pacific
being the largest producing nations (excluding Japan), North America the second
largest producers and Japan being the third largest. There is also chitin production in
Europe, Latin America, Middle East and Africa, albeit to a lesser extent. The chitin
market is projected to be worth 2.9 billion USD by the year 2027 (Future Market
Insights 2019). Most of the chitin produced is converted to chitosan and glucosamine
and other derivatives. The demand for chitin and chitin derivatives lies in health care,
wastewater treatment and agrochemicals with less although significant value in the
food and beverages, cosmetics and toiletries and other applications (Future Market
Insights 2019).
3.7 Applications 51
Chitin is mainly useful for conversion into other products, namely chitosan, glu-
cosamine and oligosaccharides. Some research studies have been reported in its use
as nanofillers; however, its application is largely based on the deacetylated and/or
depolymerized forms. Figure 3.3 summarizes the process from chitin to its different
derivatives.
With a world population projection of 9.7 billion by the year 2050 and presently
over 7 billion (FAO 2016) and rising, every one of these billions of people demanding
clean water, processed foods, pharmaceutical products and other daily requirement
of modern-day living standards, there is a huge demand waiting for the chitin industry
to meet as an alternative to the non-biodegradable, fossil fuel-based polymers which
are presently being employed to meet such demands. This is in line with the present
challenges of rising cost of fossil fuels from which these plastics are based, the
issue of pollution disaster as a result of dumping of plastics in aquatic and terrestrial
habitats and the financial distress being experienced globally. There is an urgent need
for an alternative versatile polymer which would replace the present ones.
In the present state of technology, the chitin-based products of, for example, food
packaging do not meet the same requirements in terms of applicability compared
to the conventional counterparts. This has limited the growth of such products. It
is therefore important to educate the consumers on the true value in biodegradable
products compared to the non-biodegradable and fossil-based options.
Presently, the highest demand for chitin lies in the wastewater treatment. Generally
due to the batch-to-batch variation in the quality and properties of chitin, large-scale
production is more suitable to low-end applications such as wastewater treatment.
For higher-end applications such as pharmaceutical or biomedical applications, each
batch needs to be tested; therefore, these are more difficult to scale up.
Chitin is the leading material toward a sustainable industrialized world. As the
world’s attention is drawn to the environmental concerns raised by petroleum-derived
polymers, there is rising demand for non-toxic, biodegradable, biocompatible, afford-
able, accessible and multifunctional alternatives. Chitin is the first polymer of choice
with potential to meet that demand. Figure 3.4 summarizes the value chain of chitin
from the raw materials to the final product.
3.7.1 Packaging Films
So far, chitosan has proven to be the natural biopolymer with the best film-forming
property. On its own, chitosan forms a uniform film when cast. The figure below
shows a film of chitosan. The chitosan used here is chitosan derived from chitin
extracted from crab shells and dissolved in 1% acetic acid solution. Pure chitosan
dissolved in acetic acid or hydrochloric acid can be formed into films for applications
such as edible food packaging. Aside from forming a barrier layer, it serves as an
antimicrobial and antioxidant and helps preserve the quality of the food product.
Chitosan is applied in the packaging of meat, fish, vegetables and fish and has been
shown to preserve the quality of these foods (Kanatt et al. 2013).
52 3 Chitin
Shell waste generators
Chitin producers Waste Other shell

processors/ waste
agents applications
Chitin Chitin based

distributors product
and manufacturers
suppliers
Fig. 3.4 Illustration of the value chain of chitin production
Combining chitosan with other polymers significantly improves its applicability

as a packaging material. A recent example of this is the clear plastic film of chitosan
combined with other biodegradable polymers, cellulose and polylactic acid (PLA).
A synergistic effect can be achieved between two polymers, in this case chitosan (a
main chitin derivative) and cellulose (the most abundant natural polymer on earth).
A multilayer film made of chitosan nanofibers and cellulose nanofibers coated on
polylactic acid achieved a film with up to 73% lower oxygen permeability than when
the film is made with PLA only. This is also a significant improvement to films made
up of chitosan or cellulose alone (Satam et al. 2018).
While PLA is a naturally derived polymer sourced from lactic acid obtained
from milk or bacteria, it is non-toxic, biodegradable and biocompatible; however
on its own, it has poor film-forming properties. On the other hand, chitosan forms
excellent films; however, it has much higher oxygen and water vapor permeabil-
ity, and these are not desirable for applications such as food packaging where the
food or drug or electrical component needs to be dry and airtight at least over the
duration of storage. Cellulose, although hydrophobic, has poor film-forming prop-
erties but forms anionic nanofibers with good mechanical strength. By combining
the cationic chitosan nanofibers and anionic cellulose nanofibers and spraying layer
by layer unto polylactic acid films, a composite film with low oxygen permeability
and moderate mechanical property is formed. Alternating three layers of chitosan–
cellulose–chitosan nanofiber layers on a PLA layer produced films with the least
oxygen permeability and haze resulting in transparent packaging films with excel-
lent physicochemical properties that rival those of petroleum-sourced transparent
films. Other methods of creating chitosan composite films include solvent casting
and spin coating.
3.7 Applications 53
Transparent films are in high demand in the packaging industry. From edible food
packaging to transparent films used as screen protectors, there is huge demand for
such films. Presently, petroleum-based polymers such as polyethylene offer low cost,
easily mass produced and conveniently sourced option. For chitin to compete with
this, there needs to be a well-established low-cost production process since chitin
already meets the other application demands. Off all the biopolymers available, chi-
tosan and other chitin derivatives demonstrate the most desirable properties suitable
for production of transparent packaging films.
3.7.2 Water Treatment
It can be said that one of the best explored applications of chitosan so far is in water
treatment. Industrially produced chitosan is used in wastewater treatment for removal
of metals and dyes. It also acts as a flocculant and coagulant and in immobilization
of microorganisms. This multiple role makes it very useful in wastewater treatment.
Chitosan has the ability to remove heavy metals, lipids and overall decrease in tur-
bidity of water. Applicability of chitosan in water treatment can be attributed to its
antimicrobial properties, metal chelation and adsorption properties. The antimicro-
bial property is further discussed in another subsection. The absorption process is
widely used in water treatment and is well explored. Chitosan is a commonly used
adsorbent alongside others such as cellulose and guarana.
The amine group on the deacetylated chitin plays the role of binding to metals,
and this results in the formation of a chitosan–metal complex with multiple metal
molecules binding to one chitosan molecule (Shahidi et al. 1999). The amine group
also has high capability to bind with minerals, lipids and proteins which gives chitosan
its versatility compared to chitin. Since this metal-chelating property is dependent
on the availability of the amine group on the polymer for bonding, the higher the
degree of deacetylation, the better the chitosan polymer’s metal-chelating property.
It is important that a water treatment agent is low cost, non-toxic and biodegradable
and can be used at low quantities. Chitosan at a concentration of 0.2 g in 100 ml
of water can reduce total dissolved solid content by up to 12.87%. Chitosan also
achieved significant reduction in turbidity and conductivity and overall makes water
suitable for drinking at relatively low concentration required (Al-Manhel et al. 2018).
3.7.3 Antimicrobial
A full understanding of the mechanism by which chitosan inhibits bacterial cell is

still pending. It is believed that it does this by means of blocking the surface of the
bacterial cell by chemically binding to the lipids present on the surface. This prevents
the bacteria from taking up nutrients necessary for growth and survival and eventually
leads to cell death or inhibition. This is made possible by the reactive binding of the
54 3 Chitin
deacetylated part of the chitosan polymer chain where the amino (–NH2 ) group which
has been freed of the acetyl group can react with the negatively charged surface of the
bacteria cell. Another mechanism proposed is that the chelating property of chitosan
actually results in its antimicrobial properties. It is thought that the chitosan binds
with the trace metals on the bacteria cell and results in the production of toxins which
leads to cell death. The antimicrobial activity of chitosan could be further improved
by nanosizing. Nanoparticles of chitosan result in higher surface area of the polymer
available to bind with the microbes, hence increased effectiveness (Divya et al. 2017).
The antimicrobial activity of chitosan is dependent on the molecular weight of the
chitosan. At a sufficiently low molecular weight, chitosan is able to penetrate into
the bacterial cell wall and disrupt DNA and RNA replication which results in their
inhibition (Varum et al. 2017).
Resistance of microorganism is of huge clinical and industrial significance as
microorganisms have shown resistance to almost every antimicrobial agent that
exists. Chitosan has recently been of particular interest as there is yet to be reported
bacterial resistance to it. Great potential therefore lies in the use of chitosan in various
antimicrobial products. Antimicrobial activity of chitosan extends to a broad range of
microneedles which includes bacteria, filamentous fungi, yeast and even virus: bac-
teria such as Escherichia coli (E. coli), salmonella and staphylococcus, fungi such as
Aspergillus niger, Fusarium solani and Candida albicans and viruses such as H1N1
Influenza A and the human cytomegalovirus (9HMCV) strain AD169 (Divya et al.
2017). Chitosan acts against both gram-positive and gram-negative bacteria.
For these antimicrobial properties, chitosan has found application in, for exam-
ple, antimicrobial food packaging, anti-acne cosmetic formulations, water treatment
and antimicrobial film in wound healing. Because chitosan has such diverse char-
acteristics, in a single application more than two or more of these properties can be
implemented; for example, in its use as antimicrobial film, the excellent film-forming
property is combined with its antimicrobial property.
3.7.4 Biomedical Application
Owing to its biocompatible, non-toxic, film-forming and hydrophilic nature, there

are numerous biomedical applications of chitosan currently at different stages of
development from basic research to clinical trials. Biomedical applications of chitin
and chitosan include tissue repair, wound healing, scaffold production and biomedical
implants.
One of the novel applications of chitosan in the photochemical sutureless tissue
bonding. Post-surgery, it is required to close the incisions which have been made
in tissues such as skin, cornea or peripheral nerves. Manual suturing requires good
dexterity and can be quite time consuming with side effects. To address this, pho-
tochemical tissue bonding has been introduced. These involve the joining of tissue
through activation of chemical cross-linking of collagen fibers using rose bengal as
3.7 Applications 55
the cross-linker. An improvement to this process is to combine rose bengal, chitosan

adhesive and laser at a wavelength of 532 nm (Lauto et al. 2010; Frost et al. 2016).
In the 1988 publication by Lantos, the various applications of plastics in the
medical field were reviewed. At a time when there was decreasing price of plastics,
it was appealing to evaluate the use of plastics in the medical field and ways of
improving the performance in already existing applications as well as expanding the
areas of applications. Today 30 years later, plastics have much wider applications
in the medical field: from disposable syringe to pacemaker coatings, to waterproof
aprons, to sutures to blood bags and protective eyewear. The key issues identified
at the time were improved blood compatibility, radiation resistance and improved
degradability.
3.7.5 Gene Therapy
In gene therapy, delivery of nucleic acid (DNA/RNA) into cells is often done via
viral infection, where the gene to be delivered is planted into a virus and the cell is
infected with the virus. Another alternative is to use transpection using polyplexes.
Chitosan has been widely investigated as a polyplex in DNA and RNA transfection.
The polycationic nature of chitosan, its non-toxic and biocompatibility as well as
being of renewable natural source make it a suitable alternative to other synthetic
more toxic polymers used as polyplexes such as polyethylenimine and polyami-
doamine dendrimers. Chitosan can form non-toxic complexes with DNA and RNA
for use in gene transfection. Particularly, chitosan oligomers have been shown to
have better physical properties in terms of solution viscosity and stability at phys-
iological pH than high-purity high molecular weight chitosan. Chitosan oligomers
with molecular weight between 18 and 20 demonstrate good non-viral gene delivery
properties (Koping-Hoggard et al. 2004).
More recently, chitosan/hyaluronic acid nanoparticles have been shown to be
effective in the delivery of mRNA to cell. This is promising in the area of cancer
treatment through expanding understanding of nucleic acid uptake and metabolism of
cancer cells with the potential to send mRNA to target cancer cells to stop their repli-
cation and inhibit growth of the tumor. The chitosan/hyaluronic acid nanoparticles
were more effective under acidic conditions (Lallana et al. 2017).
3.7.6 Anticancer Application
Cancer treatment is a highly significant area of interest, and chitin finds application
even in this field. Different studies have reported antitumor effect of chitin and
chitosan as well as their derivatives on a broad range of cancers including melanoma,
carcinoma, colon cancer, lung cancer, sarcoma and prostate cancer (Gibot et al. 2015;
You-Jin and Kim 2002). The mechanism by which chitin and its derivatives act against
56 3 Chitin
cancer has been widely studied and is still presently undergoing studies. Mechanisms
reported include increasing the activities of enzymes which inhibit cancer cells or
kill them, boosting the body’s own immune response to cancerous cells, inhibiting of
certain enzymes required for cancer cell growth or altering the pathways for cancer
cell proliferation such as interfering with DNA replication. Chitin and chitosan could
also act against cancer by accumulating within the cancer cells and partaking in
reactions which result in production of toxins which eventually lead to the cancer
cell death. The activity of chitin, chitosan and their derivatives vary from preventing
cancer cells from forming, inhibiting the growth of existing cancer cells or preventing
the metastasis (spreading) of the cancer to other parts of the body thereby making
treatment more manageable.
The effectiveness of chitin and chitosan in their various applications has been
shown to be significantly affected by factors such as molecular weight and the degree
of deacetylation. In fact, one study showed that oligomers of chitosan, which are
short-chain chitosan of molecular weight between 1.5 and 5.5 kDa, had more effective
antitumor activity against sarcoma than higher molecular weight chitosan and low
molecular weight chitosan indicating a specific range of molecular weight required
(You-Jin and Kim 2002). Therefore, processing techniques such as gel permeation
chromatography to separate into different molecular weights, achieving a higher
degree of deacetylation during production and higher purity using methods such
as ultrafiltration, becomes important for optimum effectiveness particularly when
considering the high-end application such as antitumor and tissue repair application
of these biopolymers. Such processing is what adds additional processing costs as
the value of the chitin increases from crude chitin to chitosan with low degree of
deacetylation and polydisperse to more monodisperse chitosan with high degree of
polymerization. The quality criteria for chitin and chitosan are mainly molecular
weight, degree of deacetylation, purity and polydispersity. The values for each of
these will depend on the intended application.
3.7.7 Anti-inflammatory
Chitosan has been shown to have some anti-inflammatory properties. Inflamma-

tions are generally caused by inflammatory cytokines released by the immune cells
such as macrophages. In certain inflammatory diseases, things get out of hand,
there is excessive production of inflammatory cytokines and these result in pain
and discomfort.
Other than the antimicrobial property which also induces anti-inflammatory effect
by destroying the inflammatory disease-causing bacteria, chitosan has been shown
to have direct anti-inflammatory effect. The mechanism by which this happens is
by inhibiting inflammatory cytokine production in the presence of inflammation-
inducing microbes. For example in the presence of chitosan-alginate nanoparticles,
the inflammatory activity of the acne causing bacteria P. acnes which induces the pro-
duction of inflammatory cytokines by the immune cells was inhibited (Friedman et al.
3.7 Applications 57
2013). Acne therapy takes the approach of eradicating the bacteria which results in
the inflammation seen as acne spots and also preventing the formation of inflam-
matory cytokines by the immune cells in the skin which results in the formation
of acne on the skin surface. Chitosan with its antimicrobial and anti-inflammatory
property therefore is an attractive drug candidate in such therapy. Similar chitosan
has demonstrated anti-inflammatory effect on other inflammatory diseases such as
inflammatory bowel disease (Friedman et al. 2013).
3.7.8 Antioxidant
The antioxidant activity of the deacetylated form of chitin, chitosan, is attributed to

the presence of hydroxyl groups and amino groups which are available to react with
free radicals. This antioxidant property of chitosan has been demonstrated. Chitosan
extracted from crab shells achieves up to 70% antioxidant activity at a concentration
of 1 mg/ml (Yen et al. 2009).
For applicability, compounds used in pharmaceutical or food need to be water
soluble. Chitosan solubility can be improved by reducing the chain size to oligosac-
charides or dimers, converting to derivatives such as carboxymethyl cellulose or chi-
tosan salts. The antioxidant properties of chitosan have been shown to improve when
formed into salts such as chitosan acetate (Charernsriwilaiwat et al. 2012). Even high
molecular weight-based chitosan films show antioxidant properties, particularly quat-
ernized high molecular weight chitosan such as N-(2-hydroxyl)propyl-3-trimethyl
ammonium chitosan chloride with molecular weights of 400 and 1240 (Wan et al.
2013). Retaining the high molecular weight as well as the antioxidant property makes
it applicable to chitosan-based antioxidant material in food and pharmaceutics with
good mechanical properties.
3.7.9 Antimalaria
Recently, deacetylated chitin (chitosan) from crab shells combined with silver
nanoparticles has demonstrated antimalarial properties. When chitosan combined
with silver nanoparticles were spread on a water reservoir, this treatment was effec-
tive in eradicating mosquito larvae and pupa (Murugan et al. 2017). The formulation
tested non-toxic to fish and also had antibacterial effect. The antimalaria effect there-
fore comes from the ability to hinder the growth of mosquitos. Furthermore, the silver
nanoparticles could be produced using plant extract and this has been explored by
several researchers. It is therefore also worth exploring the extent of chitin application
in its crude form since this requires less use of chemicals and has less environmental
impact.
58 3 Chitin
3.7.10 Papermaking
While their wastewater treatment application already makes chitin useful to the paper-
making industry in treatment of wastewater from papermaking, chitin/chitosan has
further applications in this industry beyond water treatment, and this includes surface
coating, dye fixation and paper strengthening. Antibacterial and greaseproof paper
are also in the horizon of future applications of chitosan in the paper industry.
In the wet end application, chitosan and other chitosan derivatives find use as
retention and drainage agents. They act as flocculants and coagulants causing the
aggregation of fine particles and liquids to aid the separation process. The mech-
anism by which they do this is through electrostatic attraction facilitated by the
attraction between the amino and hydroxyl group and the fine particles thanks to the
polycationic nature of chitosan.
Between cellulose fibers exist voids due to the repulsive force between like
charges. Due to its excellent film-forming property, chitosan covers this void between
the fibers, making for a stronger paper. Chitosan and chitosan derivatives can be
applied as coatings on paper. This is aided by its affinity for cellulose. Chitosan
coatings give antimicrobial properties and improve water vapor and oxygen barrier
properties of the paper. Such papers find use in food packaging and medical uses.
Further application of chitosan in paper production includes its use in dye fixation
for improving the dyeing of paper and chitosan nanoparticle derivatives used in
producing transparent paper (Song et al. 2018).
3.7.11 Electronics
Ubiquity of chitin is further demonstrated by its applicability in electronics. Flexible

electronics, photovoltaic cells and biomedical sensors are some of the applications
which employ chitin. Most of these are using chitin in the deacetylated form (chi-
tosan). The electronics industry is one that makes use of a large amount of poly-
mers, from cable coatings to casings to flexible substrates for photovoltaic cells.
While many of the applications of chitin involve the deacetylated form, chitosan,
some recent applications have explored chitin nanoparticles, for example, in use as
biodegradable, eco-friendly flexible substrates for light-emitting diodes (Jin et al.
2016).
Flexible electronics have become desirable for lightweight, low-cost electronics
such as screens and photovoltaic cells. Being able to process these electronics on
flexible substrates allows for mass reproducible roll to roll processing techniques
at low temperatures such as solvent casting and more portable designs. Recently,
plastics such as polyethylene terephthalate and polyethylene have been used for
such. Even better would be biodegradable polymers for such applications. Chitin
and chitosan and their derivatives have demonstrated suitability for such applications
(Triyana et al. 2018).
3.7 Applications 59
3.7.12 Cosmetics and Toiletries
Chitin is mainly applied in its deacetylated form, chitosan in cosmetics. The antimi-
crobial property of chitosan makes it useful as a cosmetic anti-acne product ingredi-
ent. Its barrier properties, water vapor permeability, make it a good protective barrier
to retain skin moisture. It is also used in oral care products as thickener and emulsifier.
Due to its antimicrobial, antioxidant property combined with its viscosity-enhancing
property, chitosan can play multiple roles in cosmetics and toiletries.
Glucosamine, the monomer of chitin, also finds application in cosmetics. Deriva-
tives of glucosamine have been shown to reduce wrinkles, boost natural production
of hyaluronic acid, improve skin suppleness, moisture and rejuvenate overall skin
(Jacobs 2007).
3.7.13 Agrochemicals
In agriculture, chitin and its derivatives have found use in various aspects which
include: controlled fertilizer release excipients, protective seed coating (frost pro-
tection), stimulation of growth and aiding plant defense mechanism. This has both
economic and environmental impacts by improving crop yield and optimization of
land usage. It also adds nutrients to the soil in a sustainable way replacing the use of
energy-consuming agricultural practices.
3.7.14 Biodegradation of Chitin and Its Derivatives
In curbing, the pollution crisis caused by the inadequate dumping of synthetic plastics
which pose a serious hazard to aquatic life, research and industry has turned focus
on biodegradable plastics. The physical properties which are comparable to those
of non-biodegradable synthetic plastics are what make chitin attractive for use as a
bioplastic.
Some compounds such as minerals accumulate in nature because their production
rate is much higher than their degradation rate. Like many other biopolymers, chitin
does degrade because 10–100 billion tonnes of it is being produced annually (Beier
and Bertilsson 2014) yet there is no known accumulation of chitin anywhere in
nature. This means that biodegradation of chitin is happening at a considerable rate.
The microbial degradation of chitin by various microorganisms has been reported;
however, it is widely accepted that the predominant degradation of chitin is by bacteria
(Beier and Bertilsson 2014; Abd-Aziz et al. 2008).
The process of chitin degradation can occur via different pathways. Chitin degra-
dation enzyme has been detected in various microorganisms including fungi, bacteria,
rotifers and some carnivorous plants and digestive tract of higher animals (Gooday
60 3 Chitin
1990). Chitin degradation at physiological condition is predominantly carried out by

the enzymes within the physiologic environment. Chitin degradation under standard
room conditions is considered for products that are used under such conditions. The
degradation mechanism and rate depend on the condition of storage. Its degrada-
tion in soil has been observed to be predominant by bacteria; however, some fungi
also take part in chitin degradation. In the aquatic environment where degradation
of shells and exoskeleton of dead or worn-out aquatic chitinous aquatic organisms
occurs, bacteria have been shown to be the main degraders.
Diatoms, microalgae which have quite a sizeable presence in the oceans and
other aquatic environment, are also known to have the ability to hydrolyze chitin
oligomers (Vrba et al. 1997). Chitinous aquatic organisms and other non-aquatic
arthropods occasionally shed their outer shell in order to allow for growth such that
the old shell is removed and a new larger one is formed to better accommodate and
allow for increase in size of the organism. This process is referred to as molting,
and it is thought that the organism releases chitin degradation enzymes during this
process (Vrba and Mackacek 1994). It is however yet to be determined, the exact role
of these enzymes as they could be either involved in reactive breakdown of chitin or
used to hydrolyze dissolved chitin oligomers.
Although here we have considered the degradation of chitin in nature, degradation
of chitin when it comes to products which have been made from chitin such as
scaffolds, packaging films and water treatment membranes, the environment and
conditions within which the degradation is occurring varies significantly. Studies
which look at the development of such products have also considered the degradation
mechanisms of each of these products.
The process of degradation of chitin is referred to as chitinoclastic. The degra-
dation process could be chitinolytic, and this refers to the breaking of the (1 →
4)-β-glycosidic bonds. The deacetylation of chitin to chitosan is also a form of chitin
degradation as it involves the breaking of the acetyl group. The main difference
between deacetylated chitin and glucose is the presence of an amine group in the
place of one of the hydroxyl groups on the glucose ring. Deaminization is also another
breakdown process where the deacetylated chitin is then converted to cellulose by the
removal of the amine functional group. The breakdown of the products of the initial
degradation of chitin (chitosan and glucose) then follows their respective degradation
pathways to smaller units of glucose, glucosamine and N-acetylglucosamine which
can then be returned to the carbon and nitrogen cycle. Enzymes involved in these
processes are chitinases, chitosanase and to a lesser extent, cellulases. Lysozyme, an
enzyme involved in bacteria cell death as an immune response in animals, has also
been shown to degrade chitin (Beier and Bertilsson 2014).
Chitin degradation by microorganisms occurs for the purpose of breaking down
the chitin for their metabolism or for the growth of the organism as seen during the
molting of arthropods. In the case of lysozyme, it could also occur as an immune
response or defense mechanism. The degradation pathway of chitin is illustrated in
the chart in Fig. 3.5.
The pathway of degradation of chitin depends on the mix of microbes within the
habitat. These have been shown to have more effect than temperature. End product of
3.7 Applications 61
Fig. 3.5 Degradation pathway of chitin
hydrolysis could either be organic material, or it could be mineralized into inorganic

materials depending on the activity of the microbes present. The rate of degradation
of chitin is relatively simpler than other polymers such as lignin and cellulose.
The rate of chitin hydrolysis in different environments has been presented in
different studies over the years. In soil, the rate of degradation is reported to be
between 0.6 and 1.1% per day, in freshwater it could be as high as 30% per day, in
brackish water it could be as low as below 1% and up to 8.1%, while marine water
records the lowest degradation rate at 0.00043–0.0005 per day (Beier and Bertilsson
2014). In all environments, chitin has a much faster degradation rate compared to
fossil-based polymers.
3.8 Commercial Production
The conventional sources of chitin for commercial production are lobster, crab and
shrimp shells. However more recently, other sources such as insects and mush-
rooms are emerging. Companies involved in commercial chitin production from
these unconventional sources include KitoZyme which produces chitin from fungi,
a non-animal source. Their chitin is used in food supplements and medical products.
Another example of company producing chitin on a commercial scale from shrimp
shell is Mahtani Chitosan, a biotechnology company located on the coast of Gujarat,
India (Munoz et al. 2018). The company reports producing 300 mt of chitin, 150 mt of
glucosamine and 50 mt chitosan per annum. The company primarily produces chitin
and the derivatives from waste shells of shrimp species of Penaeus spp., Metapenaeus
spp. and Parapenaeus spp. captured from the Arabian Sea (Munoz et al. 2018). They
employ the chemical extraction method for demineralization, deproteinization and
deacetylation.
A number of companies exist globally which are actively involved in commer-
cial production of chitin and its derivatives, either for conversion into other prod-
ucts or chitin being the end product. In the environmental impact evaluation section
of the chapter, we have discussed Mahtani Chitosan and an anonymous European
62 3 Chitin
company who produce chitin from shrimp and crab shells, respectively. Another
company based in Belgium called KitoZyme S. A produces chitosan from fungi
for food and pharmaceutical applications. Another company Primex produces chi-
tosan from arctic shrimp for applications in food. Other companies involved in the
production of chitin and/or its derivatives include Asiamerica Group, Inc., Spec-
trum Chemical Manufacturing Corp, Orison Chemicals Ltd., Parachem, Shandong
Laizhou Highly Bio-Products Co. Ltd., Pure Earth Biotechnology Co. Ltd., Qing-
dao BZ Oligo Biotech Co., Ltd. and Yaizu Suisankagaku Industry Co., Ltd. (Future
Market Insights 2019).
Shell-Ex, a biorefinery company located in Canada, is dedicated to converting
shrimp waste into products such as liquid fish to serve as soil NPK nutrient and
shrimp and crab shell which is mainly targeted at chitosan production company. The
specific type of shrimp shell it supplies (Pandalus borealis) is regarded as high-
quality shell preferred in chitosan production, particularly sourced from the Atlantic
Ocean for optimal purity away from industrially polluted water.
Heppe Medical Chitosan GmBH located in Germany develops, produces and sells
chitosan alongside other biopolymers targeted at supplying the research and medical
sector. For such purpose where high-quality chitosan is required, production occurs
in smaller batches. CHitOcean is another company in North America involved in the
chitosan manufacturing.
Great opportunities exist in the present and future market for manufacturers of
chitin and its derivatives. Opportunities also exist for the raw material collectors and
agents as well as the end use industries. As chitin industry expands, this will trigger
new product development in all the various areas of applications implementing the
diverse chemistry of chitosan into numerous products. Generally, fish consumption
in any country is higher in the coastal regions than inland areas. This is an important
fact to consider in location of chitin plants near to the raw materials.
3.9 Conclusion
Chitin is almost exclusively sourced from the aquatic environment with the exception
of insects, mushrooms and land crabs which are not fully aquatic. It is a versatile
biopolymer of much industrial significance. In nature, it plays a significant role in
structure within the organisms such as shrimp shells and mushroom. It also plays a
role in metal chelation and formation of coral reefs. The amount of chitin being used
and made commercially available is much less than what is being generated as waste
and how much exists in the aquatic world. Therefore, there is much room for growth
in the chitin industry with regard to available raw material. Presently, crustacean
shells are the most widely utilized sources used for commercial chitin production
although researchers have extracted from various sources such as mushrooms, insects
and fish scales. The chemical production method is the method being mainly used in
industry for chitin extraction although chitin can also be extracted using microbial
fermentation and enzyme extraction. Molecular weight, degree of deacetylation and
3.9 Conclusion 63
polydispersity are important parameters for determining quality of chitin. These

factors are therefore important in their extraction, processing and applications.
The process of sourcing and extraction of chitin has significant environmental
impact from the carbon emission from transportation and preservation of raw mate-
rials to the chemical and energy consumed during the production process. Nonethe-
less, chitin, being the second most abundant polymer on earth, offers great potential
as a replacement or at least an alternative to fossil-derived polymers used for sim-
ilar applications such as packaging films, water treatment, flexible electronics and
biomedical products.
References
Abd-Aziz S, Sin T, Slitheen N, Shahab N, Kamaruddin K (2008) Microbial degradation of chitin

materials by Trichoderma virens UKM1. J Biol Sci 8(1):52–59
Agboh OC, Qin Y (1997) Chitin and chitosan fibers. Polym Adv Technol 8:355–365
Ahearn GA, Mandal PK, Mandal A (2004) Calcium regulation in crustaceans during the molt cycle:
a review and update. Comp Biochem Physiol A Mol IntergrPhysiol 137(2):247–257
Aili D, Adour L, Houali K, Amrane A (2019) Efect of temperature in chitin and chitosan production
by solid culture of penicillium camembertii on YPG medium. Int J Biol Macromol 133:998–1007
Al- Manhel AJ, Al-Hilphy ARS, Niamah AK (2018) Extraction of chitosan, characterisation and
its use for water purification. J Saudi Soc Agric Sci 17:186–190
Azuma K, Osaki T, Minami S, Okamoto Y (2015) Anticancer and anti-inflammatory properties of
chitin and Chitosan oligosaccharides. J Funct Biomater 6:33–49
Badawy RM, Mohamed HI (2015) Chitin extraction, composition of different six insect species and
their comparable characteristics with that of the shrimp. J Am Sci 11(6):127–134
Beier S, Bertilsson S (2014) Bacterial chitin degradation- mechanisms and ecophysiological
strategies. Front Microbiol 4(149):1–13
Berg A (2009) Aquatic insects classification. Encyclopedia of inland waters, pp 128–131
Boarin-Alcalde L, Graciano-Fonseca G (2016) Alkali process for chitin extraction and chitosan
production from Nile tilapia (Oreochromis niloticus) scales. Am J Aquat Res 44(4):683–688
Brostow W, Datashvili T (2016) Environmental impacts of Natural Polymers. In: Olatunji O (Ed).
Natural Polymers, Industry Techniques and Applications. Springer, New York. pp 315–338
Castro R, Guerrero-Legarreta I, Borquez R (2018) Chitin extraction from Allopetrolisthes punctatus
crab using lactic fermentation. Biotechnol Rep 20:e00287
Charernsriwilaiwat N, Opanasopit P, Rojanarata T, Ngawhirunpat T (2012) In vitro antioxidant
activity of chitosan aqueous solution: effect of salt form. Trop J Pharm Res 11(2):235–242
Cira L, Huerta S, Hall GM, Shirai K (2002) Pilot scale lactic acid fermentation of shrimp wastes
for chitin recovery. Process Biochem 37:1359–1366
Divya K, Vijayan S, George TK, Jish MS (2017) Antimicrobial properties of chitosan nanoparticle:
mode of action and factors affecting activity. Fibers Polym 18(2):221–230
Elieh-Ali-Komi D, Hamblin MR (2016) Chitin and chitosan: production and application of versatile
biomedical nanomaterials. J Adv Res (Indore) 4(3):411–427
FAO (2016) The state of world fisheries and aquaculture 2016. Contributing to food security and
nutrition for all. Rome, 200 pp. ISBN 978-92-5-109185-2
Ficket Z, Medunic G, Turk FM, Ivanic M, Kniewald G (2017) Influence of soil characteristics on
rare earth fingerprints in mosses and mushrooms: example of a pristine temperature rainforest
(Slavonia, Croatia). Chemosphere 179:92–100
64 3 Chitin
Flores-Albino B, Arias L, Gomez J, Castillo A, Gimeno M, Shirai K (2012) CHitin and L (+) -lactic
acid production from crab (Callinectes bellicosus) wastes by fermentation of Lactobacillus sp.
B2 using sugar cane molasses as carbon source. Bioprocess Biosyst Eng 35:1193–1200
Friedman AJ, Phan J, Schaire D, Champer J, Qin M, Pirouz A, Blecher K, Oren A, Liu P, Modlin
RL, Kim J (2013) J Invest Dermatol 133(5):1231–1239
Frost SJ, Mawad D, Higgins MJ, Ruprai H, Kuchel R, Tilley RD, Myers S, Hook JM, Lauto A
(2016) NPG Asia Mater 8(73):1–9
Future Market Insights (2019) Chitin market: agrochemical end use industry segment inclined
towards high growth—moderate value during the forecast period: global industry analysis (2012–
2016) and opportunity assessment (2017–2027). REF-GB-313, pp 1–236
Gadgey KK, Bahekar A (2017) Studies on extraction methods of chitin from crab shell and
investigation of its mechanical properties. Int J Mech Eng Technol 8(2):220–231
Gibot L, Chabaud S, Bouhout S, Bolduc S, Augner FA, Moulin VJ (2015) Anticancer properties of
chitosan on human melanoma are cell line dependent. Int J Biol Macromol 72:370–379
Gil-Duran S, Arola D, Ossa EA (2016) Effect of chemical composition and microstructure on the
mechanical behaviour of fish scales from Megalops atlanticus. J Mech Behav Biomed Mater
56(2016):134–145
Gooday GW (1990) The ecology of chitin degradation. Adv Microb Ecol 11:387–430
Gortari MC, Hours RA (2013) Biotechnological processes for chitin recovery out of crustacean
waste: a mini-review. Electron J Biotechnol 16(3):1–19
Hassainia A, Satha H, Boufi S (2018) Chitin from Agaricus bisporus: extraction and characterization.
Int J Biol Macromol 117:1334–1342
Hulsey MJ (2018) Shell biorefinery: a comprehensive introduction. Green Energy Environ 3:318–
327
Ifuku S, Nomura R, Morimoto M, Saimoto H (2011) Preparation of chitin nanofibers from
mushrooms. Materials 4:1417–1425
Jacobs E (2007) Topically applied glucosamine sulfate and all its related, precursor, and derivative
compounds significantly increases the skin’s natural production of hyaluronic acid for the reju-
venation of healthier younger-looking skin: while phosphatidylcholine is required to replace its
deficiency caused by topical dimethylaminoethanol (DMAE). US Pastent US2007/0092469 A1.
Vienna (US)
Jin J, Lee G, Im H, Han YC, Jeong EG, Rolandi M, Choi KC, Bae RS (2016) Chitin nanofiber
flexible transparent paper for flexible green electronics. Adv Mater 28(26):5169–5175
Kalut SA (2008) Enhancement of degree of deacetylation of chitin in chitosan production, B.
Chemical Engineering, Universiti Malaysia Pahang, pp 14–15
Kanatt SR, Rao MS, Chawla SP, Sharma A (2013) Effects of chitosan coating on shelf-life of
ready-to-cook meat products during chilled storage. LWT-Food Sci Technol 53:321–326
Kaya M, Mujtaba M, Ehrlich H, Salaberria AM, Baran T, Amemiya CT, Galli R, Akyuz L, Sargin
I, Labidi J (2017) On chemistry of gama- chitin. Carbohydr Polym 176:177–186
Koping-Hoggard M, Varum KM, Issa M, Danielsen S, Christensen BE, Stokke BT, Artursson P
(2004) Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes
of highly defined chitosan oligomers. Gene Ther 11:1441–1452
Lallana E, Rois de la Rosa JM Tirella A, Pelliccia M, Gennari A, Stratford IJ, Puri S, Ashford
m, Tirelli N (2017) Chitosan/hyaluronic acid nanoparticles: rational design revisited for RNA
delivery. Mol Pharm 14(7):2422–2436
Lantos PR (1988) Plastics in medical applications. J Biomater Appl 2(3):358–371
Lauto A, Mawad D, Barton M, Gupta A, Piller SC, Hook J (2010) Photochemical tissue bonding
with chitosan adhesive films. BioMed Eng Online 9(47):1–11
Liu S, Sun J, Yu L, Zhang C, Bi J, Zhu F, Jiang MQC, Yang Q (2012) Extraction and characterization
of chitin from the beetle Holotrichiaparallela Mots chulsky. Molecules 17:4604–4611
Majekodunmi SO (2016) Current development of extraction, characterization and evaluation of
properties of chitosan and its use in medicine and pharmaceutical industry. Am J Polym Sci
6(3):86–91
References 65
Majtan J, Bilikova K, Marovic O, Grof J, Kogan G, Simuth J (2007) Isolation and characterization
of chitin from bumble bee (Bombus terrestris). Int J Biol Macromol 40:237–241
Massimilian F, Ludovico P (2016) Use of non-treated shrimp shells for textile dye removal from
wastewater. J Environ Chem Eng 4(4):4100–4106
Munoz I, Rodriguez C, Gillet D, Moerschbacher BM (2018) Life cycle assessment of chitosan
production in India and Europe 23:1151–1160
Murugan K, Anitha J, Suresh U, Rajaganesh R, Panneerselvam C, Aziz AT, Tseng L-C, Kalimuthu
K, Saleh Alsalhi M, Devanesan S, Nicoletti M, Sarkar SK, Benelli G, Hwang J-S (2017) Chitosan-
fabricated Ag nanoparticles and larvivorous fishes: a novel route to control the coastal malaria
vector Anopheles sundaicus? Hydrobiologia 797(1):335–350 doi: 10.1007/s10750-017-3196-1
Olatunji O, Olsson RT (2015) Microneedles from fishscale-nanocellulose blends using low
temperature mechanical press method. Pharmaceutics 7:363–378
Rinaudo M (2006) Chitin and CHitosan: properties and applications. Prog Polym Sci (Oxford)
31(7):603–632
Roy JC, Salaun F, Giraud S, Ferri A, Chen G, Guan J (2017) Solubility of chitin: solvents, solu-
tion behaviours and their related mechanisms. In: Solubility of polysaccharide (xxx Editor).
Intechopen
Rumengan IFM, Suptijah P, Wullur S, Talumepa A (2017) Characterization of chitin extracted from
fish scales of marine fish species purchased from local markets in North Sulawesi, Indonesia.
IOP Conf Ser: Earth Environ Sci 89:012028
Satam CC, Irvin CW, Lang AW, Jallorina JC, Shofner ML, Reynolds JR, Meredith JC (2018)
Spray-coated multilayer cellulose nanocrystal—chitin nanofiber films for barrier applications.
Chemistry. https://doi.org/10.1021/acssuschemeng.8b01536
Shahidi F, Arachchi JKV, Jeon Y (1999) Food application of chitin and chitosans. Trends Food Sci
Technol 10(2):37–51
Song Z, Li G, Guan F, Liu W (2018) Application of chitin/chitosan and their derivatives in the
papermaking industry. Polymers 10(4):E389
Thannimalay L, Yusoff S, Zawawi ZN (2013) Life cycle assessment of sodium hydroxide. Aust J
Basic Appl Sci 7(2):421–431
Tolesa LD, Gupta BS, Lee MJ (2019) Chitin and chitosan production from shrimp shells using
ammonium based ionic liquids. Int J Biol Macromol 130:818–826
Triyana K, Sembiring A, Rianjanu A, Hidayat SN, Riowirawan R, Julian T, Kusumaatmaja A, San-
toso I, Roto R (2018) Chitosan-based quartz crystal microbalance for alcohol sensing. Electronics
7(9):181–190
Varum TK, Senani S, Jayapal N, Chikkerur J, Roy S, Tekulapally VB, Gautam M, Kumar N (2017)
Extraction of chitosan and its oligomers from shrimp shell waste, their characterization and
antimicrobial effect. Vet World 10:170–175
Vrba J, Machacek J (1994) Release of dissolved extracellular Beta-N-Acetylglucosaminidase during
crustacean molting. Limnol Oceanogr 39:712–716
Vrba J, Kofroˇnová-Bobková J, Pernthaler J, Simek K, Macek M, Psenner R (1997) Extracellular,
low-affinity beta- N-acetylglucosaminidases linked to the dynamics of diatoms and crustaceans
in freshwater systems of different trophic degree. Int Rev Gesamten Hydrobiol 82:277–286
Wan A, Xu Q, Sun Y, Li H (2013) Antioxidant activity of high molecular weight chitosan and N,
O-Quaternized chitosans. J Agric Food Chem 61(28):6921–6928
Yen MT, Yang JH, Mau JL (2009) Antioxidant properties of chitosan from crab shells. Carbohyd
Polym 74(4):840–844
You-Jin J, Kim S (2002) Antitumor activity of chitosan oligosaccharides produced in ultrafiltration
membrane reactor system. J Microbiol Biotechnol 12(3):503–507
Chapter 4
Alginates
Abstract Alginates are obtained from brown algae, which are mainly found in
marine aquatic environment. Alginate chemical structure is characterized by the
presence of mannuronic or guluronic acid repeating unit in either alternating or block
forms within the polymer chain. They form a part of the cell wall where they provide
strength and flexibility to the cell wall. The chapter reviews a number of extraction
methods used and then discusses the environmental impact of the extraction process.
Alginates find application in a variety of industries which includes food, biomedical,
textiles and others. Alginates can be said to be one of the well-explored aquatic
biopolymers. The chapter discusses the current state and some of the limitations to
its commercial production and presents future perspectives on alginates.
Keywords Alginates · Galactose · Polymer · Brown algae · Aquatic ·

Phycocolloid
4.1 Introduction
Alginates are natural polysaccharides which are present in the cell walls of brown
algae (brown seaweed). They are extracted mainly using chemical methods with
additional processing such as gel pressing and filtration. Alginates are a general term
which refers to salts of alginic acid as well as derivatives of alginic acids and its salts.
Within this chapter, alginates refer to salts of alginic acid: sodium alginate, calcium
alginate and magnesium alginate. Another term, alginocytes, refers to sources of
alginates. Brown marine algae are the main known alginocytes.
Various forms of alginates exist; however, sodium alginates are most common due
to its solubility in cold water which makes it easier to extract and apply. As alginates
gain more commercial value, the cultivation of alginocytes (alginate producing algae)
for alginate production is on the rise. FAO reports an annual increase of over 6% in
the cultivation of brown algae, the main source of alginates. A large proportion of the
seaweeds grown commercially is grown from aquaculture where they are grown in
controlled environment for optimal yield and profitability. The increase in demand
for seaweed can in part be attributed to increased health consciousness and demand
for more non-animal-based food and pharmaceuticals. Alginate competes with the

68 4 Alginates
human and animal edible algae as a lot of the algae being cultivated globally are also
used for human consumption and animal feed. By 2024, alginate market is predicted
to increase in value up to about 87 billion USD.
Biopolymers with diverse applications offer the producer and supplier an equally
diverse distribution channel, whereby the product is in demand in several indus-
tries. Alginate is one of such polymers, and its applications include in food, textiles,
pharmaceutics and more. Some of these applications are already being made into
commercial products while others are still at an early research stage. Alginates are
applied in textiles, cosmetics, paper, food and pharmaceutics.
Alginates are one of three phycocolloids which can be obtained from algae. The
two others are agar and carrageenan. Alginates are made from brown algae while agar
and carrageenan are obtained from red algae. Alginates are one of the commercially
well-explored biopolymers of aquatic source. Since they are produced primarily from
algae, they are one of the polymers which are exclusively sourced from the aquatic
environment. The continued availability of alginates is therefore significantly affected
by the dynamics of the aquatic environment. The production of these phycocolloids
competes with the production of other products such as biofuel, fertilizers and sewage
treatment materials from algae. It is therefore important to understand the production
and impacts of alginate production from these algae in order to assess the adequate
utilization of this particular aquatic resource.
Brown seaweeds are the only known source of alginates. Although alginates from
seaweed were not discovered until the later part of the 1800s, for centuries, coastal
communities have grown around gathering of seaweed. Seaweeds are aquatic plants
which grow naturally in different aquatic conditions, from slow-moving to more
turbulent waters. There are three classifications of seaweeds: brown, red and green.
Brown seaweeds are largest in size, up to 20 m long; however, smaller ones exist rang-
ing in 2–4 m long or even smaller 30–60 cm length (McHugh 2003). Red seaweeds
are smallest with length ranging under a meter while green seaweeds are the smallest.
Here, we focus our interest on the brown seaweed species. These grow best in cold
waters around 20 °C in countries such as South Africa, Argentina, Canada, Australia,
Chile, Norway, Ireland, Mexico, Scotland, Northern Ireland, USA and other countries
in the Southern and Northern Hemisphere. Factors which affect growth of seaweeds
include temperature, nutrient content, salinity, light penetration and turbulence of
water.
Despite aquaculture dominating the global seaweed production, most of the
alginophytes are from species which grow wild such as laminarin and Ascophyl-
lum. Most of the brown seaweeds used for alginate production have a complex
reproductive cycle which makes their cultivation less cost-effective than when har-
vested from wild stock (McHugh 2003). However, as further understanding of the
diverse reproductive cycle of these algae is developed, more effective methods for
cultivation of alginophytes in aquaculture systems have been introduced in recent

years, particularly in Asia (Charrier et al. 2017). Cultivation of some species of wild
seaweed has been restricted as a result of overexploitation. Other than being a source
of alginate and other phycocolloids, seaweed is a rich source of nutrients such as
proteins, minerals and vitamins, it is also the only non-fish source of omega-3 fatty
acid. It therefore has a high value as a food product for direct consumption by humans
and other animals.
Alginate provides seaweeds with the flexibility and strength needed to survive
at sea. Within the seaweed cell walls, alginates provide flexibility and mechanical
strength, needed by the algae to survive at sea, particularly where the water is par-
ticularly turbulent. They exist in the cell wall attached to metals such as magnesium,
calcium and sodium. The seaweed also contains cellulose which provides rigidity.
Alginate contents tend to be higher in seaweeds cultivated in more turbulent water
while seaweeds grown in calmer waters have lower alginate content. The properties
of the alginates extracted from seaweed such as viscosity and gel strength depend on
the structure of the alginate, and this in turn is affected by factors such as species of
seaweed, growth environment and the method of extraction. Alginate concentration
has also been shown to be affected by the period of harvest (Schiener et al. 2014;
Taelman et al. 2015).
While seaweeds naturally grow in aquatic environment, much of the world’s
seaweeds are grown on slow stagnant water with the right nutrients and environmental
conditions, to meet the growing demands for alginates and other products of brown
seaweed. Depending on the region and its demand, majority of the brown seaweed
cultivated is used for alginate while the rest is used for food. However, other statistics
show that in China, for example, most of the seaweed cultivated in this region is used
as food.
There are a variety of brown seaweed species which act as a source of algi-
nates. These include Laminaria spp, Macrocystis pyrifera, Durvillaea antarctica,
Lessonia flavicans, Ecklonia maxima and Ascophyllum nodosum. The most commer-
cially exploited ones are Laminaria, Macrocystis and Ascophyllum. Alginate is also
present as a protective extracellular structure of some bacteria such as Azotobacter
and pseudomonas (Aleksandra and Sanja 2017).
4.3 Chemistry of Alginates
4.3.1 Polymer Chain Structure
Alginates are linear ionic heteropolysaccharides copolymer of (1-4) linked beta-d-

mannuronic acid and alpha-l-guluronic acid arranged in block or alternating manner
along the polymer chain. Such that along the alginate polymer chain, some segments
of mannuronic acid, segments of guluronic acid and segments of mannuronic acids
and guluronic acid alternating exist. This is illustrated in Fig. 4.1.
70 4 Alginates
Fig. 4.1 A section of one possible block and alternating patterns of mannuronic acid (M) and
guluronic acid (G) within an alginate chain
The manner in which the mannuronic acid and guluronic acids are arranged along
the polymer chain varies in different seaweeds. So does the ratio of mannuronic:
guluronic acid of the alginate. The GG blocks confer more stiffening effect on the
polymer chain and are also more soluble at lower pH than the alternating GM blocks
(Aravamudhan et al. 2014). An example composition of alginate extracted from the
brown algae Laminaria digitata M and G blocks is 0.47, 0.41, 0.06 and 0.06 MM, GG,
MG and GM (Fertah et al. 2017). Where MM and GG refer to the fraction of the chain
which is made of block arrangements of mannuronic acids and guluronic acids within
the polymer chain and MG and GM refers to the fraction of the chain with alternating
arrangements. Figure 4.2 shows the structure of the alginate repeating units.
Some bacteria containing alginates have also been reported. However, the structure
of alginate obtained from bacteria is quite different from that obtained from seaweed
(algae). Bacteria alginate is generally high degree of polymerization hence high
molecular weight while that from seaweed could be a variety of molecular weight.
The G sequence also varies in bacteria (Windhues and Borchard 2003). Bacteria
derived alginate has a molecular weight in the range of 154,600–730,000 g/mol much
higher than that of seaweed sourced alginate in the range of 48,000–186,000 g/mol
(Nagarajan et al. 2016; Clementi et al. 1999; Donnan and Rose 1950). Nonetheless,
molecular weight of alginate varies widely for different sources.
Fig. 4.2 Structure of

a mannuronic acid and
b guluronic acid
4.3 Chemistry of Alginates 71
4.3.2 Rheological Properties
Polymers have the unique property of increasing the viscosity of a solvent when
they are dissolved in the solvent. Alginates increase the viscosity of the solution and
form irreversible heat-stable gels. This forms the basis of most of their applications
in food and pharmaceuticals. The viscosity-enhancing properties and the gelling
properties of alginates are dependent on the amount of mannuronic acid and guluronic
acid, respectively, within the polymer chain. Alginates with higher mannuronic acid
content have higher viscosity while those with more guluronic acid have higher
gel-forming properties.
Alginates produce non-thermoreversible ionic gels, once formed into gels, and
unlike for example gelatine, they retain these gel forms at different temperatures
unlike for example gelatine which losses its gel form when the temperature is
increased. The ability to form non-thermoreversible ionic gels serves as the basis
of much of the applications of alginates. Alginate forms a gel in the presence of biva-
lent cations through ion exchange. It does not form a gel with monovalent cations
and magnesium ion. Calcium, strontium and barium are the bivalent ions used due
to their non-toxicity. Sodium, potassium and ammonium alginates are water soluble
while the others are insoluble.
4.3.3 Characterization of Alginate
Molecular weight of extracted sodium alginate reported varies in the ten thousands
(40,680 g/mol using the chemical method with a yield of 44.32% (Helmiyati and
Aprilliza 2017). For chemical characterization of polymers, methods commonly used
include FTIR, XRD, DSC and SEM (Olatunji and Olsson 2016). Typical FTIR spec-
tra of sodium alginate show peaks at 939 and 884 cm−1 for uronic acid and man-
nuronic acid functional group, respectively. The alginate chains also include OH
functional group and the CH stretching which also appear in the FTIR scan at around
3200–3400 cm−1 for OH and 2928 cm−1 for the CH stretching. The absorbance and
transmittance depend on the composition of the functional groups within the algi-
nate structure. Pure sodium alginate has a decomposition temperature of 251.12 °C
(Helmiyati and Aprilliza 2017). This is obtained from differential scanning calorime-
try. Extracted sodium alginate should therefore show values close to this depending
on the level of purity achieved during extraction process.
Crystallinity of alginate affects properties such as solubility and thermal proper-
ties. Pure sodium alginate has a crystallinity of about 35.62%. Crystallinity of raw
extract of alginate is usually less than this, and the purer it is, the closer it gets to
the crystallinity value of pure form, for example, a crystallinity 29.292% is reported
in one study for sodium alginate extracted from brown algae (Helmiyati and April-
liza 2017). Furthermore, when the SEM of alginate containing brown seaweed is
compared to that of extracted seaweed, a distinguishing factor is the visible alginate
72 4 Alginates
fibrils which can be seen in the extract but not visible in the intact seaweed due to
the presence of the other components such as lipids, minerals and other non-alginate
carbohydrates. The appearance of distinct fibrils can also therefore be used as an
additional characterization of alginate extract (Fertah et al. 2017).
The color of alginate can vary from whitish to brown. The brown pigment indicates
the presence of fucoxanthin pigment and varies for different types of alginophytes
(Mushollaeni 2011). Another main carbohydrate present in seaweed is laminarin, a
storage polysaccharide which is also discussed in a separate chapter in this book.
4.3.4 Decomposition of Alginate
The decomposition of alginate is important for two reasons: Firstly, if the biodegrad-
able polymer is going to degrade within the body or environment, the safety of the
products of degradation is as important as the safety of the product itself. For exam-
ple, when used as a thickener in food what are the products likely to be formed in the
body after consumption. Secondly, the degradation product could be a useful way
to extend the usage of the product after the first use, for instance, using an alginate
packaging film for the production of organic acids after their use as food packaging.
According to its structure, alginate can be broken down into smaller chains of
either purely mannuronic or guluronic acid oligomers or alternating chains of man-
nuronic and guluronic acid units using enzymes which are specific to the respective
sites. For example, an alginate lyase enzyme, poly-1 → 4-alpha-l-guluronate lyase
enzyme extracted from the mollusk Lamis sp., catalyzes the cleavage of the 1 → 4
glycosidic bond of alpha-l-guluronic acids (Sil’chenko et al. 2013).
The degradation of alginate salts and gels is particularly important as these are
the forms in which alginate exists in many products. Dense alginate hydrogels have
a half-life of 4–6 days in model tissues (Shkand et al. 2016). Under hydrothermal
conditions, alginate degrades into its monosaccharides, mannuronic and guluronic
acids through the hydrolysis of glycosidic bonds. Lactic acids and glycolic acids as
well as sodium carbonate are also produced as products of alginate decomposition
(Aida et al. 2010). Controlled depolymerization of alginates can be carried out to
obtain other organic acids (Aida et al. 2012) and alginate lyase enzymes (Zhu and Yin
2015). The products of the depolymerized alginates are safe and in some cases are
of commercial importance such as the alginate oligosaccharides which are discussed
in the section on applications of alginate.
Alginate is also produced by some species of bacteria such as pseudomonas and
Azotobacter (Chen and Long 2018). These bacteria also produce the enzyme alginate
lyases to break down the alginate for use. The alginate oligosaccharides derived
from the action of alginate lyases on alginate has potential applications such as
protection against disease-causing organisms (An et al. 2009), growth-promoting
agents in plants (Iwasaki and Matsubara 2000) and antioxidants (Falkeborg et al.
2014; Nagarajan et al. 2016; Chen and Long 2018).
4.4 Availability of Raw Material 73
4.4 Availability of Raw Material
Recent years have seen significant increase in seaweed cultivation. The global sea-
weed market is projected to exceed 17.59 Billion USD by 2021 (Monagail et al.
2017) and 87 Billion USD by 2024. About 96.5% of the aquatic plant production
is dominated by seaweed. In 2016, about 30.1 million tonnes of seaweed was pro-
duced, both cultivated and caught wild, and this has shown an average increase of
over 7% between 2010 and 2016 (FAO 2018), such that there exists a very active
seaweed industry to serve the demand for raw material for alginate production. As of
2015, 99% of the global farmed seaweed output was from eight Asian nations while
150 other countries with coastal waters which could take part in seaweed farming
exist, thus indicating the under exploitation of seaweed farming in coastal waters
(Radulovich et al. 2015; Monagail et al. 2017).
Seaweed harvests go up to tens of thousands as of 2009 for example in 2009
France, UK, Norway and Ireland produced 30,500 tonnes of Laminaria spp. In the
same year, Chile and Peru produced 27,000 tonnes of Lessonia spp. A drastically
lower harvest from 35,000 tonnes in 1991 to 5000 tonnes in 2009 was recorded for
Macrocystis pyrifera in 2009. This can be attributed to the closure of the International
Specialty Products facility in San Diego. Ascophyllum also showed a sharp reduction
in harvest between the specified period going from 13,500 tonnes in 1999 to only
2000 tonnes in 2009. This is due to the increase in demand for alginates in higher gel
strength which diverted interest to seaweeds which produced higher guluronic acid
content (Bixler and Porse 2011; Hernandez-Carmona et al. 2013).
China and Indonesia are the leading producers of aquatic plants from aquaculture.
The seaweed cultivation has been on an increase from 13.1 million tonnes produced
in 1995 to 30.1 million tonnes produced in 2016. Indonesia, one of the world’s leading
seaweed producers, reportedly increased production from 4 million tonnes in 2010
to 11 million tonnes in 2016 (FAO 2018). This increase in production is credited
to the increase in demand for seaweed for production of its polysaccharides, agar,
alginate and carrageenan.
Despite the millions of tonnes of farmed seaweed available, the alginophyte
species such as Laminaria and Ascophyllum are obtained from natural stock due to
the expensive cultivation process required for these species which cannot be grown
through the vegetative method but rather must go through a more complex sexual
reproductive process which requires strict control of the environment to facilitate
the alternation of generations at different phases of the production process (McHugh
2003). Such expensive cultivation process can only be compensated by selling the
cultivated seaweed as food rather than as raw material for alginate production. This
leaves the wild seaweed annual production rate at around 1.3 million tonnes, and this
fluctuates annually (Monagail et al. 2017).
10% of the alginophytes are supplied by Chile, and these are from the wild.
Although rarely used for alginate production, when in surplus, cultivated seaweed
can be sold at a subsidized rate for alginate production. In Europe, 99% of the
seaweed produced are from wild harvest, and this makes up 1% of the global seaweed
74 4 Alginates
production with Norway leading the production in Europe at 154,230 tonnes of brown
seaweed harvested from natural stock annually. France harvested 33,919 tonnes wild
seaweed in the same year (Monagail et al. 2017). Therefore, although aquaculture
has become more dominant, wild seaweed still plays a significant role, and however,
these are more economical sold as food for direct consumption rather than as sources
for alginate. 800,000 tonnes of wild seaweed has been recorded in 2014 from a total
of 32 countries. Compared to, as of 2003, 35–42 countries, out of the 195 countries
in the world were reported to be involved in seaweed production (McHugh 2003). As
of 2016, FAO reports 30.1 million tonnes of farmed aquatic plants globally, a large
proportion of this being seaweeds and smaller proportion of microalgae.
It can therefore be concluded that a smaller proportion of the seaweed available
is diverted to alginate production while a larger proportion is used for food as has
been for centuries. This is mainly due to the need for the alginophytes to be sourced
from natural stock. Nonetheless, the alginate market is thriving on brown seaweed
production in the hundred thousand tonnes which presently meets the global demand
estimated at an annual rate of 26,500 MT (Konda et al. 2015).
4.5 Extraction of Alginates
First Isolated by Dr E. C. C Stanford in 1881 in Britain (Draget 2009; Parreidt

et al. 2018), alginate makes up about 10–45% of the dry mass of seaweed (Chen and
Long 2018). Extraction yield of alginate reported ranges from 16.93% from Padina to
30.3% from S. crassifolium (Mushollaeni 2011). Commercially chemical extraction
methods are more widely used in industries. Novel methods are being explored by
researchers which are more cost-effective and also more environmentally friendly.
This includes the use of enzymes such as alginate lyase (Chen and Long 2018).
Alginate is often extracted as sodium alginate or in other forms such as calcium
alginate. These are explored separately in the following subsections.
4.5.1 Extraction of Sodium Alginate
Within the cell walls of brown algae, alginate occurs as salts of alginic acid as calcium,
magnesium and sodium alginic acid salts. Sodium alginate is mostly extracted since
it is the only water-soluble salt of alginic acid. It is therefore of more economic
significance. This solubility in water makes it more practical to extract and also
apply. The extraction process involves isolating the sodium alginate by dissolving it
out in water. The other alginates (calcium and magnesium) must also be converted
to sodium alginates in order to have a good yield of alginate from the source. Three
methods of extractions are outlined as follows. In all three methods, the seaweed is
first prepared by washing, cutting or milling to reduce size and the pigments removed.
Removal of pigment can be achieved with formaldehyde, and the cell walls may be
4.5 Extraction of Alginates 75
softened by soaking in 0.2 M HCL overnight. An alkali, sodium carbonate is then

added under high temperature (~80 °C) to extract sodium alginate in an aqueous
mixture with the seaweed residue. The solid residue is then separated by filtering
(Fertah et al. 2017). The process then proceeds using either of the two methods below
to recover the sodium alginate from the aqueous solution.
4.5.2 Method 1
The first method involves the addition of calcium salt to the liquid extract which
results in the formation of calcium alginate which is insoluble in water. This can
therefore be separated from the mixture by filtration or pressing. This is followed
by adding acid to form alginic acid. Alcohol is also added to render the sodium
alginate to be formed, insoluble. Sodium alginate is then formed by the addition of
sodium carbonate which precipitates and is separated and dried followed by further
processing depending on the end use.
4.5.3 Method 2
In this method, acid is first added to the liquid extract. This results in the formation
of alginic acid. This alginic acid is insoluble in water forming a soft gel which can
be separated from the aqueous mass. This is followed by addition of alcohol and
then an alkali, sodium carbonate to form sodium alginate from the alginic acid. The
alcohol added to the water allows the sodium alginate to precipitate out of the water
once formed, thus making it easier to separate. The product, sodium alginate can
then be dried and used for further processing and application. This method makes
use of mainly acid and alcohol.
4.5.4 Method 3
In this method following the sodium carbonate extraction, the sodium alginate is
precipitated using ethanol (Fertah et al. 2017). This method is more commonly used
for laboratory-scale extraction where dewatering processes might be impractical at
the small scale and alginate of high purity is required. Furthermore, this method can
be used where cost saving is not of concern, and the alginate is being extracted,
mainly for research purposes.
All these methods generally involve the addition of acid to dissolve the sodium
alginate from the seaweed mass. This alkaline extraction takes about two hours and
requires a hot solution of sodium carbonate. The seaweed residue is then separated
from the liquid. The main task that follows is therefore to separate the sodium alginate
76 4 Alginates
from the water. Water has a rather high latent heat of evaporation and therefore
requires lots of energy to evaporate such that less energy-consuming and economic
methods are employed to recover the sodium alginate. This is either method 1 where
acid is added followed by dewatering and reforming of sodium alginate precipitate or
method 2 where calcium salt such as calcium chloride is added followed by reforming
and precipitation of sodium alginate. Figure 4.3 summarizes the extraction process
in a flowchart.
In between, the methods as outlined above exist some major challenges mainly
in separation. The alkali treatment process yields a very thick slurry made up of the
sodium alginate solution and the solid residue of the seaweed, comprising mostly
cellulose. This cellulose can also be extracted for other uses (discussed in chapter
dedicated to cellulose) (Lakshmi et al. 2017). To ease filtration of the thick slurry,
large volume of water is added to reduce the viscosity and improve flow in the
filter. Filtration aids are also often used to improve the filtration process and prevent
clogging of the filter as the slurry also contains small size particles which can clog
the filter pore.
Depending on whether it is method 1 or 2 that is used to recover the sodium
alginate the process of separation varies. If the calcium chloride method is used,
the fibrous calcium alginate formed can be separated by filtration with a sieve and
washed. Additional step involves conversion to a fibrous form of alginic acid which
is then further screw pressed to reduce water content. Conversion to sodium alginate
Brown algae pretreatment
Solid residue to
Sodium Carbonate Separation waste or further
treatment use/processing
Sodium alginate
Method 1 Method 2
solution
Calcium Chloride Method 3

Acid
Acid addition
Ethanol precipitation Dewatering
Sodium carbonate
Sodium carbonate
Centrifugation
Sodium alginate
Fig. 4.3 Flow chart illustrating alginic acid extraction from brown algae using method 1 and 2
4.5 Extraction of Alginates 77
then follows as outlined in method 2. If the acid method is used, alginic acid formed
is gelatinous and cannot be separated by filtration, and flotation is used in this case.
The gelatinous alginic acid is then dewatered followed by reconversion to sodium
alginate as outlined in method 1.
The color of the sodium alginate extract is very important in enhancing or dimin-
ishing its market value. Lighter colored alginate attracts better price. This necessitates
a decoloring process either before or after the alkali treatment. Decoloring can be
achieved by either bleaching the final product with sodium hypochlorite to remove
color or soaking the seaweed in formalin prior to extraction in order to adhere the
pigment to the cellulose such that the pigment is retained in the cellulose while the
sodium alginate is extracted.
4.5.5 Extraction of Calcium Alginate and Other Salts

of Alginic Acid
The process of extracting sodium alginate also involves formation of calcium algi-
nate, and therefore, if calcium alginate is the desired product, following the initial
treatment of the seaweed with hot sodium carbonate, the calcium salt route of method
1 can be taken (McHugh 1987). Addition of calcium salt such as calcium chloride
will result in the formation of calcium alginate which is an insoluble fibrous alginate
that can be separated by filtration. Although sodium alginate is more widely pro-
duced, calcium alginate finds increasing applications in food and pharmaceuticals.
The other salts of alginate are obtained following neutralization of alginic acid using
alkali like potassium carbonate and ammonium hydroxide. The alkali used depends
on the desired salt.
However, propylene glycol alginate is made in a different way. It is an ester of
alginic acid, and its application varies from that of the alginate salts. The process of
producing propylene glycol alginate begins from the moist alginic acid which has
partially reacted with sodium carbonate. This partially reacted alginic acid is then
treated with propylene oxide at a temperature of 80 °C for two hours under pressure.
4.5.6 Enzyme Extraction of Alginate
Although the chemical extraction method is almost the only method used in com-
mercial production of alginate, other methods of extraction are possible greener
alternatives to obtain alginates. For such process, alginate lyases can be employed,
and however, these alginate degrading enzymes are primarily used in breaking down
the alginate chains into oligosaccharides of alginate, mannuronic and guluronic acids
(Rhein-Knudsen et al. 2015). These have been shown to have some bioactive proper-
ties and show potential for applications in biomedicine and biofuel production. The
78 4 Alginates
enzyme extraction method is based on the alginate lyase breaking down the alginate
within the cell walls into shorter chains and monomers which are more water soluble.
These are then released in the aqueous form and can be separated from the rest of
the solid. Enzyme extraction is rarely used for alginate.
Having established an understanding of the source, chemistry and production process

for alginate, here we evaluate the environmental impact of the stages involved in
the extraction process. Some estimates on material and energy consumption during
extraction are given based on results from extraction of alginate in reported studies.
Table 4.1 summarizes the consumption for a typical bench-scale extraction of sodium
alginate from L. digitata (Fertah et al. 2017).
4.6.1 Brown Algae Cultivation and the Environment
Since alginates are obtained from brown seaweeds which are mainly sourced from
wild, this section focuses on the environmental impact of harvesting of wild sea-
weed for alginate production. Seaweeds have a major environmental benefit being
extractive species, and they serve the benefit of extracting waste material from the
environment. They produce their food using the process of photosynthesis like other
Table 4.1 Typical

consumption for bench-scale
extraction of alginate from Brown algae 1.93 g per gram alginate produced
brown algae Water 60 mla per gram algae
Formaldehyde 15 mla of a 2% solution/gram algae
HCl 15 mla of a 0.2 M solution/gram algae
Sodium carbonate 15 mla of a 2% solution/gram algae
Ethanol 15 mla of 95% solution per gram algae
Energy
Centrifugation 3500 rpm for 15 minb
Drying 60 °C for 8 h
Heat for extraction 40 °C for 3 hb
Agitation 200 rpm for 5 hb
a Assumes a 1:15 g/ml, mass-to-volume ratio of algae dry mass-to-
volume of liquid; b typical values of time and rpm are used where
not specified in the reported study
plants, and they synthesize their food from nutrients extracted from the aquatic sys-
tem they grow in. These could be the waste produced by other species, thereby
cleaning up the environment which helps to promote and sustain the growth of other
species. Such that farming of seaweeds alongside other fed species of aquatic animals
within the same culture is encouraged in order to benefit from the synergy between
extractive and fed species for optimal use of resources. This has become common
place as the farming of extractive species is estimated at 49.5% as of 2016 (FAO
2018).
Another advantage of wild seaweed as a source of alginate is that it requires
even much less intensive care such as watering, fertilizer application and weeding as
required by land crops. On the other hand, the cost of sending vessels out to sea and
into rocky coasts, the machinery and manpower required for harvesting contribute
to the cost of the raw material which varies around 50–100 USD per MT depending
on the quality and source (Konda et al. 2015).
Although seaweed grows naturally in the wild, meeting the demand for future
commercial alginate production cannot be sustained by the natural stocks alone.
This means new technologies need to be developed for more profitable cultivation of
alginocytes on commercial scale. There are also issues concerning the overexploita-
tion of natural seaweed resource as the demand for seaweed continues to rise. Recent
concerns of overexploitation, environmentally harvesting methods and diminishing
seaweed beds have lead to restriction of harvesting of some species of seaweeds in
countries such as Canada and Portugal (Monagail et al. 2017).
The seaweeds in nature play a significant role in sustaining the aquatic flora and
fauna. They help remove waste products of aquatic animals preventing toxic levels
which could be harmful to the animals and other organisms. They also provide
shade from light to aquatic organisms which thrive better under low light, and they
serve as habitat and refuge to some aquatic organisms, offering protection from
predators. Seaweeds also have the impact of dampening tidal waves by absorbing
the wave energy, thereby preventing or reducing coastline erosion (Monagail et al.
2017). Seaweed harvesting has also been shown to significantly alter biodiversity and
population of some species in areas such that commercial-scale harvesting of wild
seaweed for alginate production pose some significant impact on aquatic ecosystem.
Global climate change has been shown to have affected seaweed species distribu-
tion. The reported 0.6 °C increase in temperature has had notable impact on species
distributions in areas such as Spain and Portugal, while extreme weather conditions
like earthquakes, tsunamis and el nino have resulted in total eradication of seaweed
beds in areas such as Chile (Castilla et al. 2010).
Brown algae are typically harvested by cutting the upper part where much of
the useful carbohydrates are found, leaving behind the lower part which allows the
regrowth of the plant within a year or two. The rate of regeneration after harvest
depends on factors such as the efficiency of the harvest method, species, environment
and others. The harvest could be done by hand, using rakes with boats, diving, use of
cutters or mechanical means. Mechanical harvesting has been discontinued in some
areas due to adverse effect on the seaweed population and environment (Monagail
et al. 2017).
80 4 Alginates
Seaweed cultivation, although requires less land space, does not always involve
less resource usage when compared to terrestrial crops such as corn, sugar beet or
potatoes. The resource consumption varies for different methods of cultivation, and
brown algae cultivation is particularly more resource intensive as the reproductive
process of brown algae is a more complex alternating reproductive cycle compared
to those which reproduce vegetatively. Aquaculture of cush algae generally requires
more resource use than wild-sourced seaweed. Because some seaweed cultivation
involves permanent or partial occupation of aquatic environment, this could have
further impacts such as blockage of sunlight for aquatic life below and mechanical
displacement. Cultivation of seaweed, however, reduces problems such as loss of
seaweed species through overharvesting of wild stocks.
Contrary to algae grown for direct food consumption which are mostly grown on
a commercial scale in aquaculture, seaweed for algae production is best grown wild
and harvested from natural source. Seaweed harvest for algae production, therefore,
requires less resource consumption outside of transport and the much less temporary
impact of the process of harvesting.
4.6.2 Chemicals
Based on the sample case study (Konda et al. 2015) which is representative of a
typical bench-scale extraction process, producing 26 mega tones of sodium alginate
pellets requires 720 MT formalin at a concentration of 0.1%, 835 MT of HCl at 0.38%
concentration, 73 MT of sodium carbonate at 10% concentration for extraction, 500
MT of calcium chloride at 10% concentration for the formation of insoluble calcium
alginate salt and a further 4.2 MT of sodium hypochlorite at 5% for bleaching. More
HCl is then used for neutralization after bleaching requiring a further 150 MT of 3.6%
concentration. Sodium carbonate is then added again to reform the sodium alginate
salt and this requires about 80 MT at 10%. Finally, the extracted product is purified
and dried using around 140 MT of air and 281 MT of a mixture of ethanol, methanol
and acetone at around 48% or as required. Although alginate is a biopolymer from
a natural renewable source, the use of mineral-based acids and alkali and other
chemicals may have contradictory impact on the objective of alginate as a greener
alternative to synthetic fossil-based polymers.
4.6.3 Land Use
A general advantage of aquatic sourced raw materials for biopolymers is that the
raw materials do not require large land areas for production; rather, they make use of
aquatic space. This is significant as land spaces for human habitation are becoming
more limited due to the increasing population of humans and the need for more
land space to grow food crops and rear land animals for survival of the population.
Table 4.2 Compositions of

Component Composition (%)
Brown seaweed (S.
latisimma) and corn stover Brown algae (S. Alginate 32
latissima)a Protein ~0
Laminarin 15
Mannitol 18
Ash/salt 35
Corn Stover Hemicellulose 19
Lignin 16
Protein 3
Cellulose 35
Other 27
a Forthe same species, values vary significantly with harvest
period, growth conditions
In addition to this, the biomass harvested for alginate production does not remove
nutrients from the soil. On the contrary, seaweeds remove waste from the water and
carbon dioxide from the air. Therefore, their growth, whether wild or cultivated,
contributes positively to the environment.
Seaweeds also grow more rapidly than other terrestrial plants such as sugarcane
or maize as they carry out photosynthesis 3–4 times more efficiently. Seaweed could
grow between 30 and 80 dry MT per hectares annually while other terrestrial plants
grow at a rate of 3–30 dry negatonned per hectares per year. By virtue of its size only,
brown seaweed among the seaweed serves as an abundant source for carbohydrate
biomass and has been shown to have little to no lignin content. This makes extraction
of the carbohydrate content relatively less demanding than biomass with higher lignin
content. Table 4.2 compares the carbohydrate content of brown seaweed to that of
corn stover on dry basis. Note that this is composition for a specific species (S.
latissima) and the compositions also vary with season. The compositions here are
based on three harvests between December 2010 and August 2011 (Konda et al.
2015).
4.6.4 Energy Consumption
The seaweed is often processed with a starting moisture content of around 25%
drying which is energy intensive, and therefore, some of the cost of drying can be
reduced by drying in the sun. The cultivation and harvesting have been determined
to be the most energy-consuming stage. This requires sending out vessels to source
the seaweeds which could be several miles offshore.
The initial alkali extraction stage for both methods is carried out at 80 °C (Fertah
et al. 2017). Advancement in enzyme assisted alginate extraction could offer lower
82 4 Alginates
energy-consuming process in the future. This is the stage in the extraction where
the most heat is consumed. Further energy is consumed in running the equipment
for dewatering processes which could be done by filtration or gel pressing. These
consume less energy than evaporation of the sodium alginate from aqueous solution.
Assessment of algae seaweed production in Ireland revealed that 75.1% of sea-
weed sourcing resource consumption is attributed to fossil fuel consumption required
for transportation and machinery to operate aquaculture (where the algae are not from
wild stock) (Taelman et al. 2015).
4.6.5 Water Consumption and Wastewater Generation
An estimated 1500 MT of water used in the extraction process and further 3900 MT
of water for dilution in production of 26 MT of sodium alginate dry pellets (10%
moisture content) in the case study presented by Konda et al. (2015). Harvesting
brown algae from wild stocks makes little direct use of water, and alginate production
makes use of a lot of water to the extent that the survival of an alginate production
factory is largely affected by reliable availability of abundant water. The wastewater
generated has high pH from the initial extraction stage while the later extraction stage
will generate wastewater containing calcium or acid, depending on the method used.
It also contains the compounds used to decolorize. The generated wastewater can
either be recycled in-house or sent to wastewater treatment plant before releasing it
to the sea in accordance with the local laws.
4.6.6 Carbon Dioxide Emission
Commercial alginate extraction process results in CO2 emission from the running
of the alginate production facilities. On the other hand, seaweeds are photosynthetic
aquatic plants, whose rate of photosynthesis is 3–4 times more efficient than that
of terrestrial plants. The CO2 emission in the extraction process is therefore partly
compensated for by the CO2 removal during photosynthesis. This is even more
advantageous for aquatic plants as the plants do not remove nutrients from the soil.
4.6.7 Solid Waste Generated
The main solid waste generated is the seaweed residue after the alkali sodium alginate
extraction. The solid residue also contains the formalin and filtration aid if used.
Although details of the process used by commercial producers in the treatment and
deposition of the solid waste generated from alginate production from seaweed are
not provided here, some research publications have presented innovative ways of
processing and reusing such solid wastes. These include reuse of adsorption of heavy
metals in waste treatment (Romero-Gonzalez et al. 2001) and the use of the laminarin
and mannitol content for bioethanol production (Horn et al. 2000). The utilization of
the solid wastes generated following the extraction of alginate further adds value to
the feedstock which the alginate producer could either use in a coproduction process
or sell to biorefineries.
4.6.8 Diversion of Resource for Alginate Production
Seaweeds for centuries have been harvested for food, especially in Asia where they
have constituted an essential part of the cuisine for centuries such as seaweed wraps,
salads and soups. Wild seaweed has high market value as direct consumption as food.
The process of converting the harvested seaweed into food requires much less capital
investment, and the cost of production is covered by the payment in service rendered,
compared to the extraction of alginate from seaweed which requires additional facil-
ities, materials, energy and skilled labor. The justification of these additional inputs
could lie in the role alginate which plays as a food additive in improving food quality
and shelf life which contributes toward the sustainable development goal of making
more food available for more people, in cases where alginate does play such a role.
Brown seaweeds are also used as fertilizers, and therefore, the alginate indus-
try competes with these other products for the brown algae resource. For example,
in Europe brown seaweed species such as Laminaria and Ascophyllum are used
for fertilizers, and these are the same species widely used for alginate production.
Other than alginate seaweeds also have potential in biorefinery as bioethanol for fuel
production and other uses. Although requiring much more running cost and capital
investment than for example ethanol, alginate production cost is compensated for by
the relatively higher selling price estimated at around 14–30 USD/kg (Konda et al.
2015) compared to ethanol which sells at around 2.5 USD/gal.
4.7 Applications
Global demand for alginate is estimated at 26,500 MT per year (Konda et al. 2015).
Application of alginates ranges from the use as a thickener in baked food, stabilizer
of beer foam, bandages, textiles and biomaterials for tissue repair. Today the demand
from more products to be made from naturally sourced polymer encourages the
production of polymers such as alginate.
84 4 Alginates
4.7.1 Alginates in Food
One of the widest applications of alginate is in food. Its excellent gelling and
viscosity-enhancing properties make it suitable as a thickening, gelling agent, emul-
sion stabilizer and texture modifier in foods such as dressings, ice creams, noodles,
beer, jelly and others. Alginate is FAO, WHO and FDA approved for food appli-
cations. The forms of alginate used in the food industry are alginic acid, sodium
alginate, potassium alginate, ammonium alginate, calcium alginate and propylene
glycol alginate. These are labeled E400–E405, respectively Featherstone (2015).
Alginates in food industry allow storage of food in cans, jars, hard gels, etc.
for longer periods while retaining or even improving the texture and consistency.
In modifying the texture, retaining moisture and improving the texture of foods,
alginate serves the role of extending food shelf life and preserving the organoleptic
properties over the food shelf life period. This has the economic and environmental
impact of reducing food waste and serving as an alternative to more energy-intensive
preservation methods such as freeze drying or freezing. Foods such as vegetables,
fruits, cheese, seafood and meat can be stored at room temperature when preserved
with alginate in methods such as canning and sealing.
The use of alginate can also be used to reduce the amount of other components
needed due to its superior viscosity enhancing and gelling property. For example,
where 5% starch is used as a thickener, about 4% of the starch can be replaced
with 1% low viscosity sodium alginate with calcium salt. This allows processing
such as improved heat transfer during heat sterilization and mixing, unlike starch
which is more sensitive to heat. The sodium alginate then reacts with the calcium
salt over time after processing to achieve the desired viscosity in the sealed food. The
viscosity difference can be up 10x Featherstone (2015). So with sodium alginate, you
can process the food at lower viscosity for easy mixing and heat transfer and then
package and seal while the sodium alginate in the presence of calcium salt continues
to increase in viscosity over a period of several hours or days.
Sodium alginate is the alginic salt which is soluble in water and is used in the food
industry for its viscosity-enhancing property. It also forms a non-thermoreversible
stable gel at room temperature on addition of calcium salt such as calcium chloride
or other polyvalent metal ions. This property makes it useful as a thickener, gelling
agent and stabilizer in the food industry. Polypropylene glycol alginate is also used
in the food industry as an emulsion stabilizer and stabilizer.
Even better than biodegradable packaging is edible packaging in the form of
films or coatings. Being indigestible edible polysaccharide alginate packaging can
contribute to the fiber intake. Alginate can serve as a sacrificial moisture agent,
whereby the moisture which would otherwise evaporate from the food evaporates
from the alginate coating instead such that the food retains its moisture content.
Alginate is used to produce edible films with thickness no more than 0.3 mm which
can be eaten with the food or easily removed. This packaging is to be as safe as the
food itself and should not alter the organoleptic properties of the food. Methods for
producing alginate films include solvent casting and extrusion (Olatunji and Olsson
4.7 Applications 85
2016) while methods of applying coatings could be dipping, spraying and vacuum
impregnation (Parreidt et al. 2018).
Alginate coatings can also improve the aesthetic appeal of the food by improving
gloss, reducing the chances of bruising by giving a more rigid surface and even hiding
surface imperfections. Aesthetics are important in reducing food wastage as some
foods are rejected simply due to their appearance whereas the food still has all its
nutrients, taste and texture intact. Therefore, improving the aesthetics will reduce the
chance of the food being rejected and left to spoil.
To enhance other properties such as antibacterial and antioxidant properties,
alginate-based edible films and coatings usually incorporate other compounds with
these desired properties. The incorporation of these compounds in the coating or
film has the advantage of not being included in the main food in case where for
example the direct contact with the food could initiate some undesirable interactions
which alters the food property or quality. Mechanical properties such as elasticity
and film-forming ability of alginate could be improved with the use of plasticizers
or forming composites with other compounds (Parriedt et al. 2018).
4.7.2 Textiles
Alginate serves as a more environmentally friendly printing substrate for laying

down color patterns on cotton, rayon and jute in the textiles industry, as it is a
more biodegradable option. Its viscous nature allows formation of printing pastes
of good consistency and being hydrophilic as well as its ability to form a paste at
relatively moderate water temperature. Sodium alginate is commonly used in printing
pastes as it prevents the otherwise hardening of the fabric once reacted with the dye.
When sodium alginate is used as a printing paste, a better fabric feel is achieved.
Sodium alginate also achieves good color yield and maintain stability. Application
of sodium alginate in textiles has been practiced for many decades (Rompp et al.
1983; Wang et al. 2014), and as demand grows for novel fabric technologies and
more biodegradable fabrics, alginate is likely to find increasing application in the
modern textile industry. Low to medium molecular weight sodium alginate is more
suitable for textile applications. Alginate printing pastes can be applied for screen
printing, roll printing and inkjet printing. In terms of printing paste, sodium alginate
has superior properties to other options such as starch.
4.7.3 Lowering Blood Sugar Level
Diabetes has become a medical condition of increasing significance with increasing

incidence annually across the globe. Use of alginate-based dietary supplements as
either a preventive or therapeutic measure further indicates high demand for alginate-
based products in the pharmaceutical and nutraceutical industry.
86 4 Alginates
Alginates in forms such as calcium alginate, sodium alginate and alginate gels
have been shown to have the effect of reducing blood sugar level when consumed
orally (Hisni et al. 2016). The mechanism by which this is achieved has been studied
to be due to the suppression of starch digestion while calcium alginate did not affect
the permeability of glucose across membranes nor bind to the glucose to prevent
absorption into the bloodstream as is the mechanism for some blood sugar lowering
substances, calcium alginate acts by inhibiting the action of the enzyme glucosidase
which breaks down starch to sugars. It is thought that at a dosage of 5% body weight
with a particle size of 53 µm, calcium alginate can aid in reducing blood sugar levels
(Idota et al. 2018). Some studies in human subject also indicate potential for alginate
gels to aid in reducing the uptake of cholesterol as well as glucose as a means of
controlling levels in the blood at a dosage of 1.5 g, reduced cholesterol and glucose
levels were measured in the test subjects. The mechanism by which this is achieved is
thought to be through the delay of the uptake by the strong gel (Paxman et al. 2008).
Retention of the glucose and cholesterol within the gel or acting as a barrier between
the intestine wall and the glucose or cholesterol could prevent or delay their uptake
into the bloodstream resulting in more of them being passed along the alimentary
canal along with the alginate fiber.
4.7.4 Biomedical Application
In tissue engineering, different forms of alginate are applied for the production of
biopolymer-based extracellular matrix (ECM) to promote tissue regeneration. Their
ability to provide stability in hydrogel formulations and maintain an aqueous envi-
ronment by absorbing and retaining biological fluids makes alginates attractive for
such purpose. Advanced studies up to clinical trials on animals and humans have
been carried out on a number of alginate-based hydrogel implants for cardiac regen-
eration (Liberski et al. 2016). Presently, there is no cure for cardiac failure, and
treatments exist to manage cardiac illnesses. Among the potential approaches to
treat heart failure is the use of alginate-based biomaterials to achieve self-repair of
the cardiac tissue. The properties of alginate which allows them to form viscous
fluids when dissolved in water and to form hydrogel when reacted with calcium salts
such as calcium chloride make them suitable for application as injectable implants
for cardiac repair. Here, the unique hydrogel-forming property of alginate comes as
an advantage as it can form hydrogels in physiological fluid under mild temperatures
~40 °C). These hydrogels can mimic the biomechanical properties of the cardiac tis-
sue. The biodegradability of alginate is also important for such purpose as it remains
stable enough to allow formation of the ECM, and once the tissue is regenerated, it
can biodegrade. Companies such as Life Technology Inc., LoneStar Heart. Inc and
Bellerophon BCM LLC have developed alginate-based products for cardiac tissue
regeneration, some at various stages of clinical trials such as AlgiMatrix, Algisyl-
LVR and PRESERVATION. Different salts of alginates with varying structures and
molecular weight are used and tailored for specific mechanism of action. In the area
4.7 Applications 87
of cardiac tissue regeneration, alginates have also been used as a pharmaceutical

excipient for the release of angiogenesis drugs, cell transfer, and promote interaction
between implant and cardiac cell.
Alginate-based hydrogels are also used as injectable fillers in cosmetic dermato-
logical procedures to alter facial features such as lip plumpness and appearance. One
such product is commercially available since 2010 under the brand name Novabel
(Moulonguet et al. 2011).
4.7.5 Alginate Oligomers and Monomers
Oligosaccharides of alginates are of commercial significance as they offer a range

of high-value bioactive properties. These bioactivities include antioxidant, antiin-
flammatory and antiproliferative effects (Wan et al. 2018). For this reason, they have
found applications in food (animals and humans), agriculture, pharmaceutics and
cosmetics industry.
In agriculture, alginate oligosaccharides are used for their growth-promoting abil-
ities to promote endothelial cells and multiple cytokines. When used as animal feed
supplement, alginate oligosaccharides boost serum hormone levels and antioxidant
activity and also improve intestinal functions and integrity in pigs (Wan et al. 2017).
The method of degradation of alginate by alginate lyase has been shown to be
a factor in influencing the bioactivity of the resulting oligomer. Alginate lyase-
derived oligomers had superior bioactive properties such as antitumor effect while
acid hydrolyzed alginate oligomers only had the reduced chain length without the
bioactive properties (Takeshita and Oda 2016).
4.7.6 Other Applications
Other uses of alginates include use in animal feed to improve texture and stability
and in welding rods where sodium silicate is coated unto the surface of the metal
rods. Alginate is used to bind the coating and hold it in place hence aiding a uniform
coating upon drying. This is achieved by the gelling property of alginate. In the
paper, industry alginate is used as a partial replacement for rosin pulp. It is used to
achieve a smoother surface and better absorption of ink during printing. It also aids
production of crumple resistant paper by promoting absorption of wax and grease.
Alginates are attractive in micro- and nanoparticle formulation due to their ability
to form stable structure (Paques 2015). Alginate is also used in the encapsulation of
live bacteria and active enzymes (Qin et al. 2018). Another well-known application
of alginate in the cosmetic industry is for use as a color retainer in lipstick.
88 4 Alginates
Alginate was discovered in 1882, and its commercial production was first established
in San Diego in the USA in 1927. Production of alginate in the UK followed between
1934 and 1939 years later began in Norway (McHugh 1987). Today alginate is being
produced commercially in countries in Europe, Americas, Asia-Pacific and Asia.
From its first use as a binder for briquette production to now being used in various
industries from food to textiles to pharmaceuticals alginate application has advanced
through the years across different industries.
Compared to some lesser-explored biopolymers, alginate is moderately well
explored commercially in various industries. Annual production of alginate from
seaweed is estimated at 38,000 tonnes (Venkatesan et al. 2014). Alginate production
from bacteria is yet to be commercialized. The world alginate market is estimated
to be worth 624 million USD as of 2016. The highest demand for alginate to date
is in the food industry where they find various applications (note this is different
from the direct consumption of seaweed as food). As the world population increases
and the level of civilization increases, demand for processed foods is expected to
consequently increase and alongside this will be increased in demand for food pro-
cessing agents such as ice cream, beer and packaged meat, all of which require the
use of alginate of one form or the other in their processing. Propylene glycol alginate
has shown steady rise in market revenue between 2014 and 2018. It is expected to
show further increase in market size for the next 7 years. Sodium alginate which is
also in high demand in the food industry shows similar steady reported and poten-
tial increase. Although calcium and potassium have lower rate of production in the
industry, they are also expected to show increase in revenue over the coming years,
especially as more novel applications for these forms of alginate emerge.
Cost of production of algae is still relatively higher compared to the commercial
production of well-established mechanized crop farming of crops such as corn and
soya bean. Each dry tonne of L. japonica costs between 650 and 700 USD to produce
in China and producing the carrageenan-producing species of seaweeds in Mexico
costs around 689 USD. This is higher than the estimated cost of producing corn and
soybeans in the USA which are estimated at 195 USD and 408 USD, respectively
(Forster and Radulovich 2015).
Alginates with higher guluronic acid content are in higher demand in the food
industry since this offers better gelling properties. While alginates with high M offer
better viscosity-enhancing property, other polymers exist which offer similar, and
however, the superior gelling properties are more unique to alginate. The demand
for alginate with higher guluronic acid is estimated to grow at a rate of 4.8ˆ CAGR
between 2016 and 2025.
Manauronic rich alginate finds more use in the textiles and paper industry where
their viscosity-enhancing properties are more valued. The superior binding properties
of alginate also makes it useful as welding rod as more developing countries begin
to focus on the development of manufacturing and construction, easily sourced raw
materials from renewable sources such as alginate begins to increase in demand.
4.8 Commercial Production 89
Calcium alginate, more commonly used in the pharmaceutical industries for appli-
cations such as wound dressing, is estimated to reach a market value of 154.3 million
USD by 2025. The use of calcium alginate as a wound healing aid also makes it
applicable in food as a meat binder. This increased product use in newer applications
in the industry is suspected to result in increased demand and hence commercial
production.
Propylene glycol alginate, commonly used in the food industry as an emulsifier,
thickener and stabilizer, is also used in the beer industry for foam stabilization. The
beer industry is one of the largest industries from large-scale beer manufacturing
across the globe in both developed and developing countries and rising such appli-
cation further points to expected growth in the alginate industry. Propylene glycol
alginate is also used in fruit juices and foamy dairy products. 26% of the demand for
alginate is allocated to propylene glycol alginate (Grandview Research 2017).
The commercial production of alginates can be said to be well explored across dif-
ferent countries. Key players in the alginate market includes as follows: ISP Alginates
Ltd. (UK), FMC Biopolymer USA, Degussa Texturant Systems in Germany, Kimica
Corporation in Japan, Danisco Cultor in Denmark, Figu Chemical Industry Co. Ltd.
in Japan, Algisa Compania Industrial de Alginatos S.S. in Chile and China Seaweed
Industrial Association In China (McHugh 2003), Seasol International, Acadian Sea-
plants Ltd., Yan Cheng, Chase Organics GB Ltd., Indigrow Ltd., Mara Seaweed, CP
Kelco, Aquatic Chemicals and Pacific Harvest. Many others exist operating at small
and larger scales across the globe.
Commercial alginate is priced around 12USD per Kg (Hermandez-Carmona et al.
2013), making it the second most expensive seaweed sourced polysaccharide, second
to Agar. By region, Europe took the lead in alginate production as of 2003 producing
16,000 tonnes of alginate (McHugh 2003), followed closely by Asia-Pacific which
produced 15,600 tonnes and then the Americas which recorded a production of
4500 tonnes. There was no alginate production recorded in Africa. As of 2013, the
combined production of alginates, agar and carrageenan from seaweed was worth
1.018 Billion USD as of 2013 alginate was valued at 12 USD per Kg (Hernandez-
Carmona et al. 2013).
The main issue which surrounds future market of alginate is the future availabil-
ity of seaweed for alginate production as the natural stocks available are a limited
resource. If well managed, with harvest rate balanced by the rate of regeneration
and recovery, long-term availability of raw material for alginate production should
be of little concern. The sustainable strategies being adopted by the alginate market
stakeholders which includes the manufacturer, government and regulatory bodies and
environmentalists include sustainable harvesting of seaweed for alginate production
and research and development into alternative and sustainable alginate sources. Other
factors which threaten the natural stock of seaweed are environmental disaster which
could destroy large stocks of seaweed at an instance such as tsunamis. These factors
are all important when considering the prospects of commercial alginate production.
Cost analysis of alginate production is presented in Table 4.3. The estimate used
below is obtained from the scenarios presented in literature (Konda et al. 2015)
where cost analysis is carried out for simulated scenario of the coproduction of
90 4 Alginates
Table 4.3 Cost estimates for

Cost Estimates in Million USD/annum
production of
130,000–220,000 MT/year of Facilities cost 155
alginate Raw materials 300
Waste treatment 30
Utility 40
Annual operating cost 520
ethanol and alginate from brown algae. The estimated cost for alginate is obtained by
subtracting the cost estimates for ethanol production from brown algae from the cost
of coproducing of ethanol and alginate. An assumption is made that the additional
cost is from the additional process for alginate production and is independent of
ethanol production. The figures given here could overestimate or underestimate the
real cost of production, and however, it is used here merely for comparison of the
costs. The estimated output of 130,000–220,000 MT annually assumes a yield of
50–88% (extracted alginate/theoretical content).
The potential for coproduction of alginate alongside ethanol production from
seaweed biomass is also a possibility. However, assessments show that for such
biorefineries, the alginate production rate required to make the process economically
viable would exceed the current global demand for alginate which could result in a
crash of the alginate market resulting from surplus supply (Konda et al. 2015).
4.9 Conclusion
Alginates have relatively diverse applicability which cuts across several industries
and therefore has a large commercial significance. Extraction of alginate requires
use of acids, alkali and other chemicals which pose some environmental impacts.
The process of harvesting and cultivating brown algae for use in alginate production
offers some benefits to the environment such as CO2 removal from the atmosphere and
removal of nutrients from water thereby abating eutrophication, when well managed.
Future advancement could possibly see the use of alginates in bioethanol production,
and this is dependent on further advancement in developing enzymes to catalyze the
breakdown of alginate into fermentable sugars. Alginate is one of the aquatic biopoly-
mers with a well-established market, extraction and applications. Nonetheless, there
still exist several aspects where advancements are required, such as the development
of enzymes for controlled hydrolysis and more environmentally and economically
sustainable extraction processes.
References 91
References
Aida TM, Yamagata T, Abe C, Richard L, Smith J (2012) Production of organic acids from alginate
in high temperature water. J Supercrit Fluids 65:39–44
Aida TM, Yamagata T, Watanabe M, Smith RL Jr (2010) Depolymerization of sodium alginate
under hydrothermal conditions. Carbohydr Polym 80(1):296–302
Aleksandra R, Sanja SI (2017) The Influence of nanofillers on physical- chemical properties of
polysaccharide- based film intended for food packaging. In: Food packaging, pp 637–697
An QD, Zhang GL, Wu HT, Zhang ZC, Zheng GS, Luan L, Murata Y, Li X (2009) Alginate-
deriving oligosaccharide production by alginase from newly isolated Flavobacterium sp. LXA
and its potential application in protection against pathogens. J Appl Microbiol 106:161–170
Aravamudhan A, Ramos DM, Nada AA, Kumbar GS (2014) Natural polymers: polysaccharides
and their derivatives for biomedical applications. In: Kumbar SG, Laurencin CT, Deng M (eds)
Natural and synthetic biomedical polymers. Elsevier Press, pp 67–89
Bixler HJ, Porse H (2011) A decade of change in the seaweed hydrocolloids industry. J Appl Phycol.
https://doi.org/10.1007/s10811-010-9529-3
Castilla CJ, Manriquez PH, Camano A (2010) Effects of rocky shore coseismic uplift and the 2010
Chilean mega-earthquake on intertidal biomarker species. 418:17–23
Charrier B, Abreu HM, Araujo R, Bruhn A, Coates JC, De Clerck O, Katsaros C, Robaina
RR, Wichard T (2017) Furthering knowledge of seaweed growth and development to facilitate
sustainable aquaculture. New Phytol 216:967–975
Chen F, Long J (2018) Influences of process parameters on the apparent diffusion of an acid dye in
sodium alginate paste for textile printing. J Clean Prod 205:1139–1147
Clementi F, Crudele MA, Parente E, Mancini M, Moresi M (1999) Production and characterization
of alginate from Azotobacter vinelandii. J Food Sci Agric 79(8):602–610
Donnan FG, Rose RC (1950) Osmotic pressure, molecular weight, and viscosity of sodium alginate.
Can J Res 28b(3):105–113
Draget KI (2009) Alginates. Handbook of hydrocolloids, 2nd edn. Woodhead Publishing, Sarston,
pp 807–828
Falkeborg M, Cheong LZ, Gianfico C, Sztukiel KM, Kristensen K, Glasius M, Xu X, Guo Z
(2014) Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation.
Food Chem 164:185–194
FAO (2018) The state of world fisheries and aquaculture 2018—meeting the sustainable develop-
ment goals. Rome. Licence: CC BY-NC-SA 3.0 IGO. ISBN 978-92-5-130562-1
Featherstone S (2015) Ingredients used in the preparation of canned food. In: A complete course
in canning and related processes, 4th edn. 2(Microbiology, packaging, HACCP and ingredients)
pp. 147–211
Fertah M, Belfkira A, Dahmane A, Taourirt M, Brouillette F (2017) Extraction and characterization
of sodium alginate from Morooccan Laminaria digitata brown seaweed. Arab J Chem 10:53707–
53714
Forster J, Radulovich R (2015) Seaweed and food security. In: Tiwari BK, Troy DJ (eds) Seaweed
sustainability. Elsevier Academic Press, pp 289–313
Grandview Research (2017) Alginate market analysis by type (High G, High M), by product (Sodium
alginate, calcium alginate, potassium alginate, propylene glycol alginate). By application, and
segment forecasts, 2018–2025. Report ID: GVR-2-68038-244-0, pp 1–127
Helmiyati, Aprilliza M (2017) Characterization and properties of sodium alginate from brown algae
used as an ecofriendly superabsorbent. In: IOP conference series: materials science engineering,
188 012019 https://doi.org/10.1088/1757-899x/188/1/012019
Hernandez-Carmona G, Freile-Pelegrin Y, Hernandez-Garibay E (2013) Conventional and alterna-
tive technologies for the extraction of algal polysaccharides
Hisni A, Purwanti D, Ustadi A (2016) Blood glucose level and lipid profile of streptozotocin-induced
diabetes rats treated with sodium alginate from sargassum crassifolium. J Biol Sci 16(3):58–64
92 4 Alginates
Horn SJ, Aasen IM, Ostgaard K (2000) Ethanol production from seaweed extract. J Ind Microbiol
Biotechnol 25(5):249–254
Idota Y, Kato T, Shiragami K, Koike M, Yokoyama A, Takahashi H, Yano K, Ogihara T (2018)
Mechanism of suppression of blood glucose level by calcium alginate in rats. Biol Pharm Bull
41(9):1362–1366
Iwasaki KI, Matsubara Y (2000) Purification of alginate oligosaccharides with root growth-
promoting activity toward lettuce. Biosci Biotechnol Biochem 64:1067–1070
Konda M, Singh S, Simmons BA, Klein-Marcuschamer D (2015) An investigation on the economic
feasibility of Macroalgae as a potential feedstock for biorefineries. Bioenergy Res 8:1046–1056
Lakshmi SD, Trivedi N, Reddy CRK (2017) Synthesis and characterization of seaweed cellulose
derived carboxymethyl cellulose. Carbohyd Polym 157:1604–1610
Liberski A, Latif N, Raynaud C, Bollensdorff C, Yacoub M (2016) Alginate for cardiac regeneration:
from seaweed to clinical trials. Global Cardiol Sci Pract 4:1–25
McHugh DJ (1987) Production, properties and uses of alginates ln “Production and utilization of
products from commercial seaweeds. FAO Fish Tech Paper 288:58–115
McHugh DJ (2003) A guide to the seaweed industry. FAO fisheries technical paper 441. FAO,
Rome. ISBN 92-5-104958-0, Chapter 5, pp 1–12
Monagail MM, Cornish L, Morrison L, Araujo R, Critchley AT (2017) Sustainable harvesting of
wild seaweed resources. Eur J Phycol 52(4):371–390
Moulonguet I, de Goursac C, Plantier F (2011) Am J Dermatopathol 33(7):710–711
Mushollaeni W (2011) The physicochemical characterization of sodium alginate from indonesian
brown seaweeds. Afr J Food Sci 5(6):349–352
Nagarajan A, Shanmugam A, Zackaria A (2016) Mini review on alginate: scope and future
perspectives. J Algal Biomass Util 7(1):45–55
Olatunji O, Olsson RT (2016) Processing and characterization of natural polymers. In: Natural
polymers, industry techniques and applications. Springer, Switzerland. ISBN 978-3-319-26412-7
Paques JP (2015) Alginate nanospheres prepared by internal or external gelation with nanoparticles.
In: Macroencapsulation and microspheres for food application, pp 39–55
Parreidt TS, Muller K, Schmid M (2018) Alginate-based edible films and coatings for food
packaging applications. Foods 7(170):1–38
Paxman JR, Richardson JC, Pw Dettmar, Corfe BM (2008) Alginate reduces the increased uptake
of cholesterol and glucose in overweight male subjects: a pilot study. Nutr Res 28(8):501–505
Qin Y, Jiang J, Zhao L, Zhang J, Wang F (2018) Applications of alginate as a functional food
ingredient. Biopolymers for food Design. Handb Food Bioeng, pp 409–429. https://doi.org/10.
1016/b978-0-12-811449-0.00013-x
Radulovich R, Neori A, Valderrama D, Reddy CRK, Cronin H, Forster J (2015) Farming of sea-
weeds. In: Tiwari BK, Troy DJ (eds) Seaweed sustainability: food and non-food applications, pp
27–59
Rhein-Knudsen N, Ale MT, Meyer AS (2015) Seaweed hydrocolloid production: an update on
enzyme assisted extraction and modification technologies. Mar Drugs 13(6):3340–3359
Romero-Gonzalez ME, Williams CJ, Gardiner PHE (2001) Study of the mechanisms of cadmium
biosorption by dealginated seaweed waste. Environ Sci Technol 35(14):3025–3030
Rompp W, Axon G, Thompson T (1983) Sodium alginate: a textile printing thickener. Am Dyestuff
Rep 72(2):1–16
Schiener P, Black KD, Stanley MS, Green DH (2014) The seasonal variation in the chemical
composition of the kelp species Laminaria digitata, Laminaria hyperborea, Saccharina latissima
and Alaria esculenta. J Appl Phycol 27(1):363–373
Shkand T, Chizh MO, Sleta IV, Sandomirsky BP, Tatarets AL, Patsenker LD (2016) Assessment
of alginate hydrogel degradation in biological tissue using viscosity sensitive fluorescent dyes.
Methods Appl Fluoresc 4(4):044002
Taelman SE, Champenois J, Edwards MD, De Meester S, Dewulf J (2015) Comparative environ-
mental life cycle assessment of two seaweed cultivation systems in north west Europe with a
focus on quantifying sea surface occupation. Algal Res 11:173–183
References 93
Takeshita S, Oda T (2016) Usefulness of alginate lyases derived from marine organisms for the
preparation of alginate oligomers with various bioactivities. Adv Food Nutr Res 79:137–160
Venkatesan J, Nithya R, Sudha PN, Kim S (2014) Role of alginate in bone tissue engineering. Adv
Food Nutr Res 73:45–57
Wang L, Liu B, Yang Q, Lu D (2014) Rheological studies of mixed printing pastes from sodium
alginate and modified xanthan and their application in the reactive printing of cotton. Color
Technol 130(4):320–335
Wan J, Zhang J, Chen D, Yu B, He J (2017) Effects of alginate oligosaccharide on the growth
performance, antioxidant capacity and intestinal digestion-absorption function in weaned pigs.
Anim Feed Sci Technol 234:118–127
Wan J, Zhang J, Chen D, Yu B, Huang Z, Mao X, Zheng P, Yu J, He J (2018) Alginate oligosaccharide
enhances intestinal integrity of weaned pigs through altering intestinal inflammatory responses
and antioxidant status. RSC Adv 8:13482–13492
Windhues T, Borchard W (2003) Effect of acetylation on physicochemical properties of bacterial
and algal alginates in physiological sodium chloride solutions investigated with light scattering
techniques. Carbohyd Polym 52(1):47–52
Zhu B, Yin H (2015) Alginate lyase: review of major sources and classification, properties, structure-
function analysis and applications. Bioengineered 6(3):125–131
Chapter 5
Fucoidan
Abstract Fucoidan is extracted from brown algae and echinoderms, most com-
monly, sea cucumber. It is a heteropolymer with fucose as its main repeating unit.
However, a variety of monomers and functional groups are also present within its
polymer chain. The presence of sulfate groups is attributed to many of its bioac-
tive properties. The extracts are highly polydisperse; therefore, additional processes
are often required to obtain fucoidan with uniform molecular weight. The molecu-
lar weight, degree of sulfation and monomeric unit vary significantly with species,
extraction method and growth parameters of the organisms. This diversity of fucoidan
also limits its pharmaceutical and biomedical applications. This chapter discusses
the processes involved in fucoidan production, its chemical structure, environmental
issues associated with fucoidan production, applications and industrial significance.
Keywords Fucoidan · Sulfated · Polysaccharide · Fucus · Echinoderms
5.1 Introduction
Fucoidan refers to a group of sulfated polysaccharides made up of mainly fucose

alongside deoxy sugars and other monosaccharides with varying levels of branch-
ing and acetylation. They are commonly found in brown algae and echinoderms
such as sea cucumber and sea urchins (Berteau and Mulloy 2003). Although there is
yet much unknown about the exact structure of these fucose-rich sulfated polysac-
charides, the broad range of potential applications which have been demonstrated
by fucoidans provide very promising prospects, particularly in the biomedical and
pharmaceutical industries. While fucoidans have gained approval as functional foods
commercially available as supplements with health benefits marketed including can-
cer prevention, the limitation in their advancement for clinical use, despite find-
ings that fucoidans possess such bioactivities, lies in the variation in the chemical
structures which result in a variation in the bioactivity of fucoidans from different
sources, seasons, fractions and extraction forms as well as expensive purification
requirements. Current research efforts are therefore exploring different approaches
to address these limitations. Such attempts toward developing standard fucoidans
include purification techniques, development of axenic cell culture for production

96 5 Fucoidan
of fucoidans with controlled chemical structure and development of enzymes for

hydrolysis of fucoidans toward controlling of molecular weight and attaining better
understanding of fucoidan structure.
Brown algae which serve as the main known source of fucoidan production are
relatively available but are also used in direct consumption as food and production
of other polysaccharides of commercial value such that fucoidan is not a main pri-
ority extract presently. Strategies to promote commercial-scale fucoidan production
from algae could be developing special strains with increased fucoidan content for
commercial fucoidan production.
The fact that the structural variation in fucoidans results in variation of bioac-
tivity provides opportunity for tuning fucoidan bioactivity for improved specificity
of fucoidan-based drugs. This is only achievable after gaining a full understand-
ing of the structure of fucoidan and mapping out the correlation between factors
such as source and extraction methods on the chemical properties such as molecular
weight, branching, monomeric structure, acetylation and sulfation. In the present
state of technology on fucoidans, these parameters vary with no particular identified
correlation or trend.
Sea cucumbers which are increasing in demand particularly in China are now
declining in population in different seas. In China, they are no longer available in the
sea, while in the coastal areas in countries like Madagascar, they are fast declining
in population. They usually inhabit the epipelagic (sunlight) zone of the sea which
makes them a relatively easy catch for fishers who can simply dive into catch them.
They are very valuable for their bioactive properties which can be attributed to the
fucoidan content among other compounds such as lactones and triterpenes (Qin et al.
2018).
Understanding the active compounds in these sea cucumbers such as fucoidan as
being present in other more available resources such as brown algae could decrease
the pressure on sea cucumber by providing more sources for the same active com-
pound which yields the same health benefits. To this end, this chapter explores
fucoidan, another valuable aquatic biopolymer.
Fucoidan can be regarded as a relatively recently discovered and naturally occur-

ring biopolymer. Unlike, for example, cellulose, which has been in use for centuries,
fucoidan appeared in the literature for the first time in 1913 (Kylin 1913). Fucoidan
occurs in the extracellular matrix of brown algae and marine invertebrates, where
it plays important biological functions such as maintaining cell wall integrity, tis-
sue hydration, aiding in communications between cells, regulating osmotic pres-
sure and serving as a defense mechanism for the organism (Deniaud-Bouët et al.
2017). Fucoidan distinguishes from the other biopolymers in the ECM of brown
algae—alginates and cellulose as the sulfated fucose-rich polysaccharide. Like other
polymers present in algae, the fucoidan content in brown algae shows seasonal vari-
ation. Fucoidans further show more complex seasonal variation in their structure and
bioactivities (Fletcher et al. 2017).
Although not conventional sources of fucoidan, one study discovered the pres-
ence of fucoidan-like compounds in sea grasses (Kannan et al. 2013). The sulfated
polysaccharide extracted from the sea grass Halodule pinifolia contained fucoidan-
related monomers such as mannuronic acid, fucose and high level of uronic acid and
showed antioxidant activity, thus suggesting the possibility that fucoidans might not
be limited to brown algae and echinoderms alone.
Sea cucumbers are marine invertebrates which produce sulfated fucoidan as one
of their main polysaccharide components, the other being fucosylated glycosamino-
glycan (Qin et al. 2018). Species of sea cucumber include Phylloporus proteus (Qin
et al. 2018) and Holothuria tubulosa (Chang et al. 2015).
5.3 Chemistry of Fucoidans
Fucoidans are sulfated polysaccharide with a sulfate group attached to some of the
monosaccharide units. The polysaccharide backbone is made up of mainly 1 → 3
linked or alternating 1 → 3 and 1 → 4 linked fucose units with some sulfate groups
attached to the oxygens. Branching occurs linking the mainly fucose backbone with
other monosaccharides such as rhamnose, xylose, arabinose, galactose and uronic
acid (Weelden et al. 2019). The level of branching, sulfation and polymer chain
configuration and monosaccharide units present within a given fucoidan depends on
the species, growth condition of the species and extraction method. These structural
variations also result in significant variation in the activities of the fucoidans.
Peng et al. (2018) reported fucoidan from Kjellmaniella crassifolia which contains
71.68% carbohydrate and 20.04% sulfate with 31.89% of the monosaccharides being
fucose and 23.54% galactose. Wei et al. (2019) reported fucose and galactose contents
of 77.4% and 13.9%, respectively, in fucose extracted from brown algae. Figure 5.1
shows different backbone structures of fucoidans from different sources showing
1 → 3 and 1 → 4 linkages and example of a sulfate attachment (Weelden et al.
2019).
Fucoidans are polydisperse such that within any given sample, the molecular
weight and degree of polymerization of each fucoidan polymer chain varies. This
is not uncommon characteristics of polymers. Methods such as mass spectrometry,
gas chromatography, MALDI-TOF and NMR are available for characterization of
fucoidan, and these have been employed in understanding the chemical structure
of fucoidan from different sources. In addition to the polydispersity, the average
molecular weight, configuration and degree of branching vary for fucoidans from
different sources (Fitton et al. 2015a, b). The fucoidans from sea cucumber tend to
have less complex structure and species relationship. For example, fucoidans from
98 5 Fucoidan
Fig. 5.1 Different backbone structures of fucoidans showing a 1 → 3 inked fucose units, b alter-
nating 1 → 3 and 1 → 4 linked fucose units and c showing 1 → 4 fucose linkages and sulfate group
attachments. Here R could be any monosaccharide units found in fucoidans or a sulfate group
sea cucumber of species Isostichopus badionotus, Thelenota ananas and Acaud-

ina molpadioides are all linear, and those from sea cucumber species of Aposti-
chopus japonicus and Stichopus japonica are all branched. Fucoidans from sea-
weeds have more complex and branched structures which could vary within the
same species (Chang et al. 2015). The monomer units of the fucoidan polymer struc-
ture also vary for different brown algae sources. For instance, fucoidan from the
fucaceae family consists of high fucose content relative to other monosaccharides
present within the polymer structure, while in another fucoidan source, Undaria
pinnatifida, the fucoidan chains contain more of galactose. The sulfite and acetyl
contents as well as uronic acid component also vary in different fucoidan sources
(Fitton et al. 2015a, b).
This wide variation presents a diverse range of properties of fucoidans from vari-
ous sources. The properties of different fractions of fucoidans from the same source
also vary. Fucoidans of different properties can be isolated and separated from the
same batch of extract. For example, batch of fucoidan from Sargassum muticum from
5 to 100 kDa showed a range of properties. While fractions with molecular weight
above 100 kDa contained the highest level of sulfates and phenolics, the fractions
with molecular weight between 50 and 100 kDa contained 25% of the solubles and
highest oligosaccharide contents. The fraction with the highest sulfates and pheno-
lics also showed the highest antiradical activity, while the fraction with molecular
weight in the range 10–30 kDa had the highest cytotoxic effect on cervical cancer
cells (Alvarez-Vinas et al. 2019). Although several studies have reported relation-
ships between bioactive properties such as anticoagulant activity and antitumoral
activity of fucoidan and their structure and molecular weight, there is yet to be direct
correlations between the structural properties of fucoidans and their bioactivities.
Fucoidans can be classified according to their molecular weights such as low
molecular weight, middle molecular weight and high molecular weight fucoidans
(LMWF, MMWF and HMWF). Low molecular weight fucoidans have molecular
5.3 Chemistry of Fucoidans 99
weight less than 10 kDa, medium molecular weight fucoidans have molecular weights
in the range 10–10,000 kDa, while high molecular weight fucoidans have molecu-
lar weights above 10,000 kDa. The bioactivities are strongly affected by molecular
weight; for instance, high molecular weight fucoidans have been associated with
better anticancer effects (Miyazaki et al. 2018) and low molecular weight fucoidans
offer better therapeutic enhancement and abate side effects when combined with
chemotherapy (Chen et al. 2015). Fucoidans obtained by hydrothermal treatment
of Sargassum muticum are separated into different molecular weights through frac-
tionation which results in fractions with different sulfate and phenolics contents and
with different radical scavenging properties (Alvarez-Vinas et al. 2019). The medium
molecular weight fraction (50–100 kDa) had the highest oligosaccharide content, the
>100 kDa fraction had the highest amount of sulfates and phenolics, and this fraction
also showed highest antiradical properties, while the low molecular weight fractions
with molecular weights in the range 10–30 kDa were more toxic against cervical can-
cer. Furthermore, it is possible to obtain low molecular weight fucoidan from high
molecular weight fucoidan through enzyme hydrolysis. This method of obtaining
LMWF leads to better bioactivity than acid-extracted LMWF from crude (Hwang
et al. 2017; Sanjeewa et al. 2017).
The presence of sulfate groups attached to the sugars within the fucoidan polymer
chain also affects the bioactivity of fucoidan. Generally, highly sulfated low molec-
ular weight fucoidans have better anticancer activity than unsulfated low molecular
weight fucoidan or sulfated high molecular weight fucoidan (Cho et al. 2011).
5.3.1 Degradation of Fucoidan
The biodegradation of fucoidan is important for several reasons such as production of

fractions of fucoidans with improved or varied bioactivity from the crude fucoidans,
understanding the structure of fucoidan and determining the biodegradation products
and timeframe of such degradation. There are no known enzymes in the human body
which degrade fucoidans. They are also not degraded by the enzymes present in the
intestine. However, there are different means by which fucoidan can be degraded and
then further utilized by humans for its bioactive properties.
Fucoidan is degraded by acid hydrolysis into smaller molecular weights and then
into its sugar units. Orally ingested fucoidan can be detected in the urine (Tokita
et al. 2010; Michel et al. 1996), indicating that it can withstand the conditions in
the alimentary canal and still retain its chemical structure. Fucoidan deacetylase
has been identified for partial degradation of fucoidan. This enzyme can deacetylate
fucoidan and, however, is not able to desulfate or degrade fucoidans into lower
molecular weight or fractions. That is, the enzyme activity is specific to the acetyl
and sulfate bonds on the fucoidan. This enzyme was identified in the marine bacteria
which utilized fucoidan, Luteolibacter algae H18 (Nagao et al. 2017). Such enzyme
specificity is desirable, where a fucoidan of specific degree of acetylation is required
to achieve a specific bioactivity.
100 5 Fucoidan
A number of fungi (Rodriguez-Jasso et al. 2010), bacteria (Chang 2010) and

certain invertebrates (Bilan et al. 2005) are known to contain enzymes capable of
degrading fucoidan. The level of activity and mode of activity vary for different
enzymes. For instance, the marine bacteria species such as Formosa algae produce
the enzyme fucoidanase which has the ability to hydrolyze fucoidan (Sichenko et al.
2013). This enzyme can optimally function in a wide range of pH value (6.5–9.1).
The level of activity of fucoidanase has been shown to vary for different fucoidan
structures. For example, fucoidan-utilizing bacteria of the Flavobacteriaceae family
extracted from seawater showed a fucoidan utilization rate of 81.5% for fucoidan
extracted from sea cucumber (Chang 2010), while the enzymes from terrestrial fungi,
Aspergillus niger, can degrade fucoidan from the brown algae Laminaria japonica,
that from Fucus evanescens is hydrolyzed by another type of enzyme from marine
bacteria. Deacetylated fucoidan has been found to be hydrolyzed more readily com-
pared to desulfated fucoidan. This is attributed to the specificity of the enzymes to
the 1 → 4 bonds of the polysaccharide chains, specifically for sulfated alpha-L-
fucopyranose. Such degradation by fucoidanase is significant toward producing the
immunomodulatory-active sulfated fuco-oligosaccharide.
With the aim to achieve improved pharmacological bioactivity by producing lower
molecular weight fucoidan from higher molecular weight ones, Lahrsen et al. (2018)
degraded fucoidan from a molecular weight of 4.9–38.2 kDa using hydrogen per-
oxide. However, the lower molecular weight fucoidans produced, lost their antiox-
idant and antiproliferative activities. It is, therefore, important that the activities of
fucoidan extracts can be tailored to meet desired bioactivities by optimizing the right
combination and conditions of enzyme activities.
Certain microbes, mainly marine bacteria or mollusks, contain endo- and exo-
enzymes which can break down fucoidans (Kusaykin et al. 2001). These are impor-
tant for either understanding the breakdown of the fucoidan-based products when
exposed to the environment at the end of use or in the body or as source of enzymes
used to modify fucoidan into other forms using enzymes. An example of such enzyme
is fucoidan hydrolase, alpha-l-fucosidase. This can be used in combination with a
desulfating enzyme arylsulfatase to break down the sulfated carbohydrate fucoidan
structure (Silchenko et al. 2013). This results in the formation of sulfated oligosac-
charides as a result of cleavage of the fucoidan chain into shorter chains. Fucose
is also produced in the process. Interestingly, some enzymes which are capable of
cleaving or hydrolyzing fucoidans from some species are not able to do the same
for fucoidans from other species. This is due to the diverse structure of fucoidans
as they vary for different sources. For example, the enzyme extracted from marine
bacteria which degrade fucoidan from Fucus evanescens and Fucus vesiculosus does
not hydrolyze fucoidan from another species Saccharina cichorioides (Rodriguez-
Jasso et al. 2010). This difference in the degradation process limits the large-scale
modification of fucoidans to obtain more standard batches, for example, to control
the chain length or degree of sulfation.
Brown algae, one of the two sources of fucoidan, are dominant in many coastal
regions and are already readily available as a food product in many Asian cuisines.
It is also gaining popularity in Western and other economies for its health benefits.
The chapter on alginate has discussed the global availability of brown algae. Taking
an example crude yield of 4.02% fucoidan by dry weight of brown algae (Sinurat
et al. 2015) and using Norway, the highest producer in Europe, as an example brown
algae producing country where 154,230 tonnes of brown algae is harvested from
wild stocks annually (Monagail et al. 2017), this means 6200 tonnes of fucoidan is
attainable from brown algae resource in Norway annually. As we will discuss in the
latter sections of this chapter, the yield, structure and bioactivity of fucoidans are
very species specific. This variation means that while biopolymers such as alginate
from brown algae could be mass extracted from a mixture of species of brown algae,
fucoidans need to be extracted in species-specific batches to obtain more specific
bioactivity.
Echinoderms, which comprised of species such as starfish, sea urchins and sea
cucumbers, are aquatic animals which also are a source of fucoidans. Some echino-
derms such as sea cucumbers are consumed by humans as food. Sea cucumbers can be
found in the Mediterranean Sea and the eastern parts of the Atlantic Ocean (Chang
et al. 2015). Species which have been explored for fucoidan production include
Holothuria tubulosa, Stichopus japonicus, Apostichopus japonicus and Acaudina
molpadioides among several others. The reproductive rate of algae is much faster
than that of sea cucumber which although is moderate compared to other inverte-
brates in the sea and is currently declining in population due to increasing interest
as a functional food with bioactivities such as anticoagulant and antioxidant effects
(Chang et al. 2015; Qin et al. 2018). Commercial fucoidan production should there-
fore focus more on extraction from brown algae or at least diversify production of
fucoidan from more than a single source.
In the bid to achieve standardized fucoidan-based product with consistent and
controllable chemical structures and hence bioactivity, some research work has gone
into developing enzymes involved in fucoidan synthesis and development of cell
cultures which express the genes to produce the desired fucoidan chemical structure
(Kasai et al. 2015).
5.5 Extraction of Fucoidans
Various methods exist for extraction of fucoidan, and here we shall look at some
of them. The type of extraction and purification method strongly determines the
structure and bioactivity of fucoidan (Ponce et al. 2003).
102 5 Fucoidan
5.5.1 Acid Extraction
The use of acid for extraction of fucoidan is the oldest known method. This involves
solubilizing the fucoidan in an aqueous acid solution at high temperature. This is then
followed by precipitation of the dissolved fucoidan with ethanol. This process is based
on the fact that fucoidan is soluble in polar liquids at low pH and elevated temperature
but insoluble in less polar solvents. Subsequent solubilization and precipitation can
then be carried to further purify the extracted fucoidan (Fitton et al. 2015a, b). The
proteins and alginate within the algae or sea cucumber will also be dissolved at
low pH and high temperature. The alginates can be separated from the fucoidan by
precipitation.
An example process is as follows (Sinurat et al. 2015; Lu et al. 2018): The brown
seaweeds are first soaked in a mixture of methanol, chloroform and water in the ratio
4:2:1 over a duration of 12 h. This process removes the fats and depigments the brown
algae. This is then followed by washing in acetone, and the defatted and depigmented
algae are then air-dried. The dried biomass is then treated with 0.01M HCl at a pH
of 4 with a weight-to-volume ratio of 1:10 (g:ml) under continuous stirring for 6 h.
The liquid extract obtained after filtering contains fucoidan as well as acid-soluble
proteins, alginates and smaller particles which get through the filter pore space. The
alginate is separated by precipitation with 4M of calcium chloride (CaCl2 ) under
incubation for a period of 30 min after which the precipitates are filtered off. The
precipitation is repeated to further remove more alginates. This is then followed by
centrifugation at 1544 g for 15 min in order to remove the smaller particles. Crude
fucoidan is then obtained from the solution by precipitation with ethanol and further
purified by dialysis at a cutoff point of 10 kDa. This process is based on method
reported for research studies which can also be scaled up for industrial fucoidan
production. A flowchart of the extraction process is provided in Fig. 5.2.
Here an example extraction process of extraction of fucoidan from sea cucumber

is used to explain the enzyme-based extraction of fucoidan. The enzyme extraction
process involves breaking down of the proteins in the tissue using papain. The body
of the sea cucumber which can be purchased from fishermen or from the market is
dried and milled into smaller particle sizes. The pH is adjusted using hydrochloric
acid, and the papain is added at a concentration of 0.15%. The extraction is carried out
at 50 °C for a period of 2 h at a ph of 6 and a solid-to-liquid ratio of 1 g:16.26 mL—
based on optimum conditions reported by Qin et al. (2018). This process results in the
breaking down of the tissue proteins into peptides. The pH is then adjusted to a lower
pH of 2.8 at a temperature of 4 °C over a period of 4 h. This allows the acidic proteins
to be removed and separated by centrifugation (7441 × g for 20 min). The liquid
supernatant obtained after the centrifugation contains a mixture of compounds. The
5.5 Extraction of Fucoidans 103
Brown Defatting and Drying Acid

algae depigmentation extraction
biomass
Fats and
Retentate
pigments
to waste
Calcium Filtrate
treatment
Alginate Chloride Filtration
precipitation
and filtration
Filtrate
Liquid
supernatant
Centrifugation Ethanol precipitation
Fucoidan
Fig. 5.2 Extraction of fucoidan
fucoidan is then separated by precipitation with alcohol followed by centrifugation

at 4816 × g for 15 min. The fucoidan obtained as the solid is then dried (Qin et al.
2018).
5.5.3 Microwave Extraction
Although the microwave extraction process allows for shorter extraction times and
in some cases has demonstrated better control over structure, this process is lim-
ited to small-scale laboratory extractions. There are reports of microwave-extracted
fucoidan having reduced anticancer activity compared to the conventional acid
extraction (Rodriguez-Jasso et al. 2011), while other reports reported fucoidans
with antioxidant property from Ascophyllum nodosum using the microwave-assisted
extraction method (Yuan and Macquarrie 2015). As the bioactive properties of
fucoidan vary from species to the other, it is not yet concluded that this incidence
of reduced anticancer property using microwave extraction applies to all species.
This process is nonetheless useful for high-value, low-quantity fucoidans with more
controllable bioactive properties such as their antioxidant property.
104 5 Fucoidan
5.5.4 Subcritical Water Extraction
This method of extraction has recently shown to result in fucoidan yield of 25.98%
from Nizamuddinia zanardinii when used at optimal temperature of 150 °C for 29 min
at a 21 g/mL biomass-to-water ratio (Alboofetileh et al. 2019). This is much higher
that yield from conventional extraction from a similar species. Evaluation of the
monosaccharide components of the extract of Nizamuddinia zanardinii using the
subcritical water extraction method showed 34.13% fucose, 30.70% mannose, 9.35%
xylose, 23.19% galactose and 2.65% glucose. The extracted fucose had an average
molecular weight of 694 kDa, indicating a medium molecular weight fucoidan. The
fucoidan extracted using this method showed some anticancer properties.
The effect of extraction methods on the structure of fucoidan could potentially
give more control over the structure of fucoidan. Following extraction of the crude
fucoidan, it is important to separate the non-fucoidan components from the crude
extracts and where desired separate the fucoidans into fraction in order to have
fractions of more uniform chain length and structures with similar bioactivities.
Table 5.1 shows the yield and molecular weight of fucoidan extraction using different
methods from different studies. Note that the extraction yield also depends on the
efficiency of the individual process and the purity of the final product; therefore, the
values presented within the table are not absolute values for the particular method or
source.
Table 5.1 Yield and molecular weight of fucoidan from different extraction methods
Fucoidan source Yield % Molecular weight Extraction method References
dry
weight
Sea cucumber 6.83 40–400 kDa Enzyme Qin et al. (2018)
Nizamuddinia 25.98 694 kDa Subcritical water Alboofetileh et al.
zanardinii (2019)
Brown algae NR 2–440 kDa Hot water Lu et al. (2018)
(Undaria
pinnatifida)
Brown algae (U. 12.9 NR Hot water Zhao et al. (2018)
pinnatifida)
Sea cucumber 2.5% 1567.6 ± 34.1 kDa Enzyme Chang et al.
(Holothuria (2015)
tubulosa)
Brown algae 4.02 NR Acid Sinurat et al.
(Sargassum (2015)
sp.)
Brown algae 18.2% NR Microwave-assisted Rodriguez-Jasso
et al. (2011)
NR Not reported in study
Like many biopolymers, the production of fucoidan, from the process of harvesting
the raw material to the final product, is not completely benign. Here we explore some
of the environmental issues associated with the extraction of fucoidan from brown
algae.
5.6.1 Resource Utilization of Brown Algae and Sea

Cucumber
Brown algae play a significant role in the aquatic environment, serve as hosts to some
microbes with which it has a symbiotic relationship, are a source of food to animals
and humans, fix nitrogen and CO2 and also play a key role in water remediation.
Removing such from the environment results in an imbalance in the ecosystem,
and this must be weighed against the economic and environmental significance of
harvesting brown algae for fucoidan production. Already the heavy harvesting of
brown algae is causing some environmental concerns in parts of the world. On the
other hand, algae cultivation and harvesting at a sustainable rate could have some
benefits on the environment.
Furthermore, brown algae as an alternative source of fucoidan could potentially
reduce overfishing of sea cucumber which have recently suffered from severe decline
in population is some seas in area such as Madagascar and China. In these regions,
they are seen as highly priced for their medicinal purpose. Fishermen often free dive
into the sea to collect seaweeds and sea cucumber within the sunlight zone of the sea.
The brown algae and sea cucumber are sold in local markets and restaurants as food
and as an important source of income to the coastal communities. Cultivation of both
seaweed and sea cucumber should only be carried out at a rate balanced by the rate
of their reproduction in the wild; otherwise, these aquatic resources considered as
renewable resources will become depleting resources. Harvesting brown algae and
sea cucumber for large-scale extraction of products such as fucoidan particularly
where yields could be as low as 4% requires a strain on these natural resources.
Alternatives to reduce the environmental impact of overheating of these resources
from the wild include genetically modified brown algae strains which can be culti-
vated in controlled environment to yield desired structure of fucoidan and cultivation
of sea cucumber in aquaculture (Jour et al. 2012). Limitation of aquaculture of sea
cucumber and brown algae includes costs of facilities and the risk of losing whole
batches to infection outbreak.
106 5 Fucoidan
5.6.2 Use of Mineral Acids
HCl is used at a relatively low concentration of 0.01M in the acid extraction method.
Since the acid is not chemically altered, it is washed out at the end of the process
and goes to the wastewater treatment. Even where it is neutralized, this eventually
results in increasing the mineral content of the aquatic environment where it does
not lead to direct acidification.
One way of eliminating or minimizing the need for acids in the process of the
extraction is to use enzymes. Enzyme extraction has been more commonly reported
for extraction of fucoidan from sea cucumber. This is due to the fact that sea cucumber
involves mainly proteolytic enzymes to dissolve the proteins within the tissue. Papain
is commonly used at a concentration of 15% (Qin et al. 2018). Enzyme extraction of
fucoidan from brown algae is less feasible as this would require the use of a variety
of enzymes such as alginate lyase enzymes to break down the alginate, proteolytic
enzymes to break down the protein and then cellulases to remove the cellulose.
The amount of energy consumed varies with extraction method. The enzyme method
using papain can be carried out at 50 °C which is lower than the temperature required
for the hot water extraction at 70–80 °C for longer time period. Meanwhile, the acid
extraction process can be carried out at room temperature (Sinurat et al. 2015).
Further energy is consumed in centrifugation process which for the enzyme process
is required at two different stages at 7441 × g for 20 min and at 4816 × g for 15 min.
The amount of energy and materials consumed for a typical acid extraction of
fucoidan from brown algae is given in Table 5.2. The values are based on values
Table 5.2 Typical

consumptions for fucoidan
extraction from brown algae Brown algae 24.87 g/g fucoidan
using acid extraction Methanola 5.71 ml/g fucoidan
Chloroforma 2.86 ml/g fucoidan
Watera 1.43 ml/g fucoidan
HCl 10 ml 0.01M per gram fucoidan
Calcium chloridea 4M 20 ml
Ethanola 10 ml 95%
Energy
• Stirring 200 rpm 4 h
• Centrifuge 1544 g for 15 min
a indicates estimated based on typical values
from studies carried out at laboratory scale (Sinurat et al. 2015). When the precise
values are not provided in the particular study, estimates are used based on typical
values.
5.7 Applications of Fucoidan
Fucoidans have been identified with a range of bioactive properties which give them
potential applicability in food, cosmetics, pharmaceutical and biomedical industries.
Presently, clinical applications of fucoidans are limited as they are yet to be approved
by regulatory organizations such as FDA for such use. Part of the reason for this
is that fucoidans vary significantly from batch to batch, and this variation affects
the bioactivity significantly. For instance, while several fucoidan-producing species
demonstrate ability to inhibit cancer cell adhesion to platelets (Cumashi et al. 2007),
which in turn inhibits their ability to metastasize, other fucoidan-producing species
like the Cladosiphon okamuranus do not demonstrate this property, in fact fucoidans
extracted from the latter showed increased tumor growth (Azuma et al. 2012). There-
fore, approving fucoidans, in general, for specific applications could mean approving
forms of fucoidans which might not be suitable for the same applications and vary
significantly in effectiveness and safety. There needs to be set standards for extrac-
tion, characterization and processing of fucoidans which guides their approval for
therapeutic applications. Furthermore, although there are many studies pointing to
the bioactive properties of fucoidan which makes it suitable for many applications
such as anticancer agent, the exact mechanism of much of these bioactivities is
yet unknown. Nonetheless, fucoidan’s broad range of bioactivities open up great
potentials for this biopolymer to be of significant and economic impact.
5.7.1 Biomaterials in Biomedicine and Tissue Engineering
Fucoidans either in the neat form or as a composite with other materials such as
chitosan, alginates, hydroxyapatite and polycaprolactone have been tested in various
biomedical applications. Fucoidan–polycaprolactone composites have been used as
macroporous sutures which in cellular mineralization, similarly fucoidan–chitosan–
alginate and fucoidan–hydroxyapatite composites aid in cellular mineralization (Lee
et al. 2012; Venkatesan et al. 2014; Jeong et al. 2013). Scaffold formed from fucoidan
is able to inhibit the activities of osteoclasts (Kim et al. 2014a, b), while promoting
the activities of osteoblast cells (Park et al. 2012; Pereira et al. 2014) such that the
breakdown of new cells forming in the scaffold is prevented while new cell growth
is promoted, therefore leading to faster tissue repair. Fucoidan-based scaffolds also
promote growth and cell differentiation of mesenchymal cells based on results from
studies in vitro (Han et al. 2015) and in vivo using laboratory mice (Huang and Liu
108 5 Fucoidan
2012). These various reports indicate that fucoidan has robust structural compatibility
to form functional composites with other polymers and non-polymers.
5.7.2 Anticancer Therapies
Studies of coculture cells and animals have confirmed fucoidan has some anticancer
properties. The key issues requiring further investigation include sufficient bioavail-
ability to deliver the potential anticancer effect at physiologically relevant rate and
reproducibility of the anticancer effect in different fucoidan extracts across different
species. Fucoidan from Sargassum muticum with molecular weights ranging from 5
to 100 kDa showed a range of anticancer properties. While fractions with molecular
weight above 100 kDa containing the highest level of sulfates and phenolics showed
the highest antiradical activity, the fraction with molecular weight in the range 10–
30 kDa had the highest cytotoxic effect on cervical cancer cells (Alvarez-Vinas et al.
2019). Fucoidans from different sources can therefore be used as a multiple mech-
anisms for cancer therapies since different fractions from the same source show
different forms of antitumoral activities.
The use of fucoidan in treatment of cancer is so far focusing on orally delivered
fucoidan as an adjunct either to enhance the effectiveness of conventional cancer
therapies, prevent side effects or to act as an alternative medication for cancers where
no known medications exist (Fitton et al. 2015a, b). It is hypothesized that fucoidan
takes multiple routes to act against cancer; this includes prevention of blood vessel
formation by cancer cells (angiogenesis), prevention of metastasis (spread of cancer
cells beyond the point of origin), boosting the body’s immune response to cancer
cells, making cancer cells more vulnerable, antioxidant activity and interfering with
the cancer cell metabolism pathways (Kwak 2014). For example, some study has
found that cancer cell apoptosis could be induced in breast cancer and colon cancer
using fucoidan. The mechanism through which fucoidan achieved this is thought to
be through modulation of endoplasmic reticulum stress cascades (Chen et al. 2014).
Fucoidan ingested orally can have some anticancer effect within the gastroin-
testinal tract before absorption into the bloodstream and before reaching the stom-
ach enzyme, while much of the fucoidan remains intact. This could be in different
preparations such as food supplements or processed foods with fucoidan added as a
functional food ingredient. This would, however, be use of fucoidan as a preventive
approach to cancer through its many pathways such as prevention of blood vessel
formation and mobilization of the body’s immune cells, thus preventing the forma-
tion of cancerous cells in the first place. There are already some natural products
which are used in this manner (Esmaeelian et al. 2014).
Fucoidan can be used to minimize or prevent the side effects attached to cancer
therapy such as chemotherapy. A study which showed this used low molecular weight
fucoidan extracted from Acaudina molpadioides, a sea cucumber. When administered
to mice with cyclophosphamide-induced intestinal mucositis, these mice showed
restored levels of immunoglobulin A in the mucosa and moderated cytokine levels.
5.7 Applications of Fucoidan 109
The side effects associated with chemotherapy drug cisplatin, such as weight loss
and changes in levels of hormones gastrin and serotonin, were prevented with similar
effectiveness as drugs which are used for such purpose (Zuo et al. 2015), proving that
fucoidan can act as an adjunct to anticancer drugs for reducing or eliminating the side
effects associated with such drugs. This will contribute significantly to improving
the patients’ quality of life during such treatments.
As an adjunct therapy, fucoidan could work in synergy with conventional
chemotherapy and radiotherapy for improved treatment effectiveness (Zhang et al.
2014a). However, this is still under investigation as other studies also suggest that
fucoidan could have antagonistic effects on conventional cancer therapy (Oh et al.
2014). This could be attributed to the wide variation in the structure, hence bioactiv-
ities of fucoidans for different sources. Therefore, further investigations are required
to validate this concept of fucoidan as an adjunct cancer therapy.
At a recommended dose of 4.0 g per day, human subjects ingesting fucoidan
from the Cladosiphon okamuranus species during chemotherapy treatment of unre-
sectable colon cancer showed improved tolerance of repeated rounds of chemother-
apy compared to those who were not taking fucoidan. The patients taking fucoidan
also showed less chemotherapy-related fatigue as it is common in cancer therapy
(Azuma et al. 2012; Ikeguchi et al. 2011). Studies on mice, given the equivalent
dosage of fucoidan, also showed reduced fatigue during exercise. This was attributed
to increased levels of glucose in the serum for energy and decreased levels of ammo-
nia, lactate and triglycerides which could cause stress in the cells (Chen et al. 2015).
The relationship between reduced fatigue and fucoidan ingestion still requires further
research to understand the mechanism and extent.
Fucoidan, therefore, shows some bioactivities either as a sole anticancer therapeu-
tic agent, as an adjunct to use along with conventional chemotherapy or radiotherapy
or to abate the side effects associated with cancer therapy, or as a preventive agent
against cancer initiation or proliferation. These have been shown in studies in cell
cultures, in animal models and in humans against various cancers such as breast,
prostate, lung and liver cancers. While these are very promising, the limitations lie in
the broad variation in structure and bioactivities of fucoidan from different sources
as well as the variation in the different types of cancers. Such that, the successful
application of fucoidan for cancer awaits further development in both understanding
of cancer and fucoidan variation from species to species.
5.7.3 Drug Delivery Agent
As a biopolymeric material, fucoidans have demonstrated potential for use as drug

delivery compounds. Doxorubicin, an anticancer drug, showed better treatment
against multidrug resistance cancer cells when loaded onto fucoidan nanoparticles
compared with the drug in its free form (Lee et al. 2013a, b). The heterogeneous
polysaccharide structure of fucoidan makes it potentially robust such that various
110 5 Fucoidan
fractions can show compatibility with other materials, thus widening the range of
possible biocomposite drug delivery biomaterials attainable from fucoidans.
5.7.4 Immune Modulation
Ingestion of fruits and vegetables has been known to boost the immune system,
thereby preventing the onset of diseases (Gibson et al. 2012). One of the problems
with complaints to this is the large portions required and in some cases food shortage.
In, for example, regions suffering from draft or where human conflicts have disrupted
farming and food production activities, it is important to provide the necessary quality
of nutrient intake to maintain a healthy immune system. Fucoidan provides immune
modulation when used in a much lower quantity than a larger amount of fruits and
vegetables required for the same level of immune modulation (Negishi et al. 2013).
An example of such, fucoidan extracted from Undaria pinnatifida induced
improved antibody response to vaccine in elderly human subjects who were above
retirement age. Such findings are very important as it could reduce the quantity of
vaccine required to achieve the same immune response. This would reduce the cost
of vaccines and the amount of energy and manpower required to produce them.
The immune modulation activity of fucoidan at a lower gram requirement com-
pared to dietary consumption of fruits and vegetables is also important in providing
treatment and care for individuals who are not capable of ingesting large amounts
of food. Fucoidan intake as low as 1 g per day has been shown to have sufficient
immune modulation activity.
A series of in vivo and in vitro studies show that fucoidans from different species
such as Fucus vesiculosus and Ascophyllum nodosum promote both growth and
activities of the dendritic cells of the body responsible for coordination of immune
response (Jin et al. 2014; Zhang et al. 2014a, b). These include recognition of
pathogens, antigen presentation and initiation of immune response.
It is understood that one of the ways fucoidan acts in immune modulation is
through binding to specific receptors and such interactions resulting in activation or
mediations of specific immune responses in a manner that is not toxic to the cells.
Such interactions result in production of pathogen-destroying biochemicals such
as cytokines and chemokines. Such immune bioactivity has been demonstrated by
fucoidans extracted from species such as Laminaria japonica, Laminaria cichori-
oideae and Fucus evanescens (Makarenkova et al. 2012). Immune modulation is
one of the current commercial claims of fucoidan-based food supplements. However
fucoidan is not commercially useed as a therapeutic as there is a need for more studies
in this area to confirm clinical applicability.
5.7.5 Antipathogenic Agent
Fucoidans have shown to act against a number of pathogens which include Leish-
mania parasite (Sharma et al. 2014), influenza virus (Synytsya et al. 2014), canine
distemper virus (trejo-Avila et al. 2014) and new castle virus (Elizondo-Gonzalez
et al. 2012). The mode of action includes inhibition of entry of these pathogens into
the cell and interfering with the pathogen’s defences against immune response of
the host. Due to the complex structure of fucoidan, various fractions of the polymer
could be responsible for different antipathogenic activities. Although much is yet
to be known of the specific mode of action, there are indications of what fractions
are active against certain pathogens, for example, low molecular weight sulfated
O-acetyl fucogalactan fraction of fucoidan with average molecular weight of 9 kDa
from the species Undaria pinnatifida orally administered to laboratory mice acted
against different forms of influenza virus infection (Hayashi et al. 2013; Synytsya
et al. 2014).
Fucoidans have shown antipathogenic response to a variety of diseases causing
organisms in a variety of organisms including humans, canines and birds with no
toxic effect on the body’s own cells. These findings are consistent across different
research groups.
Although fucoidans have not been identified to directly act against bacteria, their
antibacterial effect lies in their ability to boost the effectiveness of antibiotic agents
(Lee et al. 2013a, b). Such that, they can be used as an adjunct to antibacterial thera-
peutics or simply consumed as preventive care against bacterial infection. As much
of the known bacteria strains have developed one form of immunity against antibac-
terial drugs, the search for new measures against bacteria remains active. Fucoidan
could potentially have a significant economic impact as a versatile and readily avail-
able preventive and therapeutic biopolymer for bacterial infection. Tests carried out
on pseudomonas culture showed that fucoidan extracted from Ascophyllum nodosum
resulted in upregulation of the genes responsible for immune response in the organ-
ism, while the genes for metabolism, sensing and survival in the pathogenic bacte-
ria were downregulated, showing that the particular fucoidan had, to great extent,
improved the resistance of the organism to bacterial infection (Kandasamy et al.
2015). Fucoidan has also shown to protect the body against damage by endotoxins
when taking either orally or injected subcutaneously (Kuznetsova et al. 2014).
The potential economic impact of such antipathogenic impact demonstrated by
fucoidan cuts across the human and animal healthcare industry and commercial
poultry, increasing the quality of life and compliance with an antipathogenic rem-
edy which can be sourced from oral fucoidan preparations in the form of food
supplements.
112 5 Fucoidan
5.7.6 Antiinflammatory
Fucoidan has potential application as an antiinflammatory agent in skin, gut and

aortic. These antiinflammatory effects have been demonstrated in cells and tissues
in humans and other organisms. Various antiinflammatory effects have been demon-
strated by fucoidan when delivered orally, topically or parenterally. For topical appli-
cations, this will depend on the range of molecular weight fucoidans that are able to
permeate the skin layers for bioavailability. Fucoidans from species such as Fucus
vesiculosus (Carvalho et al. 2014) and Undaria pinnatifida (Fitton 2011) demonstrate
these antiinflammatory properties.
By inhibiting the action of inflammatory compound, selectin, through prevention
of inflammatory cell entry into tissue and prevention of platelet adhesion to inflam-
matory cells (Fitton 2011), treatment of inflammatory-related conditions such as
pancreatitis (Kambhampati et al. 2014), aortic aneurysm (Alsac et al. 2013), atopic
and allergic dermatitis (Fitton et al. 2015a, b) and colitis (Lean et al. 2015) could
potentially be addressed with fucoidans as indicated by various reports. Fucoidan
could serve as a replacement for some antiinflammatory drugs with unpleasant or
chronic side effects.
5.7.7 Renal and Hepatic Disease Treatment
The renal or hepatic effect of fucoidan is demonstrated by the manner in which it

prevents the accumulation of compounds which lead to renal or hepatic damage.
Accumulation of fat in the liver and increase in overall body weight, which lead to
chronic liver diseases, are reduced by orally ingested fucoidan included in the diet
of the laboratory mice (Kim et al. 2014a, b). Laminaria japonica-sourced fucoidan
when orally administered to diabetes-induced test animals showed lowered blood
sugar, decreased blood urea nitrogen levels and preserved renal nitrogen excretion
(Wang et al. 2014). This shows a potential for use as a less toxic diabetes treatment for
diabetes-related kidney disease as fucoidan showed lower toxicity compared to more
toxic available treatments for diabetes. Although at very early stage, there have been
research studies into potential use of fucoidan in the treatment of hepatitis C. Clinical
study of fucoidan from Cladosiphon okamuranus using fifteen chronic hepatitis C
patients who were orally administered fucoidan over a period of 12 months showed
some improvements in patients treated with fucoidan. Such results point to potential
use of fucoidan as a therapeutic agent in the treatment of hepatitis C (Mori et al. 2012).
As such research advances, this would have an impact in the area of improving patient
care for a disease which presently has no vaccine and existing treatment has poor
level of effectiveness.
5.7.8 Blood Anticoagulant
Although highly effective anticoagulant drugs already exist in the market, fucoidan
shows anticoagulant activities similar to heparin with potential to overcome the
limitation of heparin. One limitation to heparin’s antithrombotic effect is the risk
of hemorrhage when administered at levels required to attain antithrombotic effect.
Fucoidan extracted from Undaria pinnatifida achieved antithrombotic effect at safe
levels without similar risk of delayed blood clot when injected intravenously in
laboratory mice. However, as is known of the variation in the bioactivity of fucoidan,
that from another species, Fucus vesiculosus showed some increase in clotting time
(Min et al. 2011).
Studies on the anticoagulant property of fucoidans have found some correlation
between the structural properties of fucoidan from Fucus vesiculosus and the antico-
agulant property (Zhang et al. 2014a, b). This promises a step closer to standardized
fucoidan formulations which can be reproduced on a large scale. Minimum charge
density of 0.5 sulfates for every sugar unit and a degree of polymerization of 70 have
been shown to be the required parameters for fucoidan from Fucus vesiculosus with
pro-coagulant property. Anticoagulant property of fucoidan from Laminaria japonica
showed molecular weight dependency as well as dependency on fucose to galactose
ratio within the polymer chain (Jin et al. 2014). Platelet aggregation activation by
fucoidans has also been reported in different studies (Manne et al. 2013; Dürig et al.
1997).
Fucoidans, therefore, have multiple bioactivities in terms of blood clotting and
coagulation; they can act as antithrombotic, anticoagulant and pro-coagulant. This
makes then applicable for the treatment of conditions such as hemophilia and air-
travel-induced deep vein thrombosis. Fucoidans delivered orally and intravenously
from a variety of species have shown these properties. This further adds to the robust
applicability of fucoidan.
Other recent potential applications of fucoidans include application in the treat-
ment of type 2 diabetes through inhibiting the breakdown of starch into sugar in the
body by inhibiting the action of the enzymes, amylase and glucosidase, which are
responsible for catalyzing the hydrolysis of starch into glucose (Senthil et al. 2019) or
through other mechanisms like improving effectiveness of insulin, as demonstrated
in experiments with mice (Sim et al. 2019). Fucoidans are also being investigated
for treatment of Alzheimer’s disease by acting as a neuroprotector (Alghazwi et al.
2019). These applications vary for fucoidan fractions from different sources.
In general, the bioactive properties of fucoidan, some of which are listed in
Table 5.3, occur in a wide range of species. Applications of fucoidan for these
bioactivities range from anticancer drug or adjuvant to antipathogenic agents. Suc-
cessful commercialization of fucoidan in medicinal applications poses significant
economic and social impact in terms of providing an easily accessible raw material
for such fucoidan-based therapeutics, a natural source which is less likely to result
in undesirable or serious side effects and less costly drug development compared to
synthetic-sourced alternatives.
114 5 Fucoidan
Table 5.3 Some reported bioactivities of fucoidans from various sources

Fucoidan source Bioactivity References
Sea cucumber Modulation of metabolic syndromes Shan et al. (2019)
and gut Microbiota dysbiosis (in vitro)
Kjellmaniella crassifolia Antioxidant activity and CCl4-induced Liu et al. (2018)
liver injury (in vitro)
Nizamuddinia zanardinii Antioxidant and anticancer properties Alboofetileh et al. (2019)
(in vitro)
Undaria pinnatifida Breast cancer cell inhibition (in vitro) Lu et al. (2018)
Turbinaria conoides Antiangiogenesis in cancer cells Matsubara et al. (2005)
Fucoidans of specified characterizations such as molecular weight and source are

commercially available as research chemicals by suppliers such as Sigma Aldrich.
Despite its broad spectrum of bioactive properties, fucoidan is yet to be FDA approved
for any of the clinical applications. The fact that fucoidans are not the major biopoly-
mers of economic importance contained in brown algae (Hahn et al. 2016) is one
of the limiting factors of their commercial exploration. The inconsistency in the
structural and bioactive properties that varies from species to species, harvest period
and extraction technique also further limits the commercial production of fucoidan.
The inconsistency in the chemical properties, extraction, purification and production
methods for different forms of fucoidan means it does not meet the good manufactur-
ing practice as set out for pharmaceutical products by the world health organization
(WHO 2014), for example, the inherent contamination with other polymers and phe-
nols due to the biological source and the low bioavailability and broad variation in
the chemical structure of fucoidans.
Although fucoidans still await regulatory approval for therapeutic applications,
it, however, is approved for use as food and food supplements since there are suf-
ficient evidence to support its safety and bioavailability when consumed as a food
or a food supplement (Fitton et al. 2015a, b). Fucoidan is detected in the serum and
urine following oral administration although more efforts are being directed toward
increasing bioavailability and having a better understanding of the mechanism of
absorption into the body. Proposed methods for improving bioavailability follow-
ing oral consumption of fucoidan include use of nanoparticles and liposome-based
formulations (Pinheiro et al. 2015; Lee et al. 2013a, b; Kimura et al. 2013). Orally
consumed fucoidan generally has a bioavailability of around 2% w/w or just within
detectable limits (Fitton et al. 2015a, b). This needs to be improved in order to utilize
the bioactive potential of fucoidan. While a food product being safe is a minimal
requirement, inability to fully absorb and utilize the bioactives within the food prod-
uct when consumed is a form of wastage in itself. The oral route has been known to
lead to loss of bioavailability due to the first-pass metabolism, where much of the
component of the ingested substance is exposed to degrading enzymes and the harsh
conditions in the stomach. Such that very little or no part of the active component
gets into the bloodstream. Therefore, to make more use of the benefits of fucoidan
as a bioactive compound, the applications need to go beyond oral administration.
A number of patents exist for use of fucoidans in biomedical applications. One
of such is for use as scaffolds in tissue repair (Le Visage et al. 2015), and another
existing patent covers the formulation and use of a fucoidan-based treatment for
bleeding disorders (Dockal et al. 2014). However, obtaining a patent for a product
only protects the right of the inventor but does not grant the approval to commercial
production.
Impurities significantly affect the bioactivity of fucoidans; therefore, in addition
to the requirement for clinical application, to study the structure and bioactivity of
fucoidan extracts, the crude fucoidan must be purified in order to understand the
true structure and bioactivity of fucoidan in its pure form. Purification methods for
fucoidans such as ion-exchange chromatography (Isnansetyo et al. 2017), gel perme-
ation chromatography and biological affinity purification (Zayed and Ulber 2019),
add significant cost to the production of pure fucoidans. Because of the symbiotic
relationship marine algae have with marine microbes, some of these microbes are
deeply attached to the cell walls of the algae and there usually exist different types
of microbes (Nambisan 1999), such that a broad spectrum sterilization technique is
required to ensure these contaminants do not get to the fucoidan extract.
5.9 Conclusion
Fucoidans are presently commercially available as food supplements. Their bioactive

properties cannot be standardized and validated due to inconsistency in structure and
bioavailability in oral form. The production of brown algae for fucoidan competes
with use of brown algae as food and for production of other products from brown
algae such as alginates. With potential applications of fucoidan as a low-cost treat-
ment for diseases such as colon cancer and type 2 diabetes, these fucose sulfated
polysaccharides remain of significant research and commercial interests.
References
Alboofetileh M, Rezaei M, Tabarsa M, You S, Mariatti F, Cravotto G (2019) Subcritical water

extraction as an efficient technique to isolate biologically-active fucoidans from Nizamuddinia
zanardinii. Int J Biol Macromol 128:244–253
Alghazwi M, Smid S, Karpiniec S, Zhang W (2019) Comparative study on neuroprotective activities
of fucoidans from Fucus vesiculosus and Undaria pinnatifida. Int J Biol Macromol 122:255–264
Alsac JM, Delbosc S, Rouer M, Journe C, Louedec L, Meilhac O, Michel JB (2013) Fucoidan inter-
feres with Porphyromonas gingivalis-induced aneurysm enlargement by decreasing neutrophil
activation. J Vasc Surg 57:796–805
116 5 Fucoidan
Alvarez-Vinas M, Florez-Fernandez N, Gonzalez-Munoz JM, Dominguez H (2019) Influence of

molecular weight on the properties of Sargassum muticum fucoidan. Algal Res 38 (Article101393)
Azuma K, Ishihara T, Nakamoto H, Amaha T, Osaki T, Tsuka T, Imagawa T, Minami S, Takashima
O, Ifuku S et al (2012) Effects of oral administration of fucoidan extracted from Cladosiphon
okamuranus on tumor growth and survival time in a tumor-bearing mouse model. Mar Drugs
10:2337–2348
Berteau O, Mulloy B (2003) Sulfated fucans, fresh perspectives: structures, functions, and bio-
logical properties of sulfated fucans and an overview of enzymes active toward this class of
polysaccharides. Glycobiology 13:29–40
Bilan MI, Kusaykin MI, Grachev AA, Tsvetkova EA, Zvyagintseva TN, Nifantiev NE, Usov AI
(2005) Effect of enzyme preparation from the marine mollusk Littorina kurila on fucoidan from
the brown alga Fucus distichus. Biochemistry (Moscow) 70:1321–1326
Carvalho AC, Sousa RB, Franco AX, Costa JV, Neves LM, Ribeiro RA, Sutton R, Criddle DN,
Soares PM, de Souza MH (2014) Protective effects of fucoidan, a p- and l-selectin inhibitor, in
murine acute pancreatitis. Pancreas 43:82–87
Chang Y (2010) Isolation and characterization of sea cucumber fucoidan utilizing marine bacterium.
Lett Appl Microbiol 50(3):301–307
Chang Y, Hu Y, Yu L, McClements DJ, Xu X, Liu G, Xue C (2015) Primary structure and chain
conformation of fucoidan extracted from sea cucumber Holothuria tubulosa. Carbohyd Polym.
https://doi.org/10.1016/j.carbpol.2015.10.016
Chen S, Zhao Y, Zhang Y, Zhang D (2014) Fucoidan induces cancer cell apoptosis by modulating
the endoplasmic reticulum stress cascades. PLoS ONE 9:e108157
Chen YM, Tsai YH, Tsai TY, Chiu YS, Wei L, Chen WC, Huang CC (2015) Fucoidan supple-
mentation improves exercise performance and exhibits anti-fatigue action in mice. Nutrients
7:239–252
Cho ML, Lee BY, You S (2011) Relationship between oversulfation and conformation of low and
high molecular weight fucoidans and evaluation of their in vitro anticancer activity. Molecules
16:291–297
Cumashi A, Ushakova NA, Preobrazhenskaya ME, D’Incecco A, Piccoli A, Totani L, Tinari N,
Morozevich GE, Berman AE, Bilan MI (2007) A comparative study of the anti-inflammatory,
anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown
seaweeds. Glycobiology 17:541–552
Deniaud-Bouët E, Hardouin K, Potin P, Kloareg B, Hervé C (2017) A review about brown algal cell
walls and fucose-containing sulfated polysaccharides: cell wall context, biomedical properties
and key research challenges. Carbohyd Polym 175:395–408. https://doi.org/10.1016/j.carbpol.
2017.07.082
Dockal M, Ehrlich H, Scheiflinger F (2014) Methods and compositions for treating bleeding
disorders. U.S. Patent 8,632,991
Dürig J, Bruhn T, Zurborn K-H, Gutensohn K, Bruhn HD, Béress L (1997) Anticoagulant fucoidan
fractions from Fucus vesiculosus induce platelet activation in vitro. Thromb Res 85:479–491
Elizondo-Gonzalez R, Cruz-Suarez LE, Ricque-Marie D, Mendoza-Gamboa E, Rodriguez-Padilla
C, Trejo-Avila LM (2012) In vitro characterization of the antiviral activity of fucoidan from
Cladosiphon okamuranus against newcastle disease virus. Virol J 9:307. https://doi.org/10.1186/
1743-422X-9-307
Esmaeelian B, Abbott CA, Le Leu RK, Benkendorff K (2014) 6-bromoisatin found in muricid
mollusc extracts inhibits colon cancer cell proliferation and induces apoptosis, preventing early
stage tumor formation in a colorectal cancer rodent model. Mar Drugs 12:17–35
Fitton J (2011) Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 9:1731–1760
Fitton JH, Dell’Acqua G, Gardiner VA, Karpiniec SS, Stringer DN, Davis E (2015a) Topical benefits
of two fucoidan-rich extracts from marine macroalgae. Cosmetics 2:66–81
Fitton JH, Stringer DN, Karpiniec SS (2015b) Therapies from fucoidan: an update. Mar Drugs
13:5920–5946
References 117
Fletcher HR, Biller P, Ross AB, Adams JMM (2017) The seasonal variation of fucoidan within
three species of brown macroalgae. Algal Res-Biomass Biofuels Bioprod 22:79–86
Gibson A, Edgar JD, Neville CE, Gilchrist SE, McKinley MC, Patterson CC, Young IS, Woodside
JV (2012) Effect of fruit and vegetable consumption on immune function in older people: a
randomized controlled trial. Am J Clin Nutr 96:1429–1436
Hahn T, Schulz M, Stadtmüller R, Zayed A, Muffler K, Lang S, Ulber R (2016) A cationic dye for
the specific determination of sulfated polysaccharides. Anal Lett 49(12):1948–1962
Han YS, Lee JH, Jung JS, Noh H, Baek MJ, Ryu JM, Yoon YM, Han HJ, Lee SH (2015) Fucoidan
protects mesenchymal stem cells against oxidative stress and enhances vascular regeneration in
a murine Hindlimb ischemia model. Int J Cardiol 198:187–195
Hayashi K, Lee JB, Nakano T, Hayashi T (2013) Anti-influenza a virus characteristics of a fucoidan
from sporophyll of Undaria pinnatifida in mice with normal and compromised immunity.
Microbes Infect/Inst Pasteur 15:302–309
Huang YC, Liu TJ (2012) Mobilization of mesenchymal stem cells by stromal cell-derived factor-1
released from chitosan/tripolyphosphate/fucoidan nanoparticles. Acta Biomater 8:1048–1056
Hwang PA, Yan MD, Kuo KL, Phan NN, Lin YC (2017) A mechanism of low molecular weight
fucoidans degraded by enzymatic and acidic hydrolysis for the prevention of UVB damage. J
Appl Phycol 29:521–529
Ikeguchi M, Yamamoto M, Arai Y, Maeta Y, Ashida K, Katano K, Miki Y, Kimura T (2011)
Fucoidan reduces the toxicities of chemotherapy for patients with unresectable advanced or
recurrent colorectal cancer. Oncol Lett 2:319–322
Isnansetyo A, Lutfia LNF, Nursid M, Susidarti RA (2017) Cytotoxicity of fucoidan from three
tropical brown algae against breast and colon cancer cell lines. Pharmacogn J 9(1):14–20
Jeong HS, Venkatesan J, Kim SK (2013) Hydroxyapatite-fucoidan nanocomposites for bone tissue
engineering. Int J Biol Macromol 57(138–141):123
Jin JO, Zhang W, Du JY, Wong KW, Oda T, Yu Q (2014) Fucoidan can function as an adjuvant
in vivo to enhance dendritic cell maturation and function and promote antigen-specific t cell
immune responses. PLoS ONE 9:e99396
Jour TY, Purcell S, Hair C, Mills D (2012) Sea cucumber culture, farming and sea ranching in the
tropics: progress, problems and opportunities 368(369):68–81
Kambhampati S, Park W, Habtezion A (2014) Pharmacologic therapy for acute pancreatitis. World
J Gastroenterol 20:16868–16880
Kandasamy S, Khan W, Kulshreshtha G, Evans F, Critchley AT, Fitton JH, Stringer DN, Gardiner VA,
Prithiviraj B (2015) The fucose containing polymer (fcp) rich fraction of Ascophyllum nodosum
(l.) le jol. Protects caenorhabditis elegans against Pseudomonas aeruginosa by triggering innate
immune signaling pathways and suppression of pathogen virulence factors. Algae 30:147–161
Kannan RR, Arumugam R, Anantharaman P (2013) Pharmaceutical potential of a fucoidan-like
sulphated polysaccharide isolated from Halodule pinifolia. Int J Biol Macromol 62:30–34
Kasai A, Arafuka S, Koshiba N, Takahashi D, Toshima K (2015) Systematic synthesis of low-
molecular weight fucoidan derivatives and their effect on cancer cells. Org Biomol Chem
13(42):10556–10568
Kim MJ, Jeon J, Lee JS (2014a) Fucoidan prevents high-fat diet-induced obesity in animals by
suppression of fat accumulation. Phytother Res 28:137–143
Kim YW, Baek SH, Lee SH, Kim TH, Kim SY (2014b) Fucoidan, a sulfated polysaccharide,
inhibits osteoclast differentiation and function by modulating rankl signaling. Int J Mol Sci
15:18840–18855
Kimura R, Rokkaku T, Takeda S, Senba M, Mori N (2013) Cytotoxic effects of fucoidan
nanoparticles against osteosarcoma. Mar Drugs 11:4267–4278
Kusaykin MI, Burtseva YV, Svetasheva TG, Sova VV, Zvyagintseva TN (2001) Distribution of
o-glycosyl hydrolases in marine invertebrates. Enzymes of the marine mollusk Littorina kurila
that catalyze fucoidan transformation. Biochemistry (Mosc) 68:317–324
118 5 Fucoidan
Kuznetsova TA, Besednova NN, Somova LM, Plekhova NG (2014) Fucoidan extracted from Fucus
evanescens prevents endotoxin-induced damage in a mouse model of endotoxemia. Marine Drugs
12(2):886–898. https://doi.org/10.3390/md12020886
Kwak JY (2014) Fucoidan as a marine anticancer agent in preclinical development. Mar Drugs
12:851–870
Kylin H (1913) Zur Biochemie der meeresalgen. Z Für Physiol Chemie 83:171–197
Lahrsen E, Liewert I, Alban S (2018) Gradual degradation of fucoidan from fucus vesiculosus and
its effect on structures, antioxidant and antiproliferative activities. Carbohyd Polym 192:208–216
Lean QY, Eri RD, Fitton JH, Patel RP, Gueven N (2015) Fucoidan extracts ameliorate acute colitis.
PLoS ONE 10:e0128453
Lee JS, Jin GH, Yeo MG, Jang CH, Lee H, Kim GH (2012) Fabrication of electrospun biocomposites
comprising polycaprolactone/fucoidan for tissue regeneration. Carbohydr Polym 90:181–188
Lee KW, Jeong D, Na K (2013a) Doxorubicin loading fucoidan acetate nanoparticles for immune
and chemotherapy in cancer treatment. Carbohydr Polym 94:850–856
Lee KY, Jeong MR, Choi SM, Na SS, Cha JD (2013b) Synergistic effect of fucoidan with antibiotics
against oral pathogenic bacteria. Arch Oral Biol 58:482–492
Le Visage C, Chaubet DLF, Autissier A (2015) Method for preparing porous scaffold for tissue
engineering. U.S. Patent 9,028,857
Liu S, Wang Q, Song Y, He Y, Ren D, Cong H, Wu L (2018) Studies on the hepatoprotective effect
of fucoidan from brown algae Kjellmaniella crassifolia. Carbohyd Polym 193:298–306
Lu J, Shi KK, Chen S, Wang J, Hassouna A, White LN, Merien F, Xie M, Kong Q, Li J, Ying
T, White LW, Nie S (2018) Fucoidan extracted from the New Zealand Undaria pinnatifida-
physicochemical comparison against five other fucoidans: unique low molecular weight fraction
bioactivity in breast cancer cell lines. Mar Drugs 16(461):1–25
Manne BK, Getz TM, Hughes CE, Alshehri O, Dangelmaier C, Naik UP, Watson SP, Kunapuli SP
(2013) Fucoidan is a novel platelet agonist for the c-type lectin-like receptor 2 (clec-2). J Biol
Chem 288:7717–7726
Matsubara K, Xue C, Zhao X, Mori M, Sugawara T, Hirata T (2005) Effects of middle molecular
weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Int J Mol Med 15:695–
699
Michel C, Lahaye M, Bonnet C, Mabeau S, Barry JL (1996) In vitro fermentation by human faecal
bacteria of total and purified dietary fibres from brown seaweeds. Br J Nutr 75:263–280
Min SK, Kwon OC, Lee S, Park KH, Kim JK (2011) An antithrombotic fucoidan, unlike heparin,
does not prolong bleeding time in a murine arterial thrombosis model: a comparative study of
Undaria pinnatifida sporophylls and Fucus vesiculosus. Phytother Res 26:752–757
Miyazaki Y, Iwaihara Y, Nakamizo M, Takeuchi S, Takeuchi H, Tachikawa D (2018) Potentiating
effects of high-molecular weight fucoidan-agaricus mix (CUA) feeding on tumor vaccination. J
Immunol 200(181):22–34
Monagail MM, Cornish L, Morrison L, Araujo R, Critchley AT (2017) Sustainable harvesting of
wild seaweed resources. Eur J Phycol 52(4):371–390
Mori N1, Nakasone K, Tomimori K, Ishikawa C (2012) Beneficial effects of fucoidan in patients
with chronic hepatitis C virus infection. World J Gastroenterol 18(18):2225–2230. https://doi.
org/10.3748/wjg.v18.i18.2225
Nagao T, Kumabe A, Komatsu F, Yagi H, Suzuki H, Ohshiro T (2017) Gene identification and
characterization of fucoidan deacetylase for potential application to fucoidan degradation and
diversification. J Biosci Bioeng 124(3):277–282
Nambisan P (1999) Seaweed biotechnology. Cyanobacterial and algal metabolism and environmen-
tal biotechnology, 236–246
Negishi H, Mori M, Mori H, Yamori Y (2013) Supplementation of elderly Japanese men and women
with fucoidan from seaweed increases immune responses to seasonal influenza vaccination. J Nutr
143:1794–1798
References 119
Oh R, Kim J, Lu W, Rosenthal D (2014) Anticancer effect of Fucoidan in combination with Tyrosine

Kinase Inhibitor Lapatinib. Evid Based Complement Alternat Med. 865375. https://doi.org/10.
1155/2014/865375
Park SJ, Lee KW, Lim DS, Lee S (2012) The sulfated polysaccharide fucoidan stimulates osteogenic
differentiation of human adipose-derived stem cells. Stem Cells Dev 21:2204–2211
Peng Y, Wang Y, Wang Q, Luo X, He Y, Song Y (2018) Hypolipidermic effects of sulfated fucoidan
from Kjellmaniella crassifolia through modulating the cholesterol and aliphatic metabolic
pathways. J Funct Foods 51:8–15
Pereira J, Portron S, Dizier B, Vinatier C, Masson M, Sourice S, Galy-Fauroux I, Corre P, Weiss P,
Fischer AM et al (2014) The in vitro and in vivo effects of a low-molecular-weight fucoidan on
the osteogenic capacity of human adipose-derived stromal cells. Tissue Eng Part A 20:275–284
Pinheiro AC, Bourbon AI, Cerqueira MA, Maricato E, Nunes C, Coimbra MA, Vicente AA
(2015) Chitosan/fucoidan multilayer nanocapsules as a vehicle for controlled release of bioactive
compounds. Carbohydr Polym 115:1–9
Ponce NMA, Pujol CA, Damonte EB, Flores ML, Stortz CA (2003) Fucoidans from the brown
seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies.
Carbohyd Res 338:153–165
Qin Y, Yuan Q, Zhang Y, Li J, Zhu X, Zhao L, Wen J, Liu J, Zhao L, Zhao J (2018) Enzyme-assisted
extraction optimization, characterization and antioxidant activity of polysaccharides from sea
cucumber Phyllophorus proteus. Molecules 23(590):1–19
Rodriguez-Jasso RM, Mussatto SI, Pastrana L, Aguilar CN, Teixeira JA (2010) Fucoidan-degrading
fungal strains: screening, morphometric evaluation, and influence of medium composition. Appl
Biochem Biotechnol 162:2177–2188
Rodriguez-Jasso RM, Mussatto SI, Pastrana L, Aguilar CN, Teixeira JA (2011) Microwave-
assisted extraction of sulfated polysaccharides (fucoidan) from brown seaweed. Carbohyd Polym
86:1137–1144
Sanjeewa KKA, Lee JS, Kim WS, Jeon YJ (2017) The potential of brown-algae polysaccharides
for the development of anticancer agents: an update on anticancer effects reported for fucoidan
and laminaran. Carbohydr Polym 177:451–459
Senthil L, Raghu C, Arjun HA, Anantharaman P (2019) In vitro and in silico inhibition properties
of fucoidan against alpha-amylase and alpha-D-glucosidase with relevance to type 2 diabetes
mellitus. Carbohyd Polym 209:350–355
Shan L, Li J, Mao G, Yan L, Hu Y, Ye X, Tian D, Linhardt RJ, Chen S (2019) Effect of sulfation
pattern of sea cucumber-derived fucoidan oligosaccharides on modulating metabolic syndromes
and gut microbiota dysbiosis caused by HFD in mice. J Funct Foods 55:193–210
Sharma G, Kar S, Basu Ball W, Ghosh K, Das PK (2014) The curative effect of fucoidan on visceral
leishmaniasis is mediated by activation of map kinases through specific protein kinase c isoforms.
Cell Mol Immunol 11:263–274
Silchenko AS, Kusaykin MI, Kurilenko VV, Zakharenko AM, Isakov VV, Zaporozhets TS, Gazha
AK, Zvyagintseva TN (2013) Hydrolysis of fucoidan by fucoidanase isolated from the marine
bacterium, formosa algae. Mar Drugs 11:2413–2430
Sim S, Shin Y, Kim H (2019) Fucoidan from Undaria pinnatifida has anti-diabetic effects by
stimulation of glucose uptake and reduction of basal lipolysis in 3 t3-L1 adipocytes. Nutr Res (in
press)
Sinurat E, Peranginangin R, Saepudin E (2015) Purification and characterization of fucoidan from
the brown seaweed Sargassum binderi sonder. Squalen Bull Mar Fish Postharvest Biotechnol
10(2):79–87
Synytsya A, Bleha R, Synytsya A, Pohl R, Hayashi K, Yoshinaga K, Nakano T, Hayashi T (2014)
Mekabu fucoidan: Structural complexity and defensive effects against avian influenza a viruses.
Carbohydr Polym 111:633–644
Tokita Y, Nakajima K, Mochida H, Iha M, Nagamine T (2010) Development of a fucoidan-specific
antibody and measurement of fucoidan in serum and urine by Sandwich ELISA. Biosci Biotechnol
Biochem 74:350–357
120 5 Fucoidan
Trejo-Avila LM, Morales-Martinez ME, Ricque-Marie D, Cruz-Suarez LE, Zapata-Benavides P,

Moran-Santibanez K, Rodriguez-Padilla C (2014) In vitro anti-canine distemper virus activity of
fucoidan extracted from the brown alga Cladosiphon okamuranus. VirusDisease 25:474–480
Venkatesan J, Bhatnagar I, Kim SK (2014) Chitosan-alginate biocomposite containing fucoidan for
bone tissue engineering. Mar Drugs 12:300–316
Wang J, Liu H, Li N, Zhang Q, Zhang H (2014) The protective effect of fucoidan in rats with
streptozotocin-induced diabetic nephropathy. Mar Drugs 12:3292–3306
Weelden G, Bobinski M, Okla K, Weelden WJ, Romano A, Pijnenborg JMA (2019) Fucoidan
structure and activity in relation to anti-cancer mechanisms. Mar Drugs 17(32):1–30
Wei X, Cai L, Liu H, Tu H, Xu X, Zhou F, Zhang L (2019) Chain conformation and biological activ-
ities of hyperbranced fucoidan derived from brown algae and its desulfated derivative. Carbohyd
Polym 208:86–96
WHO (2014) Good manufacturing practices for pharmaceutical products: main principles. WHO
technical report series no. 986
Yuan Y, Macquarrie D (2015) Microwave assisted extraction of sulfated polysaccharides (fucoidan)
from ascophyllum nodosum and its antioxidant activity. Carbohydr Polym 129:101–107
Zayed A, Ulber R (2019) Fucoidan production: approval key challenges and opportunities. Carbohyd
Polym 211:289–297
Zhang W, Du JY, Jiang Z, Okimura T, Oda T, Yu Q, Jin JO (2014a) Ascophyllan purified from Asco-
phyllum nodosum induces th1 and tc1 immune responses by promoting dendritic cell maturation.
Mar Drugs 12:4148–4164
Zhang Z, Till S, Jiang C, Knappe S, Reutterer S, Scheiflinger F, Szabo CM, Dockal M (2014b)
Structure-activity relationship of the pro- and anticoagulant effects of Fucus vesiculosus fucoidan.
Thromb Haemost 111:429–437
Zhao Y, Zheng Y, Wang J, Ma S, Yu Y, White WL, Yang S, Yang F, Lu J (2018) Fucoidan extracted
from Undaria pinnatifida: source for nutraceuticals/functional foods. Mar Drugs 16(321):1–17
Zuo T, Li X, Chang Y, Duan G, Yu L, Zheng R, Xue C, Tang Q (2015) Dietary fucoidan of Acau-
dina molpadioides and its enzymatically degraded fragments could prevent intestinal mucositis
induced by chemotherapy in mice. Food Funct 6:415–422
Chapter 6
Carrageenans
Abstract Carrageenans are the sulfated polysaccharides that are obtained from red
algae. They are most commonly applied as gelling agents. They are made up of
alternating disaccharide units of 1, 3 linked beta galactose linked to either 1,4 alpha
galactopyranose or 3,6 anhydrogalactose. They are classified as either λ, κ, ι, ε, μ,
depending on the degree of sulfation. The extraction process makes use of alkali,
acids and salts and requires energy for heating and additional processes to recover
and purify carrageenan from seaweed biomass. These are considered in evaluating
the environmental impact of carrageenan production. Presently, carrageenan is more
commonly used for its rheological properties as a gelling agent in food and other
consumer goods. Some studies have also presented potential application as a bioactive
compound and in renewable energy system components.
Keywords Carrageenan · Polymer · Polysaccharide · Phycocolloids · Algae
6.1 Introduction
Red algae are a source of polysaccharides. The polysaccharides serve as structural

as well as functional compounds within the red algae cell wall. Carrageenans are the
sulfated polysaccharides present in the cell walls of red algae where they play mainly
structural roles. They are one of the aquatic sourced polymers which are exclusive to
the aquatic ecosystem. The only known source of carrageenan is the red algae which
grow mainly in the epipelagic zones of the sea, and more rarely some species grow
in littoral zones of freshwater with moderate flow rate (Sheath and Vis 2015). The
harvesting of feedstock for carrageenan production is therefore from within these
regions of the aquatic ecosystem or simulation of these environments in aquaculture
for the cultivation of the feedstock.
Familiar consumer products within which carrageenans are commonly applied
include toothpastes, air fresheners and pet food. The important role carrageenan
plays in these fast moving consumer goods puts it at a value of around 300 million
USD (McHugh 2003). The value of the aquatic environment is extended to not only
the market value of the resources directly obtained from it but also the products
obtainable from these resources. While the present commercial value of carrageenan

122 6 Carrageenans
is mainly attributed to its use for its rheological properties, further modifications
of carrageenan result in the form of carrageenans with important bioactivities thus
increasing the potential market value of carrageenans to beyond just food additives.
Carrageenans are classified as galactans which are generally characterized by
the galactose repeating units. It is one of three hydrophilic phycocolloids obtained
from algae, and it is a sulfated polysaccharide found in the cell wall of red algae
species such as Kappaphycus alvarezii (Manuhara et al. 2016), Hypnea musciformis
(Souza et al. 2018) and Palisada flagellifera (Ferreira et al. 2012). Carrageenan has
relatively well-established application in the food industry where it is used for texture
modification and stabilization.
The subsequent sections in this chapter discuss the occurrence of carrageenan
in nature, the abundance of the raw material for production of carrageenan and
the present state of these resources as well as past trends. A couple of extraction
methods are reviewed to give the reader an idea of what is required to produce
this aquatic biopolymer. As naturally sourced products received increasing attention
from consumers, researchers and producers, it is important to review the environ-
mental implications of these products beyond just the fact that they are biodegradable
and renewable. A section in this chapter is therefore dedicated to the environmen-
tal implications of the process of producing carrageenans and discusses some key
aspects.
Carrageenan is present in nature as one of the sulfated polysaccharides of red algae

(Rhodophyta). It is a component of the cell wall where it plays a structural role along-
side the other cell wall polysaccharides. Red algae species which contain carrageenan
include species such as K. alvarezii (Manuhara et al. 2016), H. musciformis (Souza
et al. 2018), P. flagellifera (Ferreira et al. 2012), Gigartina skottsbergii (Gonçalves
et al. 2005), Eucheuma spinosum (Ghani et al. 2019). Prior to carrageenan being
known as the sulfated polysaccharide phycocolloid of red algae, carrageenan has
been used for its gelling properties in its whole form as seaweed as far back as 1905
where the Chondrus crispus commonly referred to as Irish moss is used as a gelling
agent in recipes such as blancmange (Smith 1905). Carrageenans have been identi-
fied as the gelatinous extract obtained from boiling of the red algae species C. crispus
in hot water (Stanford 1862). Carrageenan-producing species of red algae of genus
Eucheuma have documented use for medicinal, food and adhesive purposes in the
far east regions dating back to the early 1950s (Eisses 1952; Zaneveld 1959; Stanley
1987).
In the wild, red algae such as K. alvarezii are abundant in places such as
Karimun Jawa Islands in town of Jepara located in Central Java, Indonesia, and
in Ubatuba, São Paulo in Brazil (Gonçalves et al. 2005) and species such as
H. musciformis are abundant in regions like Flecheiras Beach in Ceara, Brazil
(Souza et al. 2018). H. musciformis is valued for its kappa carrageenan content
and is one of the species of economic importance in the northeast coast of Brazil
(Souza et al. 2018). Red algae grow in the epipelagic (sunlight) zones of the sea.
Some freshwater red algae also exist (Sheath and Vis 2015), albeit rare.
Until the 1970s, carragenophytes, mainly K. alvarezii and Eucheuma dentic-
ulatum, were obtained only from wild stocks. As the technology to cultivate
carragenophytes began in the Philippines and Indonesia where they were first cul-
tivated, carragenophyte cultivation spread to other regions of the world where it
has become a significant source of livelihood for many seaweed farmers in coun-
tries such as Tanzania and Vietnam. Today, some carragenophytes are still harvested
from the wild. For example, the Betaphycus gelatinum is harvested in regions in
the Philippines China, Hainan Island and Taiwan Province of China, and G. skotts-
bergii, Sarcothalia crispata and Mazzaella laminaroides are harvested in some parts
of Chile. Gigartina skottsbergii also grows in Argentina. However, over 90% of the
carragenophytes are cultivated. In other regions such as the Prince Edward Island in
Canada, USA and France, carragenophyte commonly grown here is the C. crispus.
Gigartina canaliculata and H. musciformis grow in Mexico and Brazil, respectively.
In some cases, the occurrence of the wild-growing species can be sporabic. Chile is
one example where all of the carragenophytes produced in this region are sourced
from natural stock. Despite this reliant on naturally existing carragenophytes, there is
no report of overharvesting and sufficient aquatic space exists for further expansion
of carragenophyte cultivation.
Carrageenans yield obtained from bench-scale extractions are around 34.3%
(Manuhara et al. 2016) although the actual content of carrageenan in red algae could
be higher since the yield of extraction is highly dependent on the efficiency and
method of extraction. Seaweeds from which carrageenans are obtained are referred
to as carragenophytes. The types of red algae from which carrageenans are sourced
vary with the locations within which they exist or conditions within which they can
be cultivated. Main factors for growth include temperature, salinity, water current,
nutrients composition and water depth which in turn affect light intensity. Most
algae exist in the epipelagic zones of the water (see Chap. 2 on aquatic ecosystem
classification) where sufficient light penetrates for photosynthesis.
One of the red algae species which is the most common source of carrageenan is
K. alvarezii. This species grows in marine water where the temperature is ~21 °C and
there is sufficient bright light for growth. This species grows well in water exposed to
slow to moderate tides and rocks, sand and coral substrates. Another species which
grows in this region is the E. denticulatum prefers moderate to strong tides and B.
gelatinum grows in stronger tides close to the reef edge.
Species which grow at warmer temperatures include chondrus crispus which
grows in the littoral fringe with moderate light and large rock areas. In colder cli-
mates, it grows best in the summer periods and late spring with minimal growth in
winter. G. skottsbergii also grows in warmer seasons in the sublittoral region on the
sea. M. laminaroides and sarcothalia, G. canaliculata and H. musciformis all grow
along the seashore line while S. crispata grows in the epipelagic zone. Cultivations
in tanks and seaweed farms require mimicking these growth conditions for optimal
yield of the particular species of interest.
124 6 Carrageenans
6.3 Chemistry of Carrageenan
The structure of carrageenan is the sulfated polysaccharide polymer chain made up

of alternating disaccharide units of 1, 3 linked beta galactose linked to either 1, 4
alpha galactopyranose or 3,6 anhydrogalactose. It can therefore be said to be an
alternating copolymer of these two units. Carrageenans are linear polymer struc-
tures characterized by the monomeric units and also importantly sulfate ester groups
attached to some of the repeating units. Carrageenans can be differentiated from the
other sulfated polysaccharide present in red algae (agarans) by the stereochemistry
of the 1-4 linked alpha galactopyranose which is D form in carrageenan but L form
in agarans (Jiao et al. 2011). They are classified as either λ, κ, ι, ε, μ, depending on
the degree of sulfation which could range from 22 to 35%. The most common types
are λ, κ and ι, while κ is the most used of the three types. While k carrageenan has
alternating units of 1, 3 beta-d-galactose with a sulfate group at C4 and 1-4 linked
anhydrogalactose unit, iota carrageenan has a similar structure but with further sul-
fation on the C2 carbon of the anhydrogalactose unit such that the iota carrageenan
has two sulfate groups on the repeating disaccharide unit. The lambda carrageenan is
further sulfated by having a third sulfate group attached to the C6 carbon of the alpha
1-4 linked galactose unit, such that it has 3 sulfate groups per disaccharide unit. The
lambda carrageenan further differs from the other forms by having no 3,6 anhydride
bridge on the 1-4 linked galactopyranose (Funami et al. 2007). The types present
vary in different species of red alga; for example, K. alvarezii is the most common
source of k carrageenan and lambda carrageenan is more abundant in species such
as Gigartina and Chondrus genera (Zhou et al. 2006). The presence of the sulfate
group makes carrageenans anionic polymers. Extraction methods have significant
effect on the properties of the carrageenan obtained. The degree of sulfation in turn
affects properties such as the gel strength.
Other than the natural form in which they occur within different species, car-
rageenan of different forms can be obtained by chemically processing the original
forms. For example, theta carrageenan can be obtained from lambda carrageenan
through alkali treatment to form anhydride bridges between the units. This alkali
treatment usually follows the extraction process in order to convert the extracted
from into the desired form of carrageenan (Doyle et al. 2010). In nature, carrageenans
occur as a mix or hybrid of different forms of carrageenan. In addition to this, the
carrageenan structure can be further complicated by the presence of methyl groups,
pyruvic acetal or other sugars attached to the main chain (Yu et al. 2010). Figure 6.1
shows the different forms of carrageenan and how they can be transformed from one
form to the other.
In FTIR spectrometry, carrageenan is characterized by absorption peaks at
1234 cm−1 which is indicative of the presence of sulfate ester (S = 0) functional
group, 3,6, anhydrogalactose at 926 cm−1 and glycosidic linkages at 1072 cm−1 .
The band occurs around 840–850 cm−1 which indicates the presence of sulfated
anhydrogalactose for k type carrageenan. The vibration of the O–H hydroxyl groups
6.3 Chemistry of Carrageenan 125
Fig. 6.1 Disaccharide structures of λ, κ and ι forms of carrageenans
and the C–H alkyl group is indicated at 3441 and 2934 cm−1 , respectively (Souza
et al. 2018; Manuhara et al. 2016).
Carrageenans are soluble in water at high pH where they can form either viscous
solutions or thermoreversible gels. The temperature at which carrageenans dissolve
in water is increased as the level of sulfation decreases (Ghani et al. 2019). The inter-
action with water is dependent on the type of carrageenan, while iota and kappa form
thermoreversible gels, and lambda carrageenan forms viscous solutions (Williams
and Phillips 2003). Hence why, some forms of carrageenan are more suitable as vis-
cosity enhancers and others are suitable as gelling agents. Carrageenans form gel by
unraveling of their random coil structure to form helical secondary structures. These
helixes then form networks in water to form thermoreversible gels. The solubility
and nature of solution or gel formed also depend on the types of electrolytes present.
The properties of these carrageenans vary significantly and consequently so do their
applicabilities. For example, k carrageenan finds application in food industry as a
thickening and gelling agent, in acetic acid production (Iglauer et al. 2011) and in
industrial effluent treatment (Necas and Bartosikova 2013).
Occurence of carrageenan varies in different species of red algae. For example,
Eucheuma cottonii and K. alvarezii contain mainly kappa carrageenan, E. spinosum
126 6 Carrageenans
Table 6.1 Three main forms of carrageenan and their properties

Carrageenan
Kappa (κ) Iota (ι) Lambda (λ)
• Strong and rigid gels in • Gels have higher elasticity • Does not form gel network
potassium salts when formed with calcium • Forms highly viscous
• Brittle gels with calcium salts solution
salts • Clear gels • Species: Chondrus crispus,
• Slightly opaque gel which • Is less prone to syneresis Gigartina skottsbergii,
can be made clear when • More freeze/thaw stable Sarcothalia crispata
sugar is added • Species: Eucheuma
• Slight tendency for denticulatum
syneresis to occur
• Species present:
Kappaphycus alvarezii
(main component),
Chondrus crispus,
Gigartina skottsbergii,
Sarcothalia crispata
contains mainly iota carrageenan, while C. crispus contains a combination of lambda

and kappa. The main distinction between the different types of carrageenan is the
degree of sulfation and the positioning of the sulfate group. In kappa carrageenan,
the sulfate group is attached to the C4 of the 1-3, β-galactopyranose, iota carrageenan
has its sulfate group at C2, while lambda carrageenan is sulfated as the C2 of the
1,3, β-galactopyranose. The lambda form also has the highest degree of sulfation
of 70% (Williams and Phillips 2003). Table 6.1 summarizes the different types of
carrageenan and their properties.
The molecular weight of carrageenan has significant effect on its rheological,
thermal and mechanical properties as well as the bioactivity (Souza et al. 2011).
Although the effect of the molecular weight on the properties of carrageenan varies for
different types of carrageenan which in turn vary in the degree and pattern of sulfation
and sequence of repeating units, some deductions can be made from reported studies
to relate molecular weight to some specific parameters. For instance, lower molecular
weight carrageenan shows higher gel elasticity than higher molecular weight ones
(Souza et al. 2011). This was observed for a mixture of k and l carrageenan extracted
from Mastocarpus stellatus red algae. The solubility and dissolution temperature as
well as the conformation upon interaction with water are affected by the molecular
weight. Generally, polymers’ molecular weight is related to the solution viscosity
such that the viscosity average molecular weight can be obtained from the intrinsic
viscosity using the Mark–Houwink equation. Molecular weight of carrageenan of
commercial grade is between 200 and 800 kDa (Weiner et al. 2017).
Carrageenan is sourced from red algae (Rhodophyta), which is one of the three
main types of seaweeds. Seaweeds are grown commercially in 35 countries of the
world; as of 2003, the seaweed industry was valued at an estimated 5.5–6 billion
USD annually (McHugh 2003). Of this estimated value, about 1 billion occurs from
hydrocolloids production from seaweed with carrageenan being one of such. The
majority of seaweed production still goes toward direct consumption by humans as
food, while a smaller portion goes toward other miscellaneous uses such as fertilizers
and additives in animal feed. Species such as Undaria pinnatifida, Caulerpa spp. and
Porphyra spp. which are dominant in Eastern and Southeastern Asia are produced
mainly for direct consumption as food (FAO 2018).
As of 1995, 13.5 million tonnes of seaweed was reportedly produced globally
(McHugh 2003). This rose to over 30 million tonnes by 2016 (FAO 2018). The
seaweed production output is currently led by China and Indonesia. China’s seaweed
production rose from near 11 million tonnes in 2010 to over 14 million tonnes by
2016. Indonesia’s seaweed production rose from 4 million tonnes in 2010 to well over
11 million tonnes by 2016. Indonesia produces mainly K. alvarezii and Eucheuma
spp. which are the main sources for carrageenan production. Globally, Eucheuma spp.
is currently the highest produced seaweed species with output rising from 3.5 million
tonnes in 2010 to 10.5 million tonnes in 2016. K. alvarezii comes in as the fifth highest
output increasing from 1.2 million tonnes in 2010 to 1.5 million tonnes in 2016 (FAO
2018).
In 2018, FAO lists China, Indonesia, Philippines, Republic of Korea, Democratic
People’s Republic of Korea, Japan, Malaysia, Tanzania, Madagascar, Chile, Solomon
Islands, Vietnam, Papua New Guinea, Kiribati and India, as the major seaweed pro-
ducers in the world (FAO 2018). However, there exists a large gap between the output
quantities, ranging from over 14 million tonnes produced by the top producer, China,
to 3 thousand tonnes produced by India, the lowest producer. Seaweed production
by far predominates over aquatic plant production globally. This can be attributed to
their significant role as highly nutritious food, potential medicinal applications and
increasing interest in cultivating them for biofuel production.
Seaweeds grow in a variety of conditions from cold to warm such that they can
grow in different parts of the world from northern to Southern Hemisphere. About
7.5–8 million tonnes of seaweed is harvested annually on wet basis. These are either
obtained from wild-growing stocks or cultivated in seaweed farms (McHugh 2003).
Using artificial growth systems which replicate the sea conditions, seaweeds are
being commercially grown even in the middle of the desert.
The earliest red seaweed species which served as a source of carrageenan is the
C. crispus which is commonly referred to as Irish moss. It naturally occurs in
France, Portugal, Ireland, Spain and eastern coast of Canada. Other species from
which carrageenan is sourced are Irideae, Gigartina and Eucheuma which grow
in Chile, Spain and the Philippines, respectively, as additional species to source
carrageenan became necessary as demand for carrageenan grew. Today, the red
128 6 Carrageenans
seaweed species K. alvarezii and E. denticulatum have become the more com-
mon raw material for carrageenan production (McHugh 2003). For more diverse
range of applications, there are continuous efforts to explore the variety of struc-
tural forms of carrageenan and this involves uses of a broad range of species which
serve as sources of various forms of carrageenan. These include H. musciformis,
M. laminaroides, S. crispata, B. gelatinum, C. crispus, E. denticulatum, Eucheuma
gelatinase, E. spinosum, G. canaliculata and G. skottsbergii (McHugh 2003).
Red seaweed, which serves as the source of carrageenan, is primarily used as
food as it makes up a sizeable part of Asian cuisine which is adopted across different
parts of the world. In cold waters, red seaweed species of economic importance such
as Palmaria palmata can be sourced from cold regions such as Canada, Iceland,
Ireland and southern Chile, while in warmer waters they can be found in areas such
as Morocco, Portugal, the Philippines, and Indonesia.
Most common species of red seaweed that is harvested for food is the Porphyra
which is more commonly called nori or laver. It is commonly used in preparation for
sushi, a Japanese delicacy which comprises raw fish and boiled rice wrapped in red
seaweed which has been processed into thin sheets. The seaweed plays a structural
role as an edible food wrap and also has significant nutritional value.
A large proportion of the red seaweed harvested globally is from cultivation of red
seaweed as seaweed growing naturally in the wild is insufficient to meet increasing
demands. The cost of harvesting from wild is also higher, and in some cases access
is limiting. Cultivation in tanks requires a good understanding of the life cycle and
required growth conditions of the particular species. Republic of Korea, China and
Japan are some of the major large-scale producers of red seaweed. Red seaweed nori
is one of the most highly priced seaweeds which is valued at 1200 USD per wet
tonne, relative to, for example, brown seaweeds which are valued at 610–530 USD
per wet tonne.
The type of carrageenan present in each species of red algae varies. Some species
contain almost only one form of carrageenan, while some species contain a mixture
of two different types of carrageenan. These mixtures are referred to as hybrids.
A species of red seaweed known as K. alvarezii, for example, contains almost
entirely kappa carrageenan, and another species called E. denticulatum (E. spinosum)
contains almost entirely iota carrageenan. Species which contain hybrids include
Chondrus which contain a mixture of kappa and lambda. A large fraction of the red
seaweeds used for carrageenan production are sourced from the Philippines. Improve-
ment in seaweed harvesting technology has led to red seaweed cultivation spreading
to warmer regions such as Tanzania and Indonesia. Seaweeds which can be grown
vegetatively are preferred for commercial carrageenan production as those requir-
ing sexual reproductive cycles are more cost intensive making it non-economical
as raw materials for carrageenan production. For seaweeds sold as food, the cost
can be recovered more easily since very little further value addition occurs in the
product preparation. However for use as raw materials for carrageenan production,
further cost is involved in the extraction and purification of carrageenan such that it is
paramount to minimize the cost of raw material, thus making vegetative cultivation
a more favorable option.
According to FAO reports, production of some red algae species has increased
in the past 10 years while some have dropped. K. alvarezii, for example, showed
annual increase in production from 1285 MT in 2005 to 1963 in 2012. This was
then followed by a decline to 1726 MT in 2013 to 1527 in 2016 (FAO 2018). On the
other hand, Porphyra has shown a general annual rise from 703,000 tonnes in 2005 to
1,353,000 tonnes in 2016. This excludes a drop from 1,072,000 to 1,123,000 tonnes
between 2010 and 2012. On this basis, the future availability of red algae is quite
unpredictable.
The factors which may pose a threat to the availability of red algae are inter-
woven with the factors which pose a threat to the aquatic environment as a whole.
Exploring renewable sources of polymers used in products such as processing agents,
packaging, energy storage, printing, cosmetics and textiles is a route to abating the
threat of depleting fossil fuel reserves. The other issue is the impact of the process
of extraction of these resources on the environment. The next section explores the
environmental impact of producing carrageenan from red algae.
6.5 Extraction of Carrageenan
Carrageenan is soluble in aqueous alkali solution at high temperature and insoluble

in alcohol and certain salt solutions (Williams and Phillips 2003), and this property
serves as a basis for its extraction from red seaweed. Alkalis commonly used include
sodium hydroxide, potassium hydroxide and calcium hydroxide. Here, we use a
typical chemical extraction process as used in the literature (Manuhara et al. 2016)
and an enzyme extraction method used by Souza et al (2018). Variations in method
include change in the choice of alkali, use of enzymes and extraction conditions such
as extraction pH, temperature and time.
6.5.1 Chemical Extraction
In the chemical extraction method by Manuhara et al. (2016), the collected red algae
biomass is washed with water and then soaked in water for 24 h to soften. The wet
biomass is then pulverized until a pulp is obtained. Extraction is carried out in alkali
solution of calcium hydroxide at a pH of 9. The system is heated to 90 °C and the
extraction carried out for 2 h under continuous stirring. The alkali serves to decrease
the degree of sulfation and also increase the number of 3,6 anhydrous carrageenan
units. This makes for stronger gels (McHugh 2003). The mass is then separated by
filtration. The liquid filtrate which contains the carrageenan is retained for further
purification. Hydrochloric acid is added until the pH reduces to 7. The neutralized
liquid filtrate is then heated at 60 °C for 30 min. At this point, the filtrate contains
other soluble molecules; therefore, the carrageenan needs to be precipitated out of
the solution. This is done using potassium chloride (KCl) at a concentration which
130 6 Carrageenans
ranges between 1 and 3.5% in the volume ratio 1:1. This variation was done in
this particular study to investigate the effect of KCl concentration on the properties
of the carrageenan formed, and 2.5% was found to be the optimum concentration
(Manuhara et al. 2016). A duration of 15 min is allowed for the precipitation. The
carrageenan precipitates out as a gel which is separated from the liquid by filtration.
This is then followed by washing in 96% alcohol, drying at 70 °C in a cabinet dryer
for 24 h. The carrageenan is then milled into desired particle size. Figure 6.2 shows a
flowchart summarizing the extraction process. Here, it is shown that two routes can
be followed after filtration and initial concentration which is done by evaporation
of the solvent. The filtrate can be precipitated either with a salt or with alcohol.
When precipitated with alcohol, a solid precipitate is formed which can then be
dried and then taken through a repeated precipitation stage to improve purity. When
precipitated with potassium chloride, a gel is formed. This only happens for kappa
carrageenan which has the ability to form stronger gel potassium chloride and hence
is more suitable for gel pressing prior to drying.
After initial filtration, the filtrate usually contains about 1–2% w/v carrageenan,
following further filtration which could be done using vacuum distillation or ultrafil-
tration, and the final concentrate contains 2–3% carrageenan (w/v) (McHugh 2003).
The rest of the moisture then needs to be removed by drying to obtain a dried
carrageenan which can then be milled for more effective storage and packaging.
Red Washing Soaking Pulverization

algae
biomass
Solid residue
Neutralization Filtration Alkali

Liquid extraction
filtrate
KCl Precipitation Alcohol Precipitation
Gel pressing Drying
Drying Carrageenan
Fig. 6.2 Flowchart summarizing extraction process for carrageenan from red seaweed
6.5 Extraction of Carrageenan 131
Carrageenan can also be isolated from red algae using enzymatic digestion. This
involves breaking down the proteins within the structure to release the polysaccha-
rides. The sulfated polysaccharide, which in red algae is carrageenan, is then purified
through precipitation. This method has been used by Souza et al. (2018) to extract
carrageenan from the H. musciformis red algae. In the said study, the harvested
biomass was cleaned and washed to remove epiphytes and other unwanted matter.
The process of extraction then begins with proteolytic digestion using the protease
enzyme, papain from the papaya fruit (Carica papaya). This breaks down the pro-
tein into smaller units of peptides and amino acids. The digestion was carried out
at 60 °C in a sodium acetate buffer at pH 5 containing 5 mM of EDTA and cys-
teine. The digestion was allowed a period of 6 h. The next stage is then to isolate
the freed sulfated polysaccharide from the solution. This was done by precipitation
with cetylpyridinium chloride. This method resulted in a carrageenan yield of 28%
carrageenan per gram of dry red algae biomass. The carrageenan extracted showed
17.3% free sulfate content and a very polydisperse extract with peak molar mass of
519.1 kDa measured using gel permeation chromatography.
6.5.3 Semi-refined Carrageenan
The semi-refined form of carrageenan also finds some use. This form of carrageenan
is one, whereby other water-soluble components of the carrageenan are removed by
dissolving them of at lower water temperature such that the carrageenan alongside
the salt still remains behind in the seaweed. This process requires much lower cost
of processing and is used where the presence of cellulose alongside the carrageenan
does not affect the application. The seaweed is washed to get rid of debris. It is
then treated in a solution of potassium hydroxide at a moderately high temperature.
This allows the reduction in degree of sulfation as well as the hardening of the gel
structure, while the carrageenan is still within the seaweed biomass. The heat allows
the aqueous solution of alkali to penetrate the cell walls allowing interaction between
the OH- ions and the sulfate groups and the K+ ions and the gel networks resulting in
a stronger gel which does not dissolve in the solution. The other components of the
seaweed such as proteins, water-soluble carbohydrates and other smaller molecules,
however, are removed in this process. This leaves behind a solid mass which is
mostly carrageenan and cellulose. This can be heat sterilized and used as low-grade
gelling agent in canned pet food where a cheaper alternative to refined carrageenan is
acceptable. Alternatively, it can be sold to carrageen manufacturers as feedstock for
producing refined carrageenan. The latter option enables value addition to seaweed
from place of origin and reduces the cost of transportation and waste treatment for
the manufacturer. When semi-refined carrageenan is produced using more stringent
132 6 Carrageenans
processes which ensures a clean sterile product containing carrageenan and cellulose,
it can be sold as carrageenan suitable for human consumption (McHugh 2003).
The hardening of the carrageenan gel only occurs in kappa and to a lesser extent
in iota carrageenan; therefore, the gel formation-based extraction can only be used
for seaweeds containing these forms of carrageenan. The other main form of car-
rageenan, lambda carrageenan, does not form gels and therefore can only be precip-
itated using alcohol (McHugh 2003). The end use of the carrageenan must therefore
be considered prior to determining a suitable extraction method.
Carrageenan extraction is dependent on elevated temperature in aqueous alkaline

conditions. In the main extraction process of the crude carrageenan, the concerns
here are energy consumption, water usage and use of alkali. Other factors to consider
include land use changes, additional energy used in separation, milling, drying and
purification as well as other components such as use of salts for precipitation and
alcohol for washing and drying.
Table 6.2 gives a summary for a typical process of extraction of carrageenan
from red algae using chemical extraction method, and some of these are discussed
further in the following subsections. This method is chosen as an example since it
is more commonly used in present industry. The values here are simply the basic
consumptions for a bench-scale extraction.
Table 6.2 Typical

consumptions for extraction
of carrageenan Red algae biomass 2.92 g per gram carrageenan
Calcium hydroxidea 15 ml at pH 9 per gram carrageenan
HCl Neutralize 15 ml CaOH from pH 9 to 7
Potassium chloride 15 ml of 2.5% per gram carrageenan
Ethanol 15 ml 96% per gram carrageenan
Energy
• Heat 90 °C for 2 h
• Drying 60 °C for 3 min
• Stirring 70 °C for 24 h
200 rpm for 2 h
6.6.1 Cultivation and Harvest of Red Algae
An estimated 1180 kg of seaweed biomass is required to produce 1 kL of the car-

rageenan containing say which can then be further processed to produce carrageenan.
In addition to the benefits of growing algae as discussed in Chap. 5 and this chapter
on alginate and fucoidan, red algae compared to the brown algae are easier to cul-
tivate on a large scale since they have a vegetative reproductive cycle which is less
complex than that of brown algae. A particular type of the Rhodophyta known as
coralline algae has played a significant role in the formation of coral reefs for bil-
lions of years and still continues to do so (Moreira-Gonzalez et al. 2019). Harvesting
of red algae from the wilds or cultivating in aquaculture or open seas ensures that
the conditions in the water are equally conducive for the growth and functioning of
these coral-forming red algae. Hence, red algae cultivation for alginate production
encourages the maintenance of marine biodiversity.
The drying stage consumes much of the energy in the production process. The filtrate
from the extraction only contains about 2–3% carrageenan, and the final product
needs to be dried to at least 4% moisture content for storage and packaging. In this
dry state, the growth of microbes is minimized hence allowing a longer shelf life of
the carrageenan. Where there is intense sunlight and the weather is sufficiently hot
in the day, drying can be done outdoors to save cost from using electrical dryers.
Energy is also consumed in transportation of the cultivated biomass to the factory
for further processing. In the life cycle assessment carried out by Gosh et al. (2015),
transportation of the cultivated biomass accounted for 13% of the environmental
impact in the cultivation stage. In the processing of biomass into carrageenan and
sap, the high-density polyethylene production for packaging shed and electricity
made up 97.3% of the carbon footprint, with electricity consumption accounting for
25.2% and plastic packaging accounting for 54.2% of this.
When transportation by sea, road and rail was compared, transportation by sea for
conveying biomass to factory for processing proved to be the least energy-consuming
method while road consumed the most energy. Climate impact by road, rail and sea
was 138.5%, 51.8% and 141%, respectively (Ghosh et al. 2015). In some cases where
transportation by road is the only option, the most efficient means of transportation
needs to be sought. For instance where the place of cultivation (usually shore) is at a
relatively close distance to the processing factory, tricycle carts could be considered
as an option; however where quantities are on a large scale, this is not practical.
Energy consumption can be minimized by using more fuel efficient machinery
and transportation systems. Even where manual labor is used, it is important to have
the most efficient process in place, to make optimal use of manpower. Transportation
by sea to distances of up to 7200 km to other countries is even less energy-consuming
134 6 Carrageenans
that transportation by road within the same country by distances of about 1500 km
(Gosh et al. 2015).
6.6.3 CO2 Emission
The 118.6 kg of CO2 equivalents is produced per kL of sap produced from K. alvarezii
(Ghosh et al. 2015). To put this in perspective, cultivation of maize on one hectare of
land results in production of 599 kg of CO2 . The 142.5 L of sap which is equivalent
to a hectare produces 25.65 kg of CO2 (Gosh et al. 2015). The process for producing
the sap results in the production of semi-refined carrageenan which can then be
further refined to produce refined carrageenan of higher grade. This process was
based on using the same cultivation process for carrageenan production, and the
process of extraction of sap is part of the process of carrageenan production. This
quantity of CO2 includes the entire process of sap production from cultivation to the
factory gate. Following the process of separation of the sap from carrageenan, the
two processes then separate such that this estimated CO2 emission is not the same as
that for carrageenan production as the process of refining to final packaging differs.
Therefore, the CO2 emission for carrageenan production is expected to be greater
than 118.6 kg.
The environmental impact of the CO2 emission from the process is weighed
against the benefits to the environment of carrageenan and its by-product. For exam-
ple if sap is coproduced along with sap which is in turn sprayed on crops, this
substitutes the chemical fertilizers providing 21 g per l of potassium per sap. The
1000 L of sap substitutes 25.3 kg of chemical fertilizers (Ghosh et al. 2015).
6.6.4 Water Consumptions
Water serves the key role as the extraction solvent. It is also used in the washing of
the raw material and solvent for the salt for precipitation. Gosh et al. (2015) estimate
262.205 m3 of water used in the production of 1 kl of unrefined carrageenan (sap). In
the reported experimental extractions (Manuhara et al. 2016), if we include the water
used in washing and soaking and as solvent and continuous media for the process, an
estimated 75 ml of water is used per gram of red algae processed. Although water is
recycled, the release of mineral acids and alkali and salts into the water cycle results
in gradual acidification and alterations of water salinity as the water is returned into
the sea. The cultivation of red algae could be integrated into the wastewater treatment
from the extraction process to achieve a net zero release of minerals into the water.
This will require modification of the process such that the acids, alkali, salts and
alcohols used can be broken down into minerals which are consumed by algae such
as nitrates and sulfates.
6.6.5 Use of Plastics
In a life cycle assessment carried out by Ghosh et al. (2015), the economic and
environmental impact of cultivation of K. alvarezii for the production of biostimulant
sap alongside semi-refined carrageenan was assessed. It was deduced that 83.7% of
the impact on environment in the cultivation stage came from the use of plastics.
These include the polypropylene and high-density polyethylene ropes used for the
rafts and the use of plastic nets. The ropes and nets were assumed to have an average
life span of ten cultivation cycles. Implementing plastic reuse within the value chain
in, for example, packaging significantly reduced the carbon footprint of the process
as a whole by about 28% (Ghosh et al. 2015).
6.7 Applications
Much of the application of carrageenan is reliant on its rheological properties, gelling

property and viscosity-enhancing properties. Most common commercial application
of carrageenan is in food. However, its established and potential applications expand
to other industries which include pharmaceutical, cosmetics, printing and textiles.
Thus far, it seems all sulfated polysaccharides possess common properties which
can be attributed to the sulfated pyranose structure. Each sulfated polysaccharide,
however, distinguishes itself through the mechanism of action that results from the
variation in the monosaccharide units and sequence of monosaccharides occurring in
the polymer chain. This makes some sulfated polysaccharides more predominant in
some applications than others. Carrageenan finds its key relevance in food industry
as a thickener or gelling agent. However, other applications exist and either are
already being commercially explored or have recently come to light and are being
investigated for commercial use. In most applications in foods, carrageenans are
used commercially at concentrations ranging between 0.005 and 0.5% (Davidson
1980; Graham 1977). Such that, they are high-value additives used in relatively low
amounts.
6.7.1 Milk Stabilizer
Carrageenan is commonly added to liquid milk products as a stabilizer. The stabiliz-

ing properties of carrageenan are attributed to its ability to interact with the proteins
in milk and also with itself to form a weak gel network which prevents formation of
sediments and creaming of the milk product and by so doing prolongs the shelf life
of the milk product. For such purposes, it is important to have a balance between the
interaction the carrageenan polymer chains have with each other and the interactions
they have with the protein polymers in the milk.
136 6 Carrageenans
In milk preservation, the three types of carrageenans, λ, κ and ι, are usually

mixed together. Carrageenan is usually used in concentration ranging between 0.01
and 0.05 wt% (Towle 1973; Syrbe et al. 1998; Tijssen et al. 2007). Carrageenan is
particularly of importance in ultrahigh temperature (UHT) treated milk in which the
milk protein has been slightly denatured due to high-temperature treatment.
Carrageenan stabilizes milk by forming a secondary helical structure and then
bridges forming at different junctions of the helices. The κ- and ι-type carrageenans
usually form these helical secondary structures resulting in gelation. Gelation is
affected by the presence of certain ions, particularly calcium (Ca+) ions and potas-
sium (K+) ions. The high amount of sulfate groups present in the λ-type carrageenan
prevents it from gelation. This gelation is then further combined with complexation
which occurs as a result of electrostatic interactions between the carrageenan and
the protein. This is a result of the positive charged regions on the surface of the milk
protein and the sulfate groups of the carrageenan. In some cases, this may be further
aided by cations such as Ca+ forming bridges between the carboxyl group of the
proteins and the sulfate groups of carrageenan (Tijssen et al. 2007).
Carrageenan is also used in other dairy products such as cheese, ice creams,
chocolate milk and coffee creamers for the separation of whey and fat. 33% of the
carrageenan market is from use in dairy products (McHugh 2003). This makes up
the market where much of carrageenan is used.
6.7.2 Gelling Agent
In the presence of electrolytes such as rubidium, potassium and cesium, kappa car-
rageenan can form gels at much lower concentration (Williams and Phillips 2003).
Such that, less carrageenan is required to obtain the required gel texture. This is
important for both reducing cost of food processing and also minimizing the addi-
tive content. Hence why, kappa form of carrageenan is much preferred as a gelling
agent compared to other forms. Gels formed in the presence of potassium chloride
(KCl) show superior gel strength (William and Phillips). Iota carrageenan does not
show this same increased gel strength in the presence of potassium chloride. Although
kappa forms strong gels, they are rather brittle and more prone to syneresis in the
absence of enhancers. Konjac mannan and locust bean gum are used to improve the
gel properties and prevent syneresis (Williams and Phillips 2003).
In water-based jelly, a combination of kappa and iota carrageenan can be used as
a replacement for pectin where a little or no calorie alternative is needed. In nondairy
alternatives to ice cream such as sorbet, the creamy mouthfeel is achieved using a
mixture of iota and kappa carrageenan. For this purpose, locust bean or pectin is also
added to enhance the texture. A mixture of kappa and iota carrageenan is also used in
stabilizing oil in water emulsions in mayonnaise with reduced oil content. Xanthan
gum is also added here to aid the activity of carrageenan (McHugh 2003).
6.7 Applications 137
6.7.3 Meat and Poultry
Precooked meat and poultry are desirable as they minimize preparation time and in
some cases provide added flavor in cases such as smoked thin sliced ham or smoked
chicken. However, the cooking process often results in loss of some of the food mass
and protein as some of it is lost in the heated water. This otherwise lost protein can be
retained in the product using carrageenans. Due to their ability to bind and interact
with water molecules, carrageenans are used by the food processing industries to
control texture and retain structure of precooked meat and poultry products. The
challenge here is that once dissolved in water, carrageenan tends to increase the
viscosity of water which makes it difficult for the carrageenan to penetrate the meat
and then interact with the free water and protein within it in order to have the desired
effect. To overcome this challenge, the carrageenan is introduced in the water after
the brine has been dissolved in it. The high salt content prevents the carrageenan
from dissolving before cooking such that the carrageenan only begins to dissolve as
the meat cooks hence allowing for better penetration. In the process, the carrageenan
also acts to improve the texture of the meat or poultry product.
Carrageenans are also used to improve the otherwise dry texture of meat with
reduced fat. The fat in meat contributes a juicy and tender mouthfeel, and it also
contributes to flavor. When the fat is reduced, the meat tends to lose this appeal.
Carrageenans have been used in some cases to recover this texture and flavor in
low-fat meat (McHugh 2003).
6.7.4 Toothpastes
The key components of toothpastes are mild abrasives such as chalk, flavor, detergent
and water. These ingredients are then held together by a thickening agent which
ensures the product does not separate in storage, easily flows out of the toothpaste
tube and also can be easily laid on the toothbrush during usage. Iota carrageenan at
about 1% concentration is commonly used for this application.
6.7.5 Pet Food
Since animals have a more tolerant digestive system than humans, the requirement for
pet food-grade carrageenan is less than that for humans. Pets also have less demand
for food appearance than humans do so the slightly less clear seaweed powder which
is a more crude form of carrageenan can be successfully used in pet food. In pet
food, they are used as thickening agent for the meat gravy or as gelling agent for the
flavored jelly. They also help bind the meat pieces and provide texture since much
of the meat used in pet food is scraps from human meat cutting and processing.
138 6 Carrageenans
For such application, kappa carrageenan containing seaweed flour is often used in
combination with locust bean. The less refined form of carrageenan costs only a
fraction of the cost of refined carrageenan (one-fourth). Over 5 thousand tonnes of
seaweed powder is used for such applications.
6.7.6 Air Freshener Gels
An estimated 200 tonnes of lower-grade carrageenan in the form of seaweed flour

finds non-food applications as air freshener gels annually (McHugh 2003). The air
freshener perfume is mixed in potassium salt carrageenan and water. The gel is
sealed to prevent evaporation over its storage shelf life. Once opened, the perfume
and water begin to evaporate against the concentration gradient with the environment.
Eventually, the water and perfume completely evaporate and what remains is a hard
dry gel at the end of the product usage life.
6.7.7 Immobilized Cells and Biocatalysts
For different applications such as cell biology or tissue culture preparation, cells need
to be stored in inactive dry forms such that they are protected from the environment
and can also be easily stored, transferred and transported. The ability of kappa car-
rageenan to form hard gels makes them applicable for such purpose. When required
for use, these immobilized biocatalysts and cells can then be rehydrated and activated.
In one of the early studies on the use of carrageenan for such purpose, the ethanol-
producing bacterium Zymomonas mobilis has been entrapped in kappa carrageenan
(Luong 1985). Kappa carrageenan is preferred as it allows immobilization of the
biological component under mild conditions without damage (Chibata et al. 1987).
More recently, enzymes such as cellulase and pollunase have been immobilized using
carrageenan-based polymer complexes (Yasin et al. 2019; Long et al. 2016). Ability
to immobilize cells and enzymes makes more effective processes where the release
and activity of the enzyme can be better managed. Enzyme immobilization is further
discussed in this book in the chapter on Enzymes.
6.7.8 Antimicrobial Properties
Although not its main application, carrageenan extracted from red algae strains
such as H. musciformis has shown antimicrobial activity. Kappa carrageenan from
H. musciformis had antibacterial effect against Staphylococcus aureus and antifungal
effect against Candida albicans (Souza et al. 2018). The carrageenan extracted from
this study had a molecular weight of 519 kDa. Carrageenan molecular weight varies
between 200 and 800 kDa; therefore, this has a molecular weight in the midrange.
When tested on gram-negative bacteria S. enteritidis, E. coli and P. aeruginosa, the
carrageenan extracted in this study did not present any antimicrobial activity against
these gram-negative bacteria. However, growth inhibition was observed when tested
against gram-positive bacteria S. aureus and C. albicans (Souza et al. 2018).
6.7.9 Antioxidant
In the long polymeric form, carrageenan has little or no reported antioxidant activity;
however when processed into derivative forms with shorter chain, varied degree of
sulfation, acetylation and phosphorylation, these derivatives have significant antiox-
idant activity (Yuan et al. 2005). For instance, short-chain oligosaccharides of car-
rageenan obtained from hydrochloric acid hydrolysis of the polysaccharide form
which were then oversulfated and acetylated showed antioxidant properties in vitro.
This antioxidant property was evident as scavenging effect on hydroxy oxides and
superoxides and DPPH radicals (Yuan et al. 2005).
Although carrageenan extracted from H. musciformis showed no cytotoxic effects
on cancer cells investigated, it demonstrated anticancer effect through the inhibition
of cancer cell proliferation (Souza et al. 2018). Taking results from different studies
leads to the conclusion that if at all, carrageenan only shows very low antioxidant
property which is dependent on the chemical structure of carrageenan in terms of
sulfation pattern. Much of the anticancer property of carrageenan seems to be due
to its ability to enhance innate immunity of the body in, for example, enhancing the
activity and production of killer cells.
6.7.10 Neuroprotective
However, very few studies have investigated the neuroprotective property of

carrageenan. Results from some studies show carrageenan activity against 6-
hydroxydopamine, a neurotoxicant (Souza et al. 2018). The mechanism of action
is demonstrated to be through having an inhibitory effect on caspase enzyme activity
and affecting the mitochondrial transmembrane potential. Although these studies are
early stage studies on cell cultures, it does point to some potential neuroprotective
activity of carrageenan.
6.7.11 Immunomodulatory Activity
In cases where immune response leads to adverse reactions such as inflammations,

carrageenans have shown potential immunomodulatory effect. It is thought to do
140 6 Carrageenans
this by chemically attaching to phagocytic cells such as macrophages by binding to

Toll-like receptors or pattern recognition cells. This has been demonstrated in mouse
T cells and in vivo experiments in laboratory mice. Carrageenan showed potential
use in ameliorating the allergic reaction from immune response. When given an oral
dosage of lambda carrageenan mice that have been treated with ovalbumin to trigger
an allergic reaction, the reduction in serum histamine release and ovalbumin-specific
IgE indicates a reduction in allergic reaction (Maruyama et al. 2005).
Immune modulation is also required where the immune activity needs to be
enhanced, for example, in the case of vaccination. Evidence also indicates that
carrageenans could potentially aid innate immune response against cancer cells by
enhancing the cytotoxic effect of the immune system’s lymphocytes and macrophages
such that the anticancer effect of carrageenan can be attributed to its immunomodu-
latory effect. In studies using oligosaccharides of kappa carrageenan of carrageenan
extracted from the red algae Kappaphycus striatum administered to mice which
had been induced with cancer (sarcoma S180), carrageenan enhanced the activity
of killer cells, inhibited cancer cell growth and increased macrophage phagocytosis
(Yuan et al. 2006). It should be noted that in the aforementioned study, the short-
chain kappa carrageenans used are quite different from the high molecular weight
carrageenans which are commercially used in food industry. Sulfated polysaccha-
rides have generally shown structure-dependent bioactivity such that the difference
in molecular weight means higher molecular weight carrageenans are not likely to
show the same immunomodulatory activity as oligosaccharide carrageenans. These
oligosaccharides are prepared from the acid hydrolysis of the longer-chain polysac-
charide extracted from the red algae (Yuan et al. 2005), therefore making the polysac-
charide carrageenans relevant in understanding the immunomodulatory activity of
the oligosaccharides.
6.7.12 Antiviral Activity
Carrageenans show relatively weak antiviral activity compared to other aquatic poly-
mers which have been investigated for their potential antiviral activity. When tested
against denge viva virus DENV-1, DENV-2, DENVV-3 and DENV-4 in vero cells,
carrageenans only had antiviral effect of DENV-2. Carrageenan acted by inhibiting
the multiplication of the DENV-2 virus by preventing cell absorption and entry. This
effect was much less potent in DENV-3 and DENV-4 and completely ineffective in
DENV-1 (Talarico and Damonte 2007). However, even the activity against DENV-2
is dependent on the virus taking the normal route into the cell and being introduced
into the host cell at the same time as the virus or shortly after, once the normal entry
is bypassed or the virus is introduced at a longer time before the carrageenan, it then
has no antiviral infect and the DENV-2 virus is able to successfully infect the host.
6.7.13 Polymeric Electrolytes
Biopolymer-based electrolytes are favoured as energy storage devices for their bio-
compatibility, renewable source, relative abundance, ease of processing and relative
low cost. Iota carrageenan has been investigated for use as a polymeric electrolyte.
This particular form of carrageenan shows good prospects in this field of application
due to its level of sulfation and 25–30% anhydrogalactose component which results
in a medium gel strength with a moderate level of amorphous structure leaving a suf-
ficient amount of functional groups available for interactions with charge carriers,
making it more suitable as a polymer electrolyte (Ghani et al. 2019). Carrageenan
can be dissolved in water to form a solid-state electrolyte. This makes it possible
for low-temperature processing which is desirable in lowering processing costs in
such applications. Purification of the iota carrageenan significantly affects the elec-
trochemical stability. In the purified form, iota carrageenan attained a conductivity
of 1.57 × 10−5 S cm−1 while the unpurified form was 1.65 × 10−6 S cm−1 (Ghani
et al. 2019).
From all the applications of carrageenans discussed so far, applications based on
the rheological properties and viscosity- and gel-forming properties are the most well
established. The bioactive properties such as antimicrobial and anticancer properties
need further investigations to understand and possibly extend the effectiveness and
mechanism of action. Particularly important is to understand how these activities are
affected by variables such as extraction conditions, molecular weight and species.
It is indeed desirable to relate the different bioactivities of carrageenans with
specific chemical properties or structure. However, there is much yet to be known
about the structural activity relationships of sulfated polysaccharides. In attempts to
understand the structural relationship with the bioactivity, one approach is to make
inferences from similar but simpler structures. For example, studying more linear
simpler structures and relating the bioactivities of such to similar more complex
branched structure, such has been done for some sulfated polysaccharides such as
fucoidan (Jiao et al. 2011).
Carrageenan is one of the relatively well-explored sulfated polysaccharides of algae.

It mainly finds commercial applications in food as the largest market for carrageenan.
However, its potential applications also extend beyond food industry to others such
as cosmetics and nutraceuticals and possibly in the future pharmaceuticals. The total
market for carrageenan is valued at over 300 million USD (McHugh 2003). Demand
for carrageenan is further boosted by the need for a substitute for a replacement for
animal-sourced gelatin with the outbreak of mad cow disease (bovine Spongiform
encephalopathy). Its thermostable property makes it form more stable gels in hotter
environments compared to gelatine.
142 6 Carrageenans
Commercially, carrageenan is available either as refined carrageenan which is

assigned the E number E = 407 or as semi-refined carrageenan produced to high
standards which is referred to as PES assigned the E number E-407a (McHugh 2003).
Weiner et al. (2017) emphasized the difference between commercial carrageenan
used in food additives and other forms of carrageenan which have no commercial
applications in food but are used in other applications such as medical. The group
further highlighted that the difference in the molecular weight had significant effect
on the physicochemical and rheological properties of carrageenan and should there-
fore not be used interchangeably. Commercial carrageenan used as a food additive
is sulfated polysaccharide that has a average molecular weight between 200 and
800 kDa with alternating disaccharide repeating units of 1–3 alpha-d-galactose and
1–4 linked beta-d-galactose or 3,6 anhydrogalactose with sulfate groups attached to
some of the units at C2 or C4 depending on the type of galactan (3 main ones Iota,
kappa and lambda). The low molecular weight carrageenan which is extracted at
high temperature under low pH and has a molecular weight of less than 20–40 kDa
is referred to as degraded carrageenan of poligeenan. The significantly shorter chain
length and change in configuration due to low pH and high temperature of extrac-
tion result in a structure which does not share the typical attributes that characterize
carrageenan.
As of 2001, 42,930 tonnes of carrageenan was produced by the top 3 producers of
carrageenan in the world (McHugh 2003). Asia-Pacific contributed 2018 tonnes of
this, 9900 of this being PES grade and the rest being either gel or alcohol-precipitated
carrageenan. Europe contributed a total carrageenan production of 13,500 tonnes with
500 tonnes of this being PES and the rest refined carrageenan. The Americas produced
9150 tonnes; 1100 of this was PES grade, while the rest was refined carrageenan.
About half of the carrageenan produced is therefore PES grade with most of it coming
from the Asia-Pacific region.
Some of the companies producing carrageenans in different parts of the world
include Kelcco, Ingredients Solutions Inc, Shemberg Biotech Corporation, Marcel
Carrageenan Corporation, FMC Biopolymer, Degussa Texturant Systems, Danisco
Cultor, Rhodis Food, Gelymar S.A., CEAMSA, Hispanagar S A, Ina food Industry
Co. Ltd., Myeong Shin Chemical Ind. Co. Ltd., Soriano SA, Chuo Food Materials
Co. Ltd., Quest International Philippines Corp., FMC Corporation, Geltech Hayco
Inc., TBK Manufacturing Corp., Iberagar S.A., PT Gumindo Perkasa Industri, CV
Cahaya Cemerlang, PT Surya Indoalgas, P.T. Asia Sumber Laut Indonesia (McHugh
2003).
6.9 Conclusion
Carrageenan is a relatively well-explored aquatic biopolymer primarily sourced from

the aquatic environment with no reported terrestrial source; therefore, its availability
is highly reliant on the availability of red algae in the aquatic ecosystem. With a
potential yield of ~30% per dry mass of red algae, it can be considered a major algal
6.9 Conclusion 143
resource since it makes up a relatively large proportion of the red algae composition.
Although the majority of commercial applications of carrageenan mainly relies on
its rheological properties, carrageenan has some promising bioactivities which are
being investigated. Carrageenans with bioactive properties have a promising future in
developing multifunctional products such as food additives which also have health
benefits. While carrageenan itself as a polymer is a renewable resource produced
from red algae and the product itself has no toxic effect on the environment, the
process of production requires fossil energy consumption and use of chemicals which
might have less benign effects. Some of these environmental impacts of carrageenan
production process can be minimized by using milder and higher yielding processes
such as enzyme-assisted extraction and integration of algae cultivation process in the
wastewater treatment.
References
Chibata I, Tosa T, Sato T, Takata I (1987) Immobilization of cells in carrageenan. Methods Enzymol
135:189–198
Davidson RL (ed) (1980) Handbook of water-soluble gums. New York McGraw-Hill Book Co.
Doyle JP, Giannouli P, Rudolph B, Morris ER (2010) Preparation, authentication, rheology and
conformation of theta carrageenan. Carbohyd Polym 80:648–654
Eisses J (1952) The research of gelatinous substances in Indonesian seaweeds at the laboratory for
chemical research. Bogor J Sci Res Indon 1:44–49
FAO (2018) The state of world fisheries and aquaculture 2018—Meeting the sustainable develop-
ment goals. Rome. CC BY-NC-SA 3.0 IGO
Ferreira LG, Noseda MD, Goncalves AG, Ducati DRB, Fujii MT, Duarte MER (2012) Chemi-
cal structure of the complex pyruvylated and sulfated agaran from the red seaweed Palisada
flagellifera (Ceramiales, Rhodophyta). Carbohyd Res 347:83–94
Funami T, Hiroe M, Noda S, Asai I, Ikeda S, Nishinari K (2007) Influence of molecular structure
imaged with atomic force microscopy on the rheological behavior of carrageenan aqueous systems
in the presence or absence of cations. Food Hydrocolloids 21:617–629
Ghani NAA, Othaman R, Ahmad A, Anuar FH, Hassan NH (2019) Impact of purification on iota
carrageenan as solid polymer electrolyte. Arab J Chem 12:370–376
Ghosh A, Anand VKG, Seth A (2015) Life cycle impact assessment of seaweed based biostim-
ulant production from onshore cultivated Kappaphycus alvarezii (Doty) Doty ex Silva—is it
environmentally sustainable? Algal Res 12:513–521
Gonçalves AG, Ducatti DRB, Paranha RG, Duarte MER, Noseda MD (2005) Positional isomers
of sulfated oligosaccharides obtained from agarans and carrageenans: preparation and capillary
electrophoresis separation. Carbohydr Res 340:2123–2134
Graham HD (1977) Food colloids. AVI Publishing Co., Inc., Westport, Connecticut
Iglauer S, Wu Y, Schuler P, Tang Y (2011) Goddard III WA. Dilute iota- and Kappa-Carrageenan
Solutions with high viscosities in high salinity brines. J Petrol Sci Eng 75:304–311
Jiao G, Yu G, Zhang J, Ewart HS (2011) Chemical structures and bioactivities of sulfated
polysaccharides from marine algae. Mar Drugs 9:196–223
Long J, Xu E, Xingfei, Wu Z, Wang F, Xu X, Jin Z, Jiao A, Zhan X (2016) Effect of chitosan molec-
ular weight on the formation of chitosan- pullulanase soluble complexes and their application in
the immobilization of pullulanase onto Fe3 O4 -k-carrageenan nanoparticles 202:49–58
Luong JH (1985) Cell Immobilization in kappa carrageenan for ethanol production. Biotechnol
Bioeng 27(12):1651–1661
144 6 Carrageenans
Manuhara GJ, Praseptiangga D, Riyanto RA (2016) Extraction and characterization of refined K-

carrageenan of red algae [Kappaphycus alvarezii (Doty ex P.C. Silva, 1996)] Originated from
Karimun Jawa Islands. Aquat Procedia 7:106–111
Maruyama H, Tamauchi H, Hashimoto M, Nakano T (2005) Suppression of Th2 immune responses
by mekabu fucoidan from Undaria pinnatifida sporophylls. Int Arch Allergy Immunol 137:289–
294
McHugh DJ (2003) A guide to seaweed industry FAO fisheries technical paper 441. Rome.
Downloaded 5/1/2019, pp 1–6
Moreira-Gonzalez AR, Fernandez-Garces R, Batista MG, Leon-Perez AR, Caballero YC, Garcia-
Moya A, Fujii MT, Suarez-Alfonso AM (2019) Marine red algae from central-southern coast of
Cuba. Reg Stud Mar Sci 25(100450):1–9
Necas J, Bartosikova L (2013) Carrageenan: a review. Veterinarni Medicina 58(4):187–205
Sheath RG, Vis LM (2015) Red algae. Freshwater Algae of North America (Second Edition).
Ecology and Classification pp 237–264
Smith HM (1905) The utilization of seaweeds in the United States. Bull US Bur Fish 24:169–171
Souza HKS, Hilliou L, Bastos M, Goncalves MP (2011) Effect of molecular weight and chemi-
cal structure on thermal and rheological properties of gelling k/l-hybrid carrageenan solution.
Carbohyd Polym 85(2):429–438
Souza RB, Frota AF, Silva J, Alves C, Neugebauer AZ, Pinteus S, Rodrigues JAG, Cordeiro EMS,
de Almeida RR, Pedrosa R, Benevides NMB (2018) In vitro activities of kappa-carrageenan
isolated from red marine alga Hypnea musciformis: antimicrobial, anticancer and neuroprotective
potential. Int J Biol Macromol 112:1248–1256
Stanley N (1987) Production, properties and uses of carrageenan. FAO report, 1987, Rome
Syrbe A, Bauer WJ, Klostermeyer H (1998) Polymer science concepts in dairy systems: an overview
of milk protein and food hydrocolloid interaction. Int Dairy J 8:179–193
Talarico LB, Damonte EB (2007) Interference in dengue virus adsorption and uncoating by
carrageenans. Virology 363:473–485
Tijssen RLM, Canabadv-Rochelle LS, Mellema M (2007) Gelation upon long storage of milk drinks
with carrageenan. J Dairy Sci 90:2604–2611
Towle GA (1973) Carrageenan. In: Whistler RL (ed) Industrial gums, polysaccharides and their
derivatives. Academic Press, New York, pp 83–114
Weiner ML, McKim JM, Blakemore WR (2017) Addendum to Weiner ML (2016) Parameters and
pitfalls to consider in the conduct of food additive research, carrageenan as a case study. Food
Chem Toxicol 107:208–214
Williams PA, Phillips GO (2003) GUMS: properties of individual gums. In: Caballero B (ed)
Encyclopedia of food sciences and nutrition. Academic Press
Yasin MA, Gad AAM, Ghanem AF, Rehim MHA (2019) Green synthesis of cellulose nanofibers
using immobilized cellulase. Carbohydr Polym 205:255–260
Yuan H, Song J, Li X, Li N, Dai J (2006) Immunomodulation and antitumor activity of kappa-
carrageenan oligosaccharides. Cancer Lett 243(2):228–234
Yuan H, Zhang W, Li X, Lu X, Li N, Gao X, Song J (2005) Preparation and in vitro antioxidant activ-
ity of kappa-carrageenan oligosaccharides and their oversulfated, acetylated and phosphorylated
derivatives. Carbohyd Res 340(4):685–692
Yu G, Hu Y, Yang B, Zhao X, Wang P, Ji G, Wu J, Guan H (2010) Extraction, isolation and structural
characterization of polysaccharides from a red alga Gloiopeltis furcata. J Ocean Univ China Nat
Sci 9:193–197
Zaneveld JS (1959) The utilization of marine algae in tropical south and east Asia. Econ Bot
13(2):89–131
Zhou G, Sheng W, Yao W, Wang C (2006) Effect of low molecular [lambda]-carrageenan from
Chondrus ocellatus on antitumor H-22 activity of 5-Fu. Pharmacol Res 53:129–134
Chapter 7
Agar
Abstract Agar is obtained from red algae. It occurs as a mixture of agarose and
agaropectin with agarose having the more desirable properties. Its applications are
mainly attributed to its rheological properties. Such applications extend to the food,
biotechnology and pharmaceutical industries where it is used as a thermoreversible
gelling agent, stabilizer, texture modifier and thickener. The ability to serve as a sub-
stitute additive for the animal-sourced gelatin also contributes to its economic value.
In this chapter, the extraction, chemistry, occurrence in nature and applications are
discussed. In the process, the economic and environmental impact of agar production
is evaluated.
Keywords Agar · Agarose · Agaropectin · Polymers · Gels
7.1 Introduction
A range of polysaccharides can be sourced from the aquatic environment, and some
of these polysaccharides have well-established process technology and market pres-
ence. Agar is one of such well-explored polysaccharides. It is present exclusively
in red algae; there are yet to be other non-aquatic sources of agar to date. The most
common form of agar is agarose which is familiar with almost every biology stu-
dent and researcher. This partially sulfated polysaccharide of the red algae is most
valuable in its gel form for its rheological properties, which has earned it a potential
global annual market valued at around 172.8 million USD (Bixler and Porse 2011).
It can therefore be considered a highly valuable aquatic-derived resource.
Agar is one of the three main phycocolloids obtained from the aquatic environ-
ment, the other two being carrageenan and alginate. Agar is comprised of agarose
and agaropectin, where the composition of each within the organism and this in turn
affects the properties and applications of the agar (Armisen and Gaiatas 2009). Agar
finds applications in food, biotechnology and pharmaceutical industries where it is
used as a thermoreversible gelling agent, stabilizer, texture modifier and thickener.
It is often used as a substitute additive for the animal-sourced gelatin (Marcus 2014)
for reasons of ethical, health or dietary preferences where animal-derived gelling
agents are not desirable.

146 7 Agar
Global production of agar is estimated at 10,600 tonnes annually with the major
producing countries in the Asia-Pacific regions (Rhein-Knudsen et al. 2015). Agar
has well-established large-scale production process technology, and several compa-
nies across the world are involved in agar production. Examples of such companies
are MSC in Korea and Huey Shyang Seaweed Industrial Company in China. The
species of the genus Gracilaria remain the most preferred source for commercial agar
production.
In this chapter, we discuss the environmental and economic impact of agar as an
aquatic biopolymer. In doing so, we look at the red algae as a source, the occurrence
of red algae in nature and the process of extracting agar from red algae and the impact
of these processes on the environment. A review of the chemistry of agar and how it
relates to its rheological properties explain how agar differs from other polymers. The
applications of agar in several products are also explored to give an understanding of
the commercial value and the versatility of agar. Some of the progress and limitations
of commercialization of agar are also explored.
Agar has been in use as far back as 1658 in Japan (Armisen and Gaiatas 2009). It
occurs naturally in cell wall and within the intracellular spaces of red algae, mainly
those belonging to the genus Gracilaria, Gelidium and Gelidiella (Rhein-Knudsen
et al. 2015). These include species such as Gracilaria tikvahiae (Rocha et al. 2019).
Gelidium sesquipedale (Martinez-Sanz et al. 2019), Pterocladia, Ancatkopeltis and
Ceramium genera (BeMiller 2019; Sudha et al. 2014), Gracilaria cliftonii (Kumar
and Fotedar 2009), Gracilaria asiatica (Li et al. 2008) and Gracilaria lemaneiformis
(Li et al. 2008). Algae which serve as a source of agar are referred to as agarophytes
(Armisen and Gaiatas 2009).
Red algae species belonging to other genera are also explored for agar extraction
such as the Pyropia yezoensis (Sasuga et al. 2017) which, although more commonly
used for food as nori, is also a source of agar. The rhodophytes can be found growing
in marine, brackish and less commonly some freshwater. Although conditions of
the sea can be replicated in artificial growth environments for cultivation of these
algae as biomass source for production of agar, certain species grow naturally in
several regions across the world. For example, the Gracilaria tikvahiae is endemic
to Western North Atlantic region of the USA (Rocha et al. 2019), while the Gracilaria
lemaneiformis grows in China along the coast of Liaodong Peninsula (Li et al. 2008).
They are cultivated in ponds and estuaries and also occur naturally in larger water
bodies.
7.3 Chemistry of Agar 147
Fig. 7.1 Basic structure of agar repeating dimer unit
7.3 Chemistry of Agar
7.3.1 Polymeric Structure
Agar is a mixture of two polymers, agarose and agaropectin. These can be further
fractionated and characterized upon extraction by methods such as gel permeation,
ultracentrifugation or SDS-PAGE since they vary by molecular weight. Agarose is a
neutral linear copolymer comprising of 3-O substitute β-d-galactopyranosyl and 3,6-
anhydro-α-l-galactopyranosyl repeating units (Sudha et al. 2014), while agaropectin
is the charged branched chain comprising of the same repeating unit (BeMiller 2019)
as shown in Fig. 7.1.
Compared to other phycocolloids from red algae, agar has relatively lower
hydrophilicity and water solubility. This is in part due to the presence of some methyl
ether groups along the agaropectin polymer chain which may also include some small
amounts of sulfation (BeMiller 2019). Agarose forms gels, while agaropectin does
not form gels. The gel properties of the agar depend on the amount of agarose and
agaropectin presents. The sulfate groups are mainly present in the agaropectin, and
this has an effect on the syneresis generated in the gel (Mizrahi 2010).
7.3.2 Gel Formation
One of the desirable properties of agar is the ability to form a gel at relatively low
concentration. This means less of the product is needed as an additive; this reduces
cost and chances of altering other properties of the product. Gelation occurs in agar
as a result of hydrogen bonds between the functional groups of the polymer chains
forming bridges (Armisen and Gaiatas 2009). Because these bridges are as a result of
secondary bonds which can be easily broken as conformation of the polymer chain
varies at different temperatures, this makes this gelation a reversible one. Gelation
is enhanced by the formation of 3,6-anhydrogalactose which occurs during alkali
extraction where the sulfated galactose is converted to 3,6-anhydrogalactose (Bixler
and Porse 2011).
148 7 Agar
At concentrations between 0.5 and 2% w/v in water at around 80–100 °C, agar will
form a gel upon cooling (Rioux and Turgeon 2015). Gelatin, for example, will require
a higher concentration and much lower temperature to form a gel, and carrageenan
requires potassium or calcium salts in the solution in order to form a gel; agar forms
a gel at variable pH and without requiring the presence of cations. This makes agar
a preferred gelling agent in biotechnology applications.
Gels formed by polysaccharides are prone to syneresis. This is the release of water
from the gel as it loses its gel conformation. This is caused by the rearrangement of
the polymer chains due to different factors (Mizrahi 2010). The relationship between
degree of syneresis and agar concentration is quantified in Eq. (7.1) (Mizrahi 2010).
Degree of Syneresis = 1/Concentration2 (7.1)
The gel strength is affected by the growth method and growth conditions. For
example, a gel strength of 505 g cm−2 was measured for agar extracted from
Gracilaria grown from tissue-cultured seedlings using the broadcast method of sea-
weed cultivation, while 201 g cm−2 was obtained from the same species grown using
the off-bottom method from tissue-cultured seedlings (Rejeki et al. 2018).
The lower sulfate content in agar compared to the other more sulfated seaweed
polysaccharides gives it a higher gel strength and gel melting point. Compared to
carrageenan, for example, which has a melting point between 50 and 70 °C, agar has
a melting point between 85 and 95 °C and while gel strength of agar varies between
700 and 1000 g/cm2 for a 1.5% w/v concentration, that of carrageenan is between
100 and 350 g/cm2 (Rhein-Knudsen et al. 2015).
7.3.3 Viscosity
Viscosity of polymers is related to the molecular weight. This relationship is quanti-

fied by the Mark–Houwink equation (Eq. 7.2), where [η] is the intrinsic viscosity, M
is the average molecular weight and K and a are constants which vary for different
polymers.
[η] = K M a (7.2)
The constants K and a are obtained experimentally by plotting the values of intrin-
sic viscosity against molecular weight and fitting to the equation (Wang et al. 1997).
The viscosity is therefore an important parameter which is used in the characteriza-
tion of polymers. Agar has a lower viscosity (10–100 centipoise at 1.5%, 60 °C) than
carrageen (30–300 centipoise) Rhein-Knudsen et al. 2015). This lower viscosity is
attributed to the lower molecular weight of agar compared to carrageenan. While
number average molecular weight of agar is between 36 and 1144 kDa, that of car-
rageenan is usually at the higher end from 200 to 800 kDa (Weiner et al. 2017).
This lower viscosity and higher gel strength make processing of the gels easier by
allowing easier flow during processing of the product prior to gel formation.
7.3.4 Interaction with Sugars
Sugars are often used alongside agar-based products such as candies, icings and
creams. Other than acting as a sweetener, sugar also contributes to the physical prop-
erties of the food. When agar gel interacts with sugar in a fluid gel system, this has
an effect on the rheological properties of the resultant product and consequently the
nature of gels and foams formed in such systems. This property-related interaction
with sugar could be used as a means of reducing sugar contents of processed food
while giving consideration to the change in properties such as the texture, syneresis
and gel strength. These properties are important in food products, while the con-
sumer and health agencies demand a lower sugar content for health reasons, and
they also demand appealing taste, texture and appearance. The producer therefore
needs to understand the agar–sugar interaction and how this affects the property of
the product. Recent studies which analyzed the effect of concentrations of different
sugars: glucose, sucrose and fructose which are commonly used in food products,
have significant effect on the viscosity, gel strength and foam stability. At concen-
trations above 50% w/v of sugar (either fructose, sucrose or glucose), the gel formed
is more elastic; at lower concentration below 50% w/v of sugar, the shear viscosity
and modulus of elasticity increase, thus indicating a stronger more rigid gel formed.
Increasing sugar concentration also increased the foam stability measured as foam
half-life (Ellis et al. 2019). Earlier studies using agarose extracted from Gelidium
amansii have established that stronger agarose gels with higher melting points are
formed when sugar is added to the agarose gel solution up to a critical concentration
(Watase et al. 1990). This relationship between the sugar concentration and physical
properties of the resulting gel of agar is attributed to the interactions between the
functional groups of the sugars and those of the agar. These interactions in turn affect
the conformations during gelation. In the same study, it was observed that urea and
guanidine hydrochloride resulted in a decreased young modulus.
7.3.5 Thermal Degradation of Agar
For a product to be considered safe, the products upon degradation should pose
no harm to health while in storage or in use. One of the advantages of agar as a
phycocolloid is its ability to withstand higher processing temperatures. This makes
it suitable for heat-sterilized food products such as canned fish and meat. However,
recent studies point to potential toxic effect of the degradation of agar within a food
product when heated (Ouyang et al. 2018). Stronger agar gels tend to have higher
thermal degradation temperature such that the chances of having toxic degradation
150 7 Agar
products during heat treatment of agar-containing foods could be reduced by using

stronger gels.
Upon thermal degradation, agar forms 3,6-anhydropyran galactopyranose and
galactopyranose. These then further degrade 3,6-anhydropyranose galactopyra-
nose units break down into furyl hydroxymethyl ketone, a potentially toxic com-
pound to humans. The galactopyranose breaks down into 3,4-atrosan d-allose
and two other potentially toxic compounds, furfural and 5-(hydroxymethyl)-2-
furancarboxaldehyde (Ouyang et al. 2018). Therefore, although generally consid-
ered a safe and approved product, care should be taken during the process of food
treatment to avoid the formation of toxic compounds either from partial or complete
degradation, especially during sterilization.
The initial melting of agar-based gel varies with factors such as concentration,
presence of compounds such as sugars and urea and the agarose/agaropectin con-
tent. Earlier studies have shown that at different agarose concentration, the melting
temperature of the gel remains constant at 75 °C. However, the enthalpy of trans-
formation from gel to sol increases as the concentration of agarose decreases from
12 w/w to 2 w/w (Watase et al. 1990).
7.3.6 Biological Degradation of Agar
Biological degradation is a relatively economic degradation method, whereby a sub-

stance is broken down into its smaller units by naturally existing organisms which
produce the enzymes for digesting, hence degrading the substances which they can
then use for their metabolism and energy production. These enzymes can be extracted
from such organism for controlled degradation of the substance without the contami-
nation of microbes. The understanding of degradation of biopolymers is important in
determining their safety and better understanding of their bioactivity and applications.
Agar is naturally present in the cell walls and intracellular structures of red algae
which occur in the aquatic environment; an ideal source of the organisms which
degrade agar would therefore be in the same aquatic environment. These microbes
either exist freely in the environment or within the bodies of other bigger organisms
which feed on the red algae as these organisms will require the enzymes which
break down all components of the red algae, which include the agarose, into smaller
products which can then be used for metabolism.
Agar-degrading bacteria include Pseudomonas, Cellulophaga, Acinetobacter,
Agarivorans, Microbulbifer, Pseudoalteromonas, Saccharophagus, Bacillus, Paeni-
bacillus, Streptomyces and Zobellia (Kwon et al. 2019). There are more marine-based
bacteria which degrade agar than the freshwater-based ones. This is expected since
most of the red algae are found mostly in the marine environment; freshwater red
algae are rarer. The enzyme which degrades agarose is called agarase. These break
down agarose into cooligomers of 2–4 repeating units of galactose and anhydrogalac-
tose; further cleavage of the glycosidic bonds breaks down these neoagarose units
into neoagarobiose, dimers of agar which is consisting of one unit of galactose and
Agarose
Neo agarose
Neoagarobiose
D- anhydrogalactose L- galactose
Fig. 7.2 Degradation of agarose into its monomer units
one unit of anhydrogalactose. This is then finally broken down into one d-galactose
and one 3,6, anhydro-l-galactose (Kwon et al. 2019). These monosaccharides can
then be utilized for production of energy and carbon. Figure 7.2 summarizes the
degradation process of agarose.
Agarase activities are bond specific. Some α agarases cleave the α-1,3 glyco-
sidic bonds between the monomer units, while the beta agarases cleave the β-(1-4)
glycosidic bonds. Therefore, the products of degradation depend on the mix of the
enzymes being used. This has commercial relevance where, for example, neoagaro-
biose is required for specific bioactivities and the right enzymes are required for
optimal degradation of the agarose to obtain a pure product.
Agar is produced from red algae. One of the most abundant species which are com-
mercially grown for agar production is the Gracilaria verrucosa (Rejeki et al. 2018).
The Gracilaria red algae are largely grown in regions such as Indonesia. This genus
is more commonly used in food applications. The Gelidium is also a well-cultivated
genus used more in pharmaceuticals and in biological applications. These red algae
can either be grown in aquaculture or be harvested from natural stocks, although
the majority of commercial agar production is from algae cultivated in aquaculture
where aquaculture makes up 96.5% of aquatic plants produced globally (FAO 2018).
The Gracilaria species which serves as a major commercial source for agar produc-
tion ranks third in world aquaculture production volume of aquatic plants. Quantity
of Gracilaria algae grown in aquaculture rose from 933 thousand tonnes in 2005 to
152 7 Agar
3.88 million tonnes in 2015 and 4.15 million tonnes in 2016 (weight of the live plant)
(FAO 2018).
The yield of algae toward production of agar can be improved by modifying the
growth conditions and growth methods. The algae can grow at rates between 0.5 and
1.3% per day on the low side (Rejeki et al. 2018) depending on the types of seeds
and the growth conditions. The Gracilaria red algae can contain around 9.6% agar
per weight. This particular strain of red algae contributes significantly to the export
commodity of producing countries such as Indonesia where annual export is valued
at an estimated 280 thousand USD annually from an export of 20 thousand tonnes of
Gracilaria red algae (Rejeki et al. 2018). The red algae from which agar is sourced
can be grown using the off-bottom, longline or broadcast method. Method of algae
cultivation is discussed in the chapter dedicated to aquatic environment.
As of 2010, escalation in the price of seaweed was reported. This escalation in
price was attributed to the emerging markets entering the seaweed market at a larger
scale and increase in demand for seaweed products which makes their cost as raw
materials for production of phycocolloids such as agar much higher. The value of
seaweed in its whole form was competitive enough for producers to want to focus on
a product requiring less energy and production cost, hence focusing on cultivation
and quality such as nutrient value and appearance.
For many years, Gracilaria has been the main source of agar produced globally.
The Gracilaria species are endemic to a few parts of the world, and this often results
in companies either having to outsource production or move factories to locations
nearer to the resource at some additional cost compared to if factory was located
in home country. This therefore makes a compelling case to explore alternative red
algae species to serve as sources of commercial production of agar. To this end,
research efforts have been directed at seeking alternative sources of agar with yield
and properties comparable to the agar from Gracilaria species. Pyropia yezoensis, for
example, yields agar which forms gels which are suitable for bacterial culture media
and DNA electrophoresis, two common applications of commercial agar (Sasuga
et al. 2017). Having a more diverse range of red algae species which can serve as
reliable sources of agar can improve the availability of raw material in more regions
of the world and limiting the chances or resource scarcity.
7.5 Extraction of Agar from Red Seaweed
Agar can be extracted from red seaweed using aqueous extraction which generally
involves heat treatment at boiling point temperature in water. The variable conditions
of extractions include the pH and pressure. Here we look first at the conventional
and then the more contemporary extraction methods.
7.5 Extraction of Agar from Red Seaweed 153
7.5.1 Alkali Extraction
Commercial agar extraction is more commonly carried out using alkali treatment.
The alkali treatment is more commonly used for the Gracilaria species to obtain
strong gels (Bixler and Porse 2011). The goal of the alkali treatment is the disrup-
tion of the cell walls of the red algae biomass and dissolving off of the non-agar
impurities such as proteins, minerals and polyphenols. An example of such method
involved treating dried algae Gracilaria in 80% v/v NaOH at 27 °C (±3 °C) for 12 h
at a mass-to-volume ratio of 1:10 (Rejeki et al. 2018). Under this high pH and tem-
perature, the proteins and other smaller compounds are removed and the cell wall is
much more pervious. After the alkali treatment, the biomass is washed with water
followed by water extraction by heating at 90 °C in water for 1 h under continuous
stirring. At this point, the agar is released into the aqueous media. The agar solu-
tion is then filtered followed by gel formation upon cooling. The agar can also be
recovered using alcohol precipitation. The next stages involve water removal. This is
commonly achieved using gel pressing using high-pressure membrane presses fol-
lowed by drying to obtain agar in powdered form which is better for storage and
packaging. Another example of optimum condition for alkali extraction of agar from
the red algae Gracilaria cliftonii is: pretreatment time of 1 h at 30 °C using an alkali
concentration of 5% with a mass-to-volume ratio of 1:150, extraction time of 3 h in
water at 100 °C (Kumar and Fotedar 2009).
Alkali extraction could result in the degradation of part of the agar which could
reduce the yield. However, alkali extraction method could result in agar with higher
molecular weight, crystallinity and purity which are desirable properties for com-
mercial agar application (Martinez-Sanz et al. 2019). When alkali method was used
for the extraction of agar from Gelidium sesquipedale using hot water and alkali in
combination with sonication, the yield using only hot water and sonication reduced
from 10–12% to 2–3% when alkali was used (Martinez-Sanz et al. 2019). Figure 7.3
summarizes the extraction process in a flow chart.
Here we look at a typical extraction process used in the extraction of agar from the
red seaweed of the Gelidium genera (Hernandez-Carmona et al. 2013). This method
is commonly used for Gelidium red algae to obtain agar with superior gel strength
(Bixler and Porse 2011). In this method, the alga is cooked at 100 °C in acidic water
at pH between 6.3 and 6.5. The time of extraction varies depending on the conditions
and the red algae used. Under these conditions, the agar in the red algae biomass is
dissolved in water. The liquid containing the dissolved agar is then separated from the
solid residue by filtration. Upon cooling the agar dissolved in water forms a gel. This
gel contains 99% water and must be dehydrated for easy storage and longer shelf life.
Dehydration can be achieved by thawing the gel and drying in oven or through high
154 7 Agar
Washing of Alkali
Cultivation biomass pretreatment
Extraction
Separation in hot Washing
water
Gel
Precipitation pressing Purification
Packaging Milling Drying
Fig. 7.3 Schematic of the extraction of agar from red algae
pressure pressing to separate the water from the solid. This can then be followed by
further drying through heating to eliminate the remaining water. Further processing
of the extract includes bleaching with sodium hypochlorite and subsequent washing
to remove the bleach (Hernande-Carmona et al. 2013). Other studies have reported
higher extraction temperature, for example, in earlier studies. Watase et al. (1990)
reported extraction of agarose with a molecular weight of 78,000 (±320) kDa from
the red seaweed Gelidium amansii at a temperature of 130 °C.
Sulfate groups, although present in relatively lower amounts, can be removed
by treatment with sodium hydroxide. The extraction can be carried out at elevated
pressure for better yield and/or faster extraction. Increasing the pressure allows more
effective disruption of the cell walls, thus allowing for better isolation of the agarose
from the cell walls. A compromise often needs to be made between extraction yield
and quality of agarose obtained. The increased pressure and pH result in not only
the disruption of the cell walls but also the partial degradation of the agar extracted
(Hernandez-Carmona et al. 2013). The high-pressure extraction process therefore
needs to be optimized toward an acceptable agar quality and yield.
7.5.3 Hot Water Extraction
However, this form of extraction will yield an impure form of agar which contains
proteins, polyphenols and minerals removed along with the agar. These have some
7.5 Extraction of Agar from Red Seaweed 155
beneficial properties in terms of bioactivity such as antioxidant property. An extrac-

tion yield of 10–12% is achievable using the hot water method, albeit with brownish
color and impurities (Martinez-Sanz et al. 2019). This method is relatively simpler;
it involves boiling the washed red algae biomass in hot water for an extended period
of time. The liquid extract is then separated from the solid algae residue by filtration.
Although alkali extraction is more commonly used for extraction of agar, enzyme-
assisted extraction is also attractive prospect for the potential of low energy requir-
ing and more environmentally friendly extraction process. More recently, enzyme
extraction of agar has been explored. Gel strength of 1521 g/cm2 , 29.2% 3.6-anhydro-
l-galactose and 0.84% sulfate content were obtained for agar extracted using the
enzyme-assisted method. The agarase enzyme is capable of breaking the glycosidic
linkages in agar which makes it more readily dissolve in the aqueous medium of
extraction (Xiao et al. 2019). Using the enzyme extraction method to extract agar
from Gracilaria caudata, agar with an average molecular weight of 116.51 kDa and
a degree of sulfation of 0.14% was obtained (Alencar et al. 2019). Although alkali
extraction results in agar with superior properties than enzyme-based extraction of
agar, when considering the environmental impact, the enzyme-based extraction pro-
vides a more eco-friendly alternative. The biodegradation of agarose can be used
as a means of extracting agarose oligomers, dimers or monomers from red algae.
Although it results in short-chain degraded agar, these may be useful, for exam-
ple, the more bioactive oligomeric forms of agar in cosmetics and pharmaceutical
applications.
7.5.5 Ultrasound-Assisted Extraction
Ultrasound is commercially used in the food industry in the extraction and process-
ing of various food compounds in the form of sonication. The ultrasound acts by
creating multiple cavities in the process of formation, expansion and bursting of
these microcavities; they exert pressure on the cell walls causing its disruption. The
combination of the alkali extraction method with sonication results in more effective
extraction process. Although alkali and heat are still required, the time of extrac-
tion could be effectively reduced, hence resulting in optimal use of time by up to
fourfold (Martinez-Sanz et al. 2019). It is also possible to reduce the amount of the
alkali required by combining the process with sonication. Unlike in alkali extraction
which results in degradation of some of the agar upon extraction thereby resulting
in reduced yield, use of ultrasound does not alter the yield or properties of the agar
obtained.
156 7 Agar
Other extraction methods which have been explored include extraction by photo-
bleaching process (Li et al. 2008). Photobleaching extracts of agar from Gracilaria
asiatica and Gracilaria lemaneiformis showed good gel strength of 1913 g cm−2
which is higher than that obtained from using the conventional methods. For exam-
ple using microwave-assisted method, a gel strength of 1331 g/cm2 was obtained
(Sousa et al. 2010). A yield of 14.4%, sulfate content of 1.73% and 3.6-anhydro-
l-galactose content of 39.4% are achievable using the microwave-assisted method
from Gracilaria vermiculophylla (Sous et al. 2010).
Although agar is a biodegradable and biocompatible polymer, the process of extrac-

tion of agar requires energy and materials consumption, and the release of substances
into the environment in the process. This therefore needs to be weighed against its
advantages to the environment and commercial value. Agar degradation goes through
two stages of degradation before finally degrading into its monomer which can then
be broken down by microbes for use as energy or as carbon source. The product itself
is therefore not toxic or a nuisance to the environment unlike the non-biodegradable
petrochemical-derived plastics which over time have accumulated in the oceans. An
estimated 10–20 million tonnes of plastics accumulate in the ocean (Ubanek et al.
2018) annually to the point that these form what is now known as plastic islands
(Lebreton et al. 2018). In developing solutions and alternatives to the plastic man-
agement problem, it is important to evaluate the environmental implications of the
process involved in the production of these biopolymers and identify areas where
improvements are required to make these biopolymers as environmentally benign as
possible.
7.6.1 Cultivation of Red Algae
Red algae are a renewable natural resource as they grow freely in the wild, and
when harvested, they are replenished naturally within a number of days. Unlike fos-
sil fuels which are formed over billions of years and are therefore constantly being
depleted, demand for fossil products increases with increasing population. The grow-
ing demand for algae as food and as a feedstock for the production of phycocolloids
can potentially reduce overfishing of some aquatic species being experienced in some
parts of the world. By serving as an alternative source of income, it reduces the pres-
sure on the fish resource as people in the fishing industry diversify their commercial
interests.
Naturally, algae are able to extract nutrients from the environment for their growth
and metabolism. This is advantageous where they can be used to clean polluted
waters. While uncontrolled algae growth can have adverse impacts such as harmful
algae blooms, well-controlled algae growth in a balanced ecosystem can be an effi-

cient means of keeping the water bodies clean and safe. In some parts of the world,
biological methods of cleaning water bodies include the use of aquatic plants and
algae for biological nutrient uptake. The process involves an extraction system built
into the flow channel which is comprised of aquatic plants and algae. As the water
flows through the channel, it passes through the extraction system where the plants
and algae extract the nutrients and the water exits the channel cleaner than it entered.
This process of extraction of nutrients from the water can be integrated into the cul-
tivation of biomass for the production of agar (Rocha et al. 2019). The limitation of
such systems is controlling the nutrient level in the water coming from homes and
industry to achieve the desired growth conditions for agar production and ensuring
that the agar produced is not contaminated and safe for intended applications.
Another approach of integrating seaweed cultivation is integration of integrated
multi-trophic aquaculture (IMTA). This process capitalizes on the synergy between
fish growth and algae growth and nutrient requirement. The algae take up the nutrients
produced by the fish and in turn produce the oxygen required by the fish. An example
of an IMTA system cultivates Gracilaria alongside the milkfish (Bixler and Porse
2011).
Agar production therefore adds value to red algae beyond food. This is particularly
important as outside of Asia; algae are not commonly consumed as food in other parts
of the world. There are therefore less incentives for them to be cultivated or preserved
as a natural resource. Creating awareness of the value of red algae encourages better
preservation of the resource and diversifies interest from aquatic species which are
currently being fished at unsustainable rates.
7.6.2 CO2 Emission
While agar in itself is a biodegradable polymer with no direct adverse impact on the
environment, the process of production and the chemicals used in the production can
result in considerable generation of CO2 , use of fossil energy and the toxification
of human and aquatic environment. The growth and cultivation of algae for the
production of agar mitigate against some of these environmental impacts as algae
remove CO2 from the environment for the production of polysaccharides and other
organic compounds which can then be used by humans, agar being one of such.
Algae also remove nutrients from the water and are used for cleaning up polluted
water as a biological treatment method.
The cultivation of red algae removes CO2 from the environment and can therefore
be considered as carbon negative. 260 thousand tonnes of carbon can potentially be
converted into biomass by macroalgae annually, assuming that all of this carbon is
generated from the CO2 fixation from the environment (Chung et al. 2011; Sahho
et al. 2012). The process of extraction does not involve direct release of CO2 into the
environment. However, other processes attached such as transportation, packaging
158 7 Agar
and running of the plants are processes which result in CO2 emission into the envi-
ronment. It is therefore important that the CO2 emissions in the processes involved
in the extraction as well as other ancillary processes are minimized.
Much of the water used in the extraction process can be recovered during dewatering
using the gel press method. This water then needs to be treated to remove impuri-
ties. The process of water treatment requires further use of energy and resources.
During the drying process, there is return of water to the environment, especially in
less sophisticated small or medium-scale production processes where the evaporated
water is not recondenced. The water used in the extraction varies for different process
and choice of processor. An example process for extraction of agar uses 150 ml of
water per gram of algae biomass in the alkali pretreatment stage (Rejeki et al. 2018).
7.6.4 Use of Acid and Alkali
In the acid extraction method, a low pH is required to aid the process of cell wall
disruption, a slightly acidic solution of pH ~6 at a temperature of 90–100 °C. In
the alkali extraction method, a NaOH concentration of 5% w/v is used at a lower
temperature of 30 °C, where a weight-to-volume ratio of 1:150 is used, that means
for every gram of agar produced from red algae, 7.5 g of NaOH is used (Table 7.1).
Sodium hypochlorite is also used to improve the appearance and optical properties
of the agar extracted by bleaching it. Depending on the purification process used,
alcohol may also be used for precipitation. Ethanol used can be sourced from non-
fossil source such as from corn. The production of these acids, alkali and other
chemicals involves further consumption of energy and CO2 emissions and release of
toxins. For example, in a life cycle assessment of sodium hydroxide production, it
was deduced that 3.5 MJ of energy was consumed, much of which is fossil based, and
0.6329 kg of CO2 was emitted per gram of sodium hydroxide produced (Thannimalay
Table 7.1 A summary of

estimates of consumption in
agar extraction process from CO2 260 kt year−1
red algae Energy consumption 100 °C for 3 h
Acids pH 6.5
Alkali 7.5 g per gram algae
Water 150 ml per gram algae
Biomass 10 g
et al. 2013). The process also leads to release of toxins to human and aquatic life
and acidification of the atmosphere, although these adverse effects are not directly
linked to the production of sodium hydroxide, but to the consumption of electricity
and energy required for the process. This should therefore be considered in the real
cost of producing biopolymers to the environment. Table 7.1 gives a summary of the
estimated consumption for agar production based on reviewed works thus far.
7.7 Applications
7.7.1 Food
As an additive agar is required in relatively low amounts, a teaspoon of agar powder

can achieve thickening effect in approximately 250 ml of liquid food (Marcus 2014),
and at a concentration of 0.03 g per ml, agar fluid gels achieve optimal stability and
half-life of up to 6 days (Ellis et al. 2017). Agarose is used in icings and frosting. This
particular application is possible due to its compatibility with sugar and its stability
at the relatively high room storage temperature used for such products, especially
during transportation. It is used as a substitute for low-fat products such as oil-free
salad dressings, creams and low-fat yoghurts. Other applications include in canned
meat and fish to retain texture, in pie fillings to improve mouthfeel and in candies for
gel strength. Agar can remain stable at the heat sterilization temperatures; hence, this
makes it applicable for processed foods requiring heat sterilization such as canned
foods.
Colloidal systems such as emulsions, gels and foams are used widely in the food
industry. Some food products are aerated to form foam; these are dispersion of air
within a continuous phase. Foams are used in some food products such as whipped
cream and aerated chocolates. This foam structure gives a desirable texture to these
foods and also serves to reduce mass per volume and calorie since less product is
required within a pack. Typically, foams require a stabilizer to ensure even disper-
sion of air within the continuous phase, hence maintaining stability. Such stabilizer
could be in the form of a surfactant such as Tween 20 (Ellis et al. 2017). Foams
are even much less stable since the dispersed phase is air with low viscosity and
surface tension, hence a higher chance of coalescence. Agar fluid gels are used in the
stabilization of foams. Fluid gels, particles of agar gels suspended in a continuous
phase, can be used to form stable foams. These fluid gels can serve as substitutes for
colloidal systems which are conventionally made using fat. Agar gels stabilize foams
via two mechanisms: absorption of water from the foam cavities hence preventing
the release of water leading to destabilization of the foam and by acting as a viscosity
enhancer. This increased viscosity prevents separation of the phases through coales-
cence of the dispersed phase. The presence of the gel particles also serves as a barrier
to fluid drainage from the foam (Ellis et al. 2019). Agar therefore has a significant
160 7 Agar
role to play in the preparation of foam-based food as a food ingredient stabilizer and
also providing a healthier option to fat-based foams.
7.7.2 Tissue Mimicry
The ability to either test products or procedures on artificial tissue saves much time
and cost in the sciences and medical research and training. Use of real tissue for
in vitro studies often requires freshly excised tissue and/or constant refrigeration and
freezing of the tissue for preservation. For example, where porcine skin is used in
transdermal studies, the neonatal porcine obtained from the abattoir once slaughtered
are stored in the freezer until required. In less developed regions where electricity is
limited and access to fresh cadaver or animal models is more challenging, artificial
tissue systems are required for either training, research or product in vitro testing
and trials. Gelatine is commonly used in tissue mimetics, for example, as artificial
skin for friction testing (Dabrowska et al. 2017). In mimicking tissue for ultrasound
procedure training, gelatine gel used requires refrigeration and this poses a challenge
in less developed region. Agar can retain its gel structure at room temperature without
need for refrigeration and also has a longer period before spoilage since agar is
particularly more resistant to bacterial infection (Kwon et al. 2019), thus making
it more suitable as a tissue model without need for refrigeration. Agar-based tissue
models have been tested for use in training of ultrasound medical imaging procedure.
Agar concentration at 7.5–10% mixed with flour can serve as effective tissue models
for training of medical practitioners in the use of ultrasound tissue imaging (Earle
et al. 2016).
Access to cost-effective training of medical professionals has significant eco-
nomic impacts, particularly in regions where resources such as electricity are scarce.
Such low-cost training materials ensure the development of technical capacity to use
advanced medical facilities such as ultrasound which could significantly improve
the quality of health care rendered to the community. The quality of health in turn
affects the economic activities such as farming and education. Furthermore, trials
and training carried out on real animal or human tissue might violate some ethics
and religious beliefs. Therefore, a plant-based tissue mimetic is often preferred over
real animal or human tissue and is also a step better than the animal-based gelatin
where a tissue model completely free of animal use is required.
7.7.3 Biotechnology
The ability to form a strong gel and serve as a moist, relatively unreactive and clear gel
environment, makes agar applicable in several aspects of biology and biotechnology.
In the biology, agar, particularly agarose, is very commonly used in preparation of
culture media for microorganisms. It can be easily prepared from the powdered form
when mixed with water and dissolved at high temperature forming a gel upon cooling.
Some novel agarose preparations can form gels at lower temperature below 100 °C
(BeMiller 2019), allowing for even easier gel preparations. Agarose is also used
in electrophoresis for DNA analysis and some chromatography techniques (Sasuga
et al. 2017).
7.7.4 Preservation of Post-Hatchery Chicks
When chicks are hatched, they undergo a period when there is no access to feed or
water for a period of about 24 h as they are transported to production facilities where
they will continue their life and be processed into poultry products. The period
after hatching is a critical stage where the moisture content has an effect on the
future productivity of the chicks in terms of weight, growth rate and quality of egg
produced. As a means to address this moisture issue, agar-based aqueous system has
been introduced. Then, this aqueous agar which contains 95% water and 5% agar
is applied to the chick after hatching; they show better productivity and quality. For
example, post-hatched chicks which were packed with aqua agar had body weight
of 710 g, while those that were not packed with aqua agar weighed 670 g 21 days
after hatching (Incharoen et al. 2015). The aqueous agar covering maintains adequate
moisture content for the chicks during the period such that they retain their viability
at this important stage of their growth. This application of agar is significant toward
contribution to food security as a way of optimizing the poultry food resource for
optimal yield.
7.7.5 Cosmetics
Due to the ability to entrap water and retain moisture, agar is used in the cosmetics
industry as a moisturizing agent in skin and hair products (Rasmussen and Morrissey
2007). More recently, melanin has been loaded into agar-based composite films with
the aim of achieving antioxidant effect in skin care (Roy and Rhim 2019). Much
earlier studies have presented the moisturizing effect of short-chain neoagarobiose
which also had skin-whitening effect (Kobayashi et al. 1997). This moisturizing
effect of agar is likely due to the ability of agar to absorb water; the water is then
released into the skin. This short-chain agar is better able to penetrate the skin stratum
corneum barrier, hence why the short-chain agar forms show this moisturizing effect.
The viscosity-enhancing property of agar also makes it applicable as a thickener in
cosmetics products giving the product a more desirable texture and ease of use.
162 7 Agar
7.7.6 Antioxidant
When degraded into short-chain polymers (oligomers) referred to as agaro-

oligosaccharides and neoagaro-oligosaccharides, agar demonstrates antioxidant
activities (Chen and Yan 2005). These antioxidant activities of the short-chain agar
forms are attributed to the improved chance of the short chains to cross tissue barriers
and also the presence of some sulfate groups on the chain. Most sulfated polysaccha-
rides studied so far are attributed with some antioxidant properties. Although agar has
a relatively lower degree of sulfation (e.g., 0.14% of agar extracted from Gracilaria
caudata through enzyme extraction) (Alencar et al. 2019), more research is required
to fully understand the mechanism of its antioxidant activity. Sulfated polysaccha-
rides with agar-like chemical structure extracted from the red algae Gracilaria cau-
data show antioxidant properties which suggest that agar could have some promis-
ing application beyond its rheological properties. Agar-based oligomers also show
other bioactivities such as antitumor, prebiotic, anti-inflammatory, antidiabetic and
anti-obesity activities (Kwon et al. 2019).
7.7.7 Packaging
Currently, one of the biggest global challenges of modern times is the accumulation
of non-biodegradable plastic packaging waste. These have gone on to cause serious
environmental issues as plastics from landfill clog up drainage systems and end up
in the sea where they pose fatal risk to aquatic organisms. Their floating on the
water prevents penetration of light and oxygen, and they end up inside the digestive
systems of aquatic animals where they pose potentially fatal health risks. These
fossil-based packaging materials are used for short periods; however, they take very
long time to degrade. Degradation processes such as pyrolysis and incineration either
require additional energy input (temperatures of ~450 °C for pyrolysis) or they pose
a health risk (such as release of carcinogens from incineration of plastics). Marine
microorganisms from the arctic are being studied as potential candidates to degrade
these fossil-derived plastics; however, this is yet to be actualized as it is primarily
dependent on the microbes adapting to develop enzymes and means to degrade these
plastics (Ubanek et al. 2018).
One of the factors which make these materials attractive as packaging materials
is the fact that they are not degradable by microbes and they have superior mechani-
cal properties and water resistance; therefore, they can be used to hygienically pack
foods. Biodegradable packaging materials on the other hand are almost as biodegrad-
able as the food they are packed in. Such that, several researches have been directed
toward developing biodegradable packaging materials, which incorporate antimicro-
bial properties. The fact that agar is relatively more resistant to bacterial degradation
(Kwon et al. 2019) could contribute to its applicability in packaging materials.
Agarose has thermoplastic properties as well as moderate water resistance (Mak-

wana et al. 2018), and this has been explored in the production of packaging materials.
The limitations of agar for use in packaging materials are the brittleness at low mois-
ture content, inferior mechanical properties compared to non-biodegradable thermo-
plastics such as low-density polyethylene and insufficient solvent resistance. These
properties of agar can be improved by combining with other materials and other
polymers in the form of composites and blends. Blends of two different polymers
can result in a material with superior properties than each individual polymer alone.
Similarly, a composite which is made up of a dispersed phase within a polymer
matrix can significantly improve the polymer in its neat form. The dispersed phase
could be made up of particles such as silver nanoparticles or fibers such as cellulose
nanofibers (Olatunji and Olsson 2015). An example of such enhancement of agar’s
properties using a combination of nanocomposite and blend is a blend of agar with
carboxymethyl cellulose (CMC) which is then dispersed with silver nanoparticles to
produce a packaging film with thermomechanical properties suitable for production
of packaging films. The introduction of silver nanoparticles improved antibacterial
properties and also the mechanical properties (Makwana et al. 2018). Nanocompos-
ites can significantly boost the physical properties of polymers in nanocomposites;
however, their effect will be undermined if aggregation occurs. To prevent the aggre-
gation of the silver nanoparticles within the agar-CMC nanocomposite, the silver
nanoparticles were contacted with montmorillonite clay to prevent their aggregation
within the polymer matrix. At 3 and 5% loading, silver nanoparticle and montmo-
rillonite clay nanocomposite in agar-CMC blend resulted in 45 and 50% increase in
the tensile properties and increased the young modulus by 20 and 89%, respectively
(Makwana et al. 2018). Therefore, modified forms on agar can act as effective pack-
aging materials with properties comparable to those of petroleum-based packaging
materials and thus serve as a more environmentally friendly packaging material.
Agar can also be cross-linked to improve its applicability as a biodegradable
plastic packaging. Cross-linking increases the water resistance, tensile and thermal
properties of agar. Cross-linkers such as diisocyanates can be used. Better cross-links
are achieved with aromatic diisocyanates than aliphatic ones. Non-cross-linked agar
has a water uptake value of 206%; when cross-linked with aromatic diisocyanate,
this is reduced to 33.6%. The tensile stress of cross-linked agar is 45.3 MPa, while
non-cross-linked agar has a tensile stress of 31.7 MPa (Sonker et al. 2018).
Recent patented technology of biodegradable thermoplastic packaging material
incorporates agar as a modifying agent. One of the requirements for packaging mate-
rials is the ability to be heat processed. They are therefore required to have thermo-
plastic characteristics. This allows for large-scale production using the conventional
continuous plastic processing techniques such as blown film extrusion. This con-
tributes to reducing the cost of production of biodegradable plastics and also requir-
ing less technological transition when the feed is changed from non-degradable to
biodegradable plastics. Biodegradable thermoplastic is a mixture of PLA and mTPS
(polylactic acid and modified thermoplastic starch). The mTPS comprises of agar,
epoxide and gum arabic. For example, formulation of such thermoplastic starch
164 7 Agar
comprises of 80% unmodified starch, 28.5% glycerol, 20% agar, 10% epoxidized
vegetable oil and 0.5% gum arabic (Justyna et al. 2016).
7.7.8 Preservation of Stone Structures
Agar has also recently been investigated for use in the desalination of limestone as a
preservative measure for stone heritage structures which overtime are destroyed by
salt accumulation. Aqueous-based methods are sometimes counterproductive as the
water may cause other forms of damages such as rusting and erosion. Agar gel can
gradually release water to remove the salt in a sponge-like manner (Martins et al.
2017). This is a gentler method than more abrasive sponges. The water released
during syneresis dissolves off the salt, while the pores within the gel absorb the
solution from the surface of the structure. The gel can then be wiped off the surface.
Other applications of agar include use in capsules for pharmaceuticals, glucose-
lowering effects, in dentistry for preparation of denture molds, in archeology where
it is used in the reproduction of ancient remains and in forensics for fingerprinting.
As demand increases for more plant-based products, agar has potentially ever-
increasing demand in food as a plant-based replacement to the animal-sourced
gelatin. The demands for the hydrocolloids sourced from algae are generally increas-
ing annually. The largest producers, which are the Asia-Pacific regions, have reported
2–3% growth in the hydrocolloids market in recent years, and this includes agar
(Rejeki et al. 2018). There seems to be a general long-term increase in production
rate since earlier years; production rate between 1999 and 2009 had shown an esti-
mated 2% increase in production volume (Bixler and Porse 2011). Although the
market has witnessed considerable price fluctuations over the past decades, the phy-
cocolloid and algae market has gone through different phases since the 1930s. The
Asia-Pacific region remains the highest producer of agar and has been for many
years.
Of the three seaweed-derived hydrocolloids, agar has the highest commercial
value in terms of commodity prices, selling at ~18USD per kg as of 2009 com-
pared to carrageenan and alginates priced at 10.5 USD/kg and 12 USD/kg, respec-
tively. As of 2009, an estimated global sale of 9600 tonnes was estimated for agar,
while carrageenan and alginate were 50,000 tonnes and 26,500 tonnes, respectively
(Bixler and Porse 2011). As of 2014, global agar production of agar was an estimated
10,600 tonnes annually (Rhein-Knudsen et al. 2015). Agar is also sold for research
and laboratory use by chemical supplies company; for example, 100 g agar powder
for microbiology from Sigma-Aldrich is priced at 117 Eur as of June 5, 2019, while
the same quantity and quality of carrageenan from the same supplier are priced at
108 Eur (Sigma-Aldrich).
At smaller scale, agar of different grades is produced for research purposes. For
example, high purity agar for laboratory research studies is supplied by Sigma-
Aldrich (Ellis et al. 2019). These sources of agar for research are important in
developing new applications of agar and also as essential materials for experimental
procedures such as preparation of cell culture and tissue mimetics.
In commercial application, agar is mostly used as agarose (Makwana et al. 2018)
where much of the agaropectin is removed since agarose has more desirable proper-
ties. Some species produce more agarose content than others such that the isolation of
high agarose-producing species could reduce the processing time and cost in deriv-
ing more agarose rich fractions. Agar is sold as dry powder, squares or strips. This
is unlike carrageenan and alginates which are almost exclusively sold in powdered
form (Bixler and Porse 2011).
Successful commercial production of agar requires a mixture of factors: reliable
supply of low-cost and good-quality agar producing red algae, an efficient production
process, steady demand and good sales and marketing. It is also important for these
factors to be fine-tuned to specific agar application. Companies involved in com-
mercial production of agar include Algas Marinas in Chile, Agarindo Bogatama in
Indonesia, MSC in Korea, Ina Food Industry Co Ltd. in Japan, Hispanagar in Spain,
Setexan in Morocco, Huey Shyang Seaweed Industrial Company in China and B &
V in Italy (outsourcing production to Indonesia and Morocco).
Production of agar from mixed species of red algae does not yield a desirable
product with uniform properties; therefore, for agar production process to be better
controlled and optimized for yield and agar property, a single type of red algae is used
in the production process. Therefore, commercial cultivation of red algae for agar
production is more focused on Gracilaria. For instance, 80% of the agar produced
globally as of 2009 was produced from the Gracilaria species of red algae (Bixler and
Porse 2011). However, some research efforts are directed at exploring other red algae
species for commercial agar production toward having more diversified sources of
raw materials.
7.9 Conclusion
Agar is a well-established aquatic biopolymer commercially sourced mainly from

the Gracilaria red algae; its diverse applications in biotechnology and food make it a
highly valued biopolymer of the aquatic environment. It also evidently has a role in
the future polymer industry as more recent applications are emerging. This also points
to a potential rise in market value of agar as newer applications emerge. The raw
material is relatively available with main problem with commercial production being
the competitive price for seaweed in general due to rising competition with use of
seaweed as food. Although enzyme-based extraction and hot water extraction without
use of chemicals are possible, alkali extraction remains the most widely used process.
166 7 Agar
Therefore, more research should be focused on more advanced extraction techniques

which bring down the production cost and also minimize the environmental impact
of the production process without compromising quality or yield.
References
Alencar POC, Lima CG, Barros FCN, Costa LEC, Freitas ALP (2019) A novel antioxidant sul-
fated polysaccharide from the algae Gracilaria caudata: in vitro and in vivo activities. Food
Hydrocolloids 90:28–34
Armisen R, Gaiatas F (2009) Agar in: handbook of hydrocolloids. In: Phillips GO, Williams PA
(eds) Woodhead Publishing Series in Food Science Technology and Nutrition, pp 82–107
BeMiller JN (2019) Carrageenans in: carbohydrate chemistry for food scientists, 3rd edn. Woodhead
Publishing and AACC International, pp 279–291
Bixler HJ, Pose H (2011) A decade of change in the seaweed hydrocolloids industry. J Appl Phycol
23:321–335
Chen HM, Yan XJ (2005) Antioxidant activities of agaro-oligosaccharides with different degrees
of polymerization in cell-based system. Biochim Biophys Acta 1722:103–111
Chung IK, Beardall J, Mehta S, Sahoo D, Stojkovic S (2011) J Appl Phycol 23:877–886
Dabrowska A, Rotaru GM, Spano F, Affolter C, Fortunato G, Lehmann S, Derler S, Spencer ND,
Rossi RM (2017) A water -responsive, gelatine-based human skin model. Tribol Int 113:316–322
Earle M, De Portu G, DeVos E (2016) Agar ultrasound phantoms for low-cost training without
refrigeration. Afr J Emerg Med 6:18–23
Ellis AL, Norton AB, Mills TB, Norton IT (2017) Stabilization of foams by agar gel particles. Food
Ellis AL, Mills TB, Norton IT, Norton-Welch AB (2019) The effects of sugars on agar fluid gels
and the stabilisation of their foams. Food Hydrocolloids 87:371–381
Hernandez-Carmona G, Freile-Pelegrin Y, Hernandez-Garibay E (2013) Conventional and alter-
native technologies for the extraction of algal polysaccharides. In: Functional ingredients from
algae for foods and nutraceuticals. Woodhead Publishing Series in Food Science Technology and
Nutrition, pp 475–516
Incharoen T, Jomjanyouang W, Preecha N (2015) Effects of aqua agar as water replacement for
posthatch chicks during transportation on residual yolk-sac and growth performance of young
broiler chickens. Anim Nutr 1:310–312
Justyna K, Macie S, Helena J, Andrzej S, Katarzyna B, Aneta L (2016) Biodegradable thermoplastic
polymer composition, method for its manufacture and use thereof. EP3064542A1
Kobayashi R, Takisada M, Suzuki T, Kirimura K, Usami S (1997) Neoagarobiose as a novel
moisturizer with whitening effect. Biosci Biotechnol Biochem 61:162–163
Kumar V, Fotedar R (2009) Agar extraction process for Gracilaria cliftonii. Carbohyd Polym
78(4):813–819
Kwon GH, Kwon MJ, Park JE, Kim YH (2019) Whole genome sequence of a freshwater agar-
degrading bacterium Cellvibrio sp. KY-GH-1. Biotechnol Rep 23: e00346
Lebreton L, Slat B, Ferrari F, Sainte-Rose B, Aitken J, Marthouse R, Hajbane S, Cunsolo S, Schwarz
A, Levivier A, Noble K, Debeljak P, Maral H, Schoeneich-Argent R, Brambini R, Reisser J (2018)
Evidence that the great Pacific garbage patch is rapidly accumulating plastic. Sci Rep 8(1):4666
Li H, Yu X, Jin Y, Zhang W, Liu Y (2008) Development of an eco-friendly agar extraction technique
from the red seaweed Gracilaria lemaneiformis. Bioresour Technol 99(8):3301–3305
References 167
Makwana D, Castano J, Somani RS, Bajaj HC (2018) Characterization of agar-CMC/Ag-

MMT nanocomposite and evaluation of antibacterial and mechanical properties for packaging
applications. Arab J Chem (in press). https://doi.org/10.1016/j.arabjc.2018.08.017
Marcus JB (2014). Food science basics: healthy cooking and baking demystified. Culinary nutrition.
Academic Press, pp 51–97
Martinez-Sanz M, Gomez LG, Rubio AL (2019) Production of unpurified agar-based extracts
from red seaweed Gelidium sesquipedale by means of simplified extraction protocols. Algal Res
38:101420
Martins J, Dionisio A, Neves O (2017) Agar gel for anca limestone desalination. Procedia Earth
Planet Sci 17(2017):754–757
Mizrahi S (2010) Syneresis in food gels and its implications for food quality. In: Chemical dete-
rioration and physical instability of food and beverages. Woodhead Publishing Series in Food
Science, Technology and Nutrition, pp 324–348
Ouyang Q, Hu Z, Li S, Quan W, Wen L, Yang Z, Li P (2018) Thermal degradation of agar: mechanism
and toxicity of products. Food Chem 264:277–283
Rasmussen RS, Morrissey MT (2007) Marine biotechnology for production of food ingredients.
Adv Food Nutr Res 52:237–292
Rejeki S, Ariyati RW, Widowati LL, Bosma RH (2018) The effects of three cultivation methods
and two seedling types on growth, agar content and gel strength of Gracilaria verrucosa. Egypt
J Aquat Res 44:65–70
Rhein-Knudsen N, Ale MT, Meyer AS (2015) Seaweed hydrocolloid production: an update on
enzyme assisted extraction and modification technologies. Mar Drugs 13:3340–3359
Rioux LV, Turgeon SL (2015) Seaweed carbohydrates. In: Tiwari BK, Troy DJ (eds) Seaweed
sustainability, pp 141–192
Rocha MRC, Sousa AMM, Kim JK, Magalhaes JMCS, Goncalves MP (2019) Characterization of
agar from Gracilaria tikvahiae cultivated for nutrient bioextraction in open water farms. Food
Roy S, Rhim J (2019) Agar-based antioxidant composite films incorporated with melanin
nanoparticles. Food Hydrocolloids 94:391–398
Sahho D, Elangbam G, Devi SS (2012) Using algae for carbon dioxide capture and biofuel
production to combat climate change. Phykos 42(1):32–38
Sasuga K, Yamanashi T, Nakayama S, Ono S, Mikami K (2017) Algal Res 26:123–130
Sonker AK, Belay M, Rathmore K, Jahan K, Verma S, Ramanathan G, Verma V (2018) Crosslinking
agar by diisocyanates. Carbohyd Polym 202:454–460
Sousa AMM, Alves VD, Delerue-Matos MC, Goncalves MP (2010) Agar extraction from inte-
grated multi trophic aquacultured Gracilaria vermiculophylla: evaluation of a microwave assisted
process using response surface methodology. Biores Technol 101(9):3258–3267
Sudha PN, Gomathi T, Vinodhini PA, Nasreen K, (2014) Marine carbohydrates of wastewater
treatment. In: Kim SK (ed) Marine carbohydrates: fundamentals and applications, part B. Elsevier,
pp 103–143
Thannimalay L, Yusoff S, Zawawi NZ (2013) Life cycle assessment of sodium hydroxide. Aust J
Basic Appl Sci 7(2):421–431
Ubanek AK, Rymowicz W, Mironczuk AM (2018) Degradation of plastics and plastic-degrading
bacteria in cold marine habitats 102: 7669–7678
Wang K, Huang H, Sheng J (1997) Determination of the Mark-Houwink equation parameters and
their interrelationship. J Liq Chromatogr Relat Technol 21(10):1457–1470
Watase M, Nishinari K, Williams PA, Phillips GO (1990) Agarose gels: effect of sucrose, glucose,
urea, and guanidine hydrochloride on the rheological and thermal properties. J Agric Food Chem
38(5):1181–1187
168 7 Agar
Weiner ML, McKim JM, Blakemore WR (2017) Addendum to Weiner ML (2016) Parameters and
pitfalls to consider in the conduct of food additive research, carrageenan as a case study. Food
Chem Toxicol 87:31–44. Food and Chem Toxicol 107:208–214
Xiao Q, Weng H, Xiao A (2019) Physicochemical and gel properties of agar extracted by enzyme
and enzyme-assisted methods. Food Hydrocolloids 87:530–540
Chapter 8
Ulvans
Abstract Ulvans are sulphated polysaccharides present in the cell walls of green
algae alongside other cell wall polysaccharides. Their polymer structure is charac-
terized by repeating units of disaccharide of sulfated rhamnose linked to other units
of either uronic acid, guluronic acids or xylose. They are soluble polysaccharides;
therefore, their extraction process is relatively milder compared to other insoluble
polysaccharides. Ulvans are extracted from the Ulva species of green algae. These
species of green algae as raw materials for biopolymer production have the advantage
of growing in more diverse habitats and having a rapid growth rate. Extraction of
ulvans is one way of utilizing the excess green algae resource which often results in
algae blooms, to produce high-value bioactive polymers.
Keywords Ulvans · Biopolymers · Algae · Chlorophyta · Sulfated ·

Polysaccharides
8.1 Introduction
Ulvans are one of the three sulphated polysaccharides found in algae. While the sul-
fated polysaccharide of red algae is carrageenan and that of brown algae is fucoidan,
ulvans are produced by green algae. Ulvans are found in the cell walls of green algae
alongside cellulose and insoluble polysaccharide and two other soluble cell wall
polysaccharides, xyloglucan and glucuronan, which are present in lower amounts
(Kidgell et al. 2019). Ulvans are water-soluble polysaccharides and are character-
ized by their copolymer repeating units of disaccharide of sulfated rhamnose linked
to other units such as uronic acid. The presence of sulfates, multiple sugar units make
ulvans more complex than simple polysaccharides such as cellulose and chitin.
Like the other sulphated polysaccharides of algae, ulvan has been shown to have
some bioactive properties which make it potentially applicable in diverse industries
including food, pharmaceutics, cosmetics and biotechnology. Such bioactive proper-
ties include immune modulatory (Berri et al. 2017), antibacterial (Berri et al. 2016)
and antiinflammatory effects.

170 8 Ulvans
Marine algae are of great economical significance as they are a source of valuable
biomaterials: polyphenols, polyunsaturated fatty acids, proteins, polysaccharides and
pigments. All of which are useful in the pharmaceutical, cosmetics, food, biotech-
nology and microbiology fields. Some of the advantages of polymers sourced from
algae include the higher biomass yield of algae compared to terrestrial plants sim-
ilarly used as biorefinery crops, the use of aquatic rather than land space for their
cultivation, removal of CO2 from the atmosphere and uptake of eutrophying nutrients
from the water. On the other hand, issues such as competition with algae used for
food and relatively high cost of algae as feedstock pose a challenge in commercial
production of ulvan.
This chapter reviews ulvan as an aquatic sourced biopolymer, its source, extraction
process, environmental issues surrounding its production and the present state of
commercial ulvan production. In so doing it furthers the goal of the book to highlight
the diverse range of biopolymers in the aquatic ecosystem, the present issues as well
as their environmental and economic significance.
Green marine algae such as Ulva amoricana and Ulva rigida and Ulva enteromorpha
are the known sources of ulvan which forms a part of the cell wall alongside other cell
wall polysaccharides such as mannan, xylan and cellulose (Misurcova et al. 2012).
These green algae contain around 38–54% Ulvan by dry weight (Michel and Czjzek
2014) (8–29% Lahaye and Robic 2007), while others report between 9 and 36% by
dry mass in Ulva species (Kidgell et al. 2019). Ulvan producing species include U.
rigida, Ulva pertusa, Enteromorpha, Ulva intestinalis (Tabarsa et al. 2018), E. linza,
E. clathrata (Tabarsa et al. 2018), Ulva ohnoi (Fernández-Díaz et al. 2017). The
green algae Ulva species are rapid-growing species to the extent that they can result
in algae blooms under uncontrolled growth in the wild aquatic habitat (Kidgell et al.
2019). Figure 8.1 is a schematic representation of Ulva within the green algae cell
wall. Ulvan exists in a matrix with other cell wall polymers such as cellulose proteins
and glucuronan.
Sulfated polysaccharides are also found in animals, and however, plants do not
produce sulfated polysaccharides. Examples of sulfated polysaccharides of animals
are glycosaminoglycans (GAGs) and proteoglycans (Scharnweber et al. 2015). Much
of the studies on bioactivity of ulvans have explored mimicking the functions of these
animal sulfated polysaccharides as it is expected that the similarity in structure will
manifest as similarity in bioactivity. The plant macromolecule rhamnogalacturonans
and rhamnolipids in phytopathogenic bacterium (Varnier et al. 2009) are the com-
ponents of these organisms which have the closest similarity with ulvans of green
algae.
8.3 Chemistry of Ulvans 171
Ulvan
Glucoronan
Xyloglucan
Protein
Cellulose
Fig. 8.1 Schematic representation of cell wall structure illustrating the location of ulvan in green
algae
8.3 Chemistry of Ulvans
Ulvans are heteropolysaccharides made up of several sugar units mainly: sulfated

rhamnose and xylose units, xylose, glucuronic acid and iduronic acid (Kim et al.
2011a). Figure 8.2 shows the structure of disaccharide units in ulvan. Ulvans are
polydisperse and molecular weight of different fractions could vary, for example,
Tabarsa et al. (2018) report weight average molecular weight (M w ) of fractions
extracted from U. intestinalis ranging between 87,100 and 194,100 g/mol following
extraction and fractionation of the crude ulvan into different molecular weights using
DEAE-Sepharose Fast Flow column.
The ulvan polymeric chain is arranged as repeating unit disaccharides of either
rhamnose and iduronic acid called the aldobiuronic acid (Lahaye and Robic 2007),
sulfated rhamnose and glucuronic acid or sulfated rhamnose and xylose (Michel and
Czjzek 2014). Arabinose and galactose are also said to be present in ulvans (Misur-
cova et al. 2012; Tabarsa et al. 2018). While the structure of the ulvan polymer could
vary depending on factors like source, species and harvest period, it is established
Fig. 8.2 Structures of a

disaccharide repeating unit
in ulvan comprising of a
glucuronic unit linked to a
sulfated rhamnose
172 8 Ulvans
that the sulfate groups of ulvan generally attaches to either the second or third carbon
of the rhamnose unit or the second carbon of the xylose unit (Tabarsa et al. 2018).
The bioactivity of ulvans is affected by the molecular weight. The molecular
weight of ulvans varies between 100 and 8000 kDa (Lahaye 1998) although others
report it between 150 and 2000 kDa (Rioux and Turgeon 2015). Lower molec-
ular weight has been shown to favor interactions with immune cells which aid
immunomodulatory and inflammatory activities (Chen et al. 2008; Jaswir and Mon-
sur 2011). This superior bioactivity of lower molecular weight ulvan over higher
molecular weight ulvan is attributed to the ease of access into the cells and ease of
proton exchange as a result of lower molecular weight (Qi et al. 2005).
Each repeating unit within the ulvan structure plays a distinct role in the bioactivity
of ulvan. Rhamnose can aid in hyaluronic and collagen production in human fibroblast
dermal cells through interacting in biosynthetic pathways in the dermis (Adrien et al.
2017). The presence of functional groups such as sulfate and amine groups also
confers certain properties on the ulvan polymer. The repeated disaccharide units of
ulvans comprising mainly of uronic acid linked to sulfated rhamnose or xylose make
them applicable for modulation of cellular activities carried out by polysaccharides
in mammals. These properties make them applicable in areas such as wound healing,
tissue repair and antiviral activities.
While each repeating unit has its contributory role to the physicochemical and
bioactive properties of the ulvan, the configuration, order with which the repeat-
ing units are arranged within the polymeric structure, is also important. This in
turn affects the conformation of the ulvan molecules and consequently the overall
properties. Branching occurs usually at the 1.2 linked glucuronic acid linked to the
rhamnose (Kidgell 2019; Lahaye and Robic 2007). As with most polymers the level
of branching is related to properties such as crystallinity, thermal and mechanical
properties of the polymer.
Ulvans are soluble polysaccharides that dissolve in water to form a gel-like solu-
tion. Like all polymers, ulvans will increase the viscosity of the solution within which
it is dissolves in. The solution viscosity of ulvan depends on the pH and the pres-
ence of salts. At low to neutral pH, ulvan takes on a bead-like conformation which
aggregates in the presence of salts such as sodium chloride. Such solutions of ulvan
usually have low viscosity. In more alkaline environment, ulvan forms more viscous
solutions with higher gel strength (Lahaye and Robic 2007).
The biochemistry of ulvans varies for different sources as a result of variation in
the primary structure, molecular weight, branching and the polydisperse nature of the
polymer. These variation correlate with their bioactive properties. For instance, low
molecular weight ulvans have been shown to stimulate kidney macrophage better than
ulvans with higher molecular weights (Fernández-Díaz et al. 2017). There have been
studies which investigate the bioactive properties of crude ulvan extract and those
which separate the ulvan into different molecular weight fractions to investigate the
properties of specific molecular weight range, and other studies have extended to
modification of ulvans to control specific properties. Chemical properties such as
degree of sulfation can be modified by removal or addition of sulfate functional
groups such as solvolysis or base hydrolysis (Qi et al. 2012). The chain length of the
8.3 Chemistry of Ulvans 173
polymer can be altered by breaking specific glycosidic bonds using either chemical
(Pengzhan et al. 2004) or enzyme hydrolysis (Reisky et al. 2018) of the ulvan polymer
chain. The solution properties at different pH can be used to develop ulvan-based
products with specific rheological properties. Ulvans therefore offer a broad range
of tuneable chemical and rheological properties which presents a range of potential
applicabilities.
8.3.1 Biodegradation of Ulvans
Understanding the biodegradation of ulvans is important in assessing their safe degra-

dation when released into the environment and when consumed as food, cosmetics
or therapeutics. It is also important in understanding the structure of ulvans. The
biodegradation products and the enzymes which catalyze the biodegradation provide
an insight into the structural configuration of the ulvan as well as an understanding
of the biological activity of the microbes which degrade ulvans.
Ulvanolytic enzymes refer to enzymes which are capable of degrading ulvans.
Microbes identified to be capable of degrading of ulvan polysaccharide such as gram-
negative marine bacterium, ochrobactrum and flavobacterium produce ulvanolytic
enzymes (Michel and Czjzek 2014). These enzymes are capable of cleaving specific
glycosidic bonds, for example, a type of ulvanolytic enzyme called ulvan lyases
catalyzes the cleavage of the bonds between sulfated rhamnose and glucuronic acid
or that between rhamnose and iduronic acid (Nyvall Collen et al. 2011). Effectiveness
of ulvanolytic enzymes therefore depends on the chain configuration of the specific
ulvan and the repeating disaccharide units within the ulvan polymeric chain. Ulvan
is not degraded by any enzyme produced by the human body, and it is also able to
survive the gastrointestinal tract and colon undigested (Misurcova et al. 2012). This
makes it a good option for dietary-soluble fiber.
Green algae are a renewable natural resource for biopolymer production. It also has
the additional advantage of growing in more diverse habitat with varying abiotic
factors. Compared to red or brown algae which primarily grow in marine waters,
green algae grow in both marine and freshwater. The ulvan producing species grow
in marine and freshwater. Example of marine species is U. rigida (Tabarsa et al.
2018) and that of freshwater is Ulva thalli (Rybak et al. 2012). This occurrence in
diverse habitat means less limitation on region where the raw material for production
of ulvans can be the sources. A readily available resource from multiple regions is
always better for the economy than resources concentrated in only a few regions. This
concentration of resources in limited regions has led to some of the socioeconomic
problems of other natural resources such as crude oil and minerals.
174 8 Ulvans
Unlike brown algae with a relatively complex reproductive cycle which makes
controlled cultivation in aquaculture more labor and cost intensive to be economi-
cally viable for commercial biopolymer production, the cultivation of green algae
is relatively simpler. Due to its relatively rapid growth rate and simpler cultivation
process, the ulvan species of green algae are more promising for the cultivation of
monocultures which will result in more uniform ulvan extracts. This is important in
developing standard formulations with specific bioactivities where factors such as
molecular weight and degree of sulfation are within shorter ranges. Some Ulva green
algae grow so easily that in situations where there is an environmental imbalance
in the aquatic ecosystem such as increase in nutrient content of the water this could
result in drastic population boom of green algae resulting in what is known as green
tides (Kidgell et al. 2019). The Ulva species have been identified as one of the algae
species which form algae blooms (Young and Gobler 2016). Ulva prolifera, a highly
productive Ulva can increase by 28% in population within a period of 24 h (Zhang
et al. 2019). In a well-managed controlled environment, this rapid reproductive rate
is an advantage for generating feedstock for ulvan production.
Asian countries generally contribute a large fraction of global algae production.
Algae are also produced in some countries in Europe and the USA. The FAO data
on global commercial algae production makes no mention of the Ulva species. This
could be classified under “other” (FAO 2018). However, the Ulva green algae are
evidently abundant as they have resulted in algae blooms in areas such as the Yellow
Sea in China off the coast of Jiangsu and Shandong Province (Zhang et al. 2019) and
in Japan at Yatsu tidal flat in Tokyo Bay (Yabe et al. 2009). Green tides in the Yatsu
tidal flat of Tokyo Bay could cover an area of water up to 27.1 ha (0.271 km2 ) of a
total area of 40 ha (0.4 km2 ) at peak growth (Yabe et al. 2009). That is over 67% of
the lagoon being covered by green tide. The green tide of the Yellow Sea in China
covers an area of 20,000 km2 and has been described as the largest ever experienced
in the world (Zhang et al. 2019). Therefore, not only is the ulvan producing green
algae available, they are presently causing environmental nuisance and finding ways
to utilize these green algae is a priority.
8.5 Extraction of Ulvan
As a water-soluble fiber, ulvan can be extracted from green algae using aqueous
extraction. Typical extraction temperature ranges between 80 and 90 °C. An extrac-
tant such as ammonium oxalate is often used at a 0.02M concentration (Robic et al.
2009) as a divalent cationic chelating agent to aid the extraction by making the
biomass more susceptible to the solvent. The crude extract can be further purified
using alcohol or quaternary ammonium salt precipitation (Lahaye and Robic 2007;
Shao et al. 2011; Rioux and Turgeon 2015). High-temperature acidic extraction at
a pH of 2 and a temperature of around 80 °C are also used to extract ulvan (Yaich
et al. 2014; Rioux and Turgeon 2015).
8.5 Extraction of Ulvan 175
Here, the extraction method used by Tabarsa et al. (2018) to extract ulvan from
Ulva intestinalis is used as an example of extraction process. Algae strain U. intesti-
nalis were sourced from the Iranian coast, Noor. The collected biomass was then
washed using tap water followed by drying at a temperature of 60 °C. To reduce
the particle size, the washed and dried algae mass was ground using a blender and
sieved to particle size below 0.5 mm. To isolate crude polysaccharide, the alcohol-
soluble pigments, lipids and low molecular weight compounds were removed from
the ground seaweed by adding 80% ethanol at a mass-to-volume ratio of 1:10 (g:mL).
This was left overnight at ambient conditions. Separation was carried out using a cen-
trifuge at 10 °C at 8000 rpm for 10 min followed by decanting off of the supernatant.
The solid residue which now contains a mix of polysaccharides and other macro-
molecules is then rinsed with acetone and dried at room temperature. The next stage
which involved extraction of the water-soluble polysaccharides was carried out at
65 °C using distilled water in the mass-to-volume ratio of 1: 20 (g:ml). The extrac-
tion time allowed in the particular study was 2 h. Following the extraction period,
the liquid extract was separated from the solid residue using centrifugation at 10 °C
operating at 10,000 rpm for 10 min. The extraction was repeated with fresh dis-
tilled water to further remove soluble sulphated polysaccharides. The extracts from
both runs were combined and evaporated under reduced pressure at a temperature
of 60 °C. The ulvan was precipitated out using 90% ethanol (when added to liquid
mixture concentration of ethanol decreases to 70%). Precipitation was allowed to
take place overnight at 4 °C followed by centrifugation at 10,000 rpm for 10 min at
10 °C. The precipitated were further washed and dehydrated with acetone and 99%
ethanol, respectively. The final product was dried at room temperature.
Following extraction of the crude ulvan, it is often desired to separate into dif-
ferent fractions. This could be for analysis or obtaining a less polydisperse product.
The bioactivity of ulvans like other polysaccharides is affected by the molecular
weight. Therefore, separating into fractions with specific range of molecular weight
is important in analyzing and utilizing these bioactivities and physicochemical prop-
erties. Fractionation can be carried out using methods such as DEAE-Sepharose Fast
Flow column (Tabarsa et al. 2018).
Co-extraction can be carried out to extract other polymers alongside the ulvan
since the cell walls always contain other useful polysaccharides such as cellulose,
xyloglucans as well as proteins. This would then involve including a separation
process after the extraction stage. The yield and quality of ulvan obtained from
any given biomass depend on the conditions of extraction. Temperature, pH, use
of extractants and concentration of extractant, particle size of biomass, duration of
extraction and pretreatment method are factors which have been identified to affect
the yield and quality of ulvan extraction (Kidgell et al. 2019). The yield is defined
in terms of the quantity of actual extract compared to the known content of ulvan in
the biomass, while the quality is quantified by the degree of polymerization; hence,
molecular weight of the ulvan extracted the purity (i.e., absence of other polymers
and contaminants) and the level of degradation as a result of depolymerization and
desulphurization of the ulvan in the process of extraction.
176 8 Ulvans
The key challenge to overcome in the extraction process is that ulvan has rel-
atively low solubility in water, and the cell wall has a rather stable structure with
strong intermolecular bonding between the cell wall polymers within which ulvan
is entangled (Robic et al. 2009). These challenges are addressed by altering the pH
which in turn affects the solubility of the ulvan and the intermolecular interactions
between ulvan and the cell wall polymers as these parameters are pH-dependent. In
other words, the intermolecular bonds between ulvan and the other molecules mak-
ing up the green algae cell wall are weakened, while its tendency to be isolated into
the solution is increased.
A further challenge in extraction of ulvan is the isolation of ulvan while excluding
the other soluble polysaccharides and proteins present in the green algae biomass.
The solubility of these other polymers is also pH- and temperature-dependent. Glu-
curonan, for example, has higher solubility in alkaline condition. This is addressed
by selecting the right pH which favors only ulvan, making the extraction process
more selective toward ulvan. The presence of salt also affects the extraction process
as salts cause aggregation of ulvan. This is addressed by treatment with warm water
to dissolve off the salt and increase osmotic pressure prior to extraction (Glasson
et al. 2017).
At lower pH, the extraction is more selective toward ulvan as the solubility of
other polysaccharides such as glucuronan and proteins is reduced at this condition.
For example, protein impurities in ulvan extracted from U. ohnoi are significantly
lower when hydrochloric acid extraction is used compared to when sodium oxalate
extraction is used. The protein content of the extract increased from 4 to 7 µg protein
per mg extract to 114–162 µg protein per mg extract. The drying and milling process
is also important to minimize the particle size and optimize contact surface between
biomass and extractant. Degradation during extraction results in an ulvan extract
with shorter chains and less option for depolymerization to obtain more versatile
fractions. While lower pH favors more selective extraction with less impurities,
under acidic conditions and high temperature the ulvan extract is likely to undergo
more degradation during extraction. The processor is therefore faced with the choice
of higher yield at the expense of a more degraded product having lower degree of
polymerization.
Recommended extraction conditions based on that used in various extractions
reported in the literature are the extraction at a pH between 2 and 4.5, temperature
between 80 and 90 °C and extraction period of 1–3 h. Some studies carry out repeat
extraction on the same biomass to further extract any ulvan still left in the sample.
Additional cost of energy and time should be considered for the second extraction
which would have lower yield than the first and a lower concentration of ulvan requir-
ing more evaporation per gram of ulvan extracted. Furthermore, since the structure
of ulvans varies for different sources and also varies with harvest season, for extract
with most uniform chemical structure, biomass from the same source and harvest
should be used in the batch.
Unit operations which follow the extraction process are required to isolate and
purify the product. These processes vary depending on availability and quality or
form of desired final product. The first step after extraction is the separation of
8.5 Extraction of Ulvan 177
Green algae biomass

Washing
Drying 333K
Pulverization Particle size

<0.5mm
Pigments, Pretreatment with 80% 1:10

ethanol (g:ml)
impurities, low
molecular
weight 8000 rpm for 10 minutes.
Centrifugation Supernatant decanted off
compounds
Solid residue, a mix of polysaccharides
Rinsing with 338K in distilled
Acetone water 1:20 (g:ml)
Drying Aqueous
Filtration Extraction
Fig. 8.3 Flowchart summarizing the extraction process of ulvans from green algae
the dissolved crude ulvan from the solid mass. This can be done using filtration or
centrifugation. The extracted liquid at his point is likely to have smaller molecules
and other polymers, etc. dissolved within it or smaller particles which are too small
to be separated. The ulvan is precipitated out by addition of ethanol. Since ulvan
will not dissolve in ethanol, this allows the ulvan to precipitate out of the solution.
The gel-like precipitate can then be evaporated or concentrated using ultrafiltration.
The process of washing, redissolving and reprecipitation can be repeated to improve
purity. Impurities of salt and small molecules can be further removed by dialysis
and ultrafiltration. For separation into different fractions with a range of molecular
weights, methods such as size-exclusion chromatography can be employed.
The extraction process used and the level of purification depend on the target
use of the ulvan and standard requirement of the industry. Figure 8.3 summarizes a
typical extraction process of ulvan.
While ulvans offer numerous benefits and potential for application in food, nutraceu-
ticals and pharmaceuticals, it is important to understand the environmental impact
the production of ulvans. This requires an understanding of the processes, materials
178 8 Ulvans
and chemicals involved in the whole process from harvesting of the raw material to
the final product leaving the production factory gate.
8.6.1 Cultivation and Harvest of Green Algae
In the wild aquatic ecosystem, green algae play a significant role in carbon and
nitrogen fixation as well as maintaining oxygen levels. These are essential to other
life forms in the aquatic ecosystem. Harvesting of green algae from the wild should
therefore give careful consideration to the impact of disrupting the existing balance
within the environment where green algae play a key role. The system of harvesting
from the wild should therefore implement a method where the total population of
algae at any given time is maintained by balancing the rate of harvesting and the rate
of replenishing.
Another issue with cultivation of green algae for commercial purpose is the danger
of introducing exotic strains into the wild, thereby propagating algae blooms or
artificially creating an environment which favors unnatural growth rate of green
algae. This is a likely outcome in algae cultivation in aquaculture systems operating
within the open sea (mariculture). Recent investigations into the cause of the green
tides experienced along the Jiangsu coast of China revealed that the U. prolifera
which is the predominant species causing the green tides in the region is genetically
similar to that growing in the Porphyra mariculture located along the same Jiangsu
coast (Zhang et al. 2019).
In China, for example, the economic loss from the green tide algae bloom has
been estimated at over 183 million USD (1.3 billion RMB) (Zhang et al. 2019). This
loss comes from the loss of income resulting from decline in aquatic organism as a
result of hypoxia (oxygen depletion in the water), loss of income from recreational
and tourist activities on the shoreline as a result of loss of aesthetics, foul smell and
health risk associated with the green tides.
The cultivation of green algae for production of ulvans must therefore be carefully
considered to prevent the risk of propagating green tides which result in serious
adverse environmental impact.
The extraction process is an aqueous one such that water serves as the solvent for
extraction. Water is also used in the washing of the raw material during cleaning
and pretreatment. Following extraction and separation of the gel, the water which
contains impurities then needs to be treated before release into the environment.
Depending on the level of treatment, this inevitably involves release with a level of
contamination into the environment.
The amount of water used in the washing of the biomass was not specified in
the said study (Tabarsa et al. 2018). Assuming a 1:10 ratio of dry biomass to tap
water, such that 10 mL of water is required per gram for washing stage. For the
main extraction, 20 mL of water is used per gram of Ulva. To therefore obtain the
crude ulvan extract, a minimum estimated 30 mL of water (10 mL tap water and
20 mL distilled water) is used. Compared to more water-consuming process such
as alginate extraction (see chapter on alginate), ulvan extraction consumed a fair
amount of water.
8.6.3 Alcohol
In the pretreatment stage, each gram of pulverized ulvan powder required 10 mL

of ethanol at 80% v/v concentration. This is used to dissolve off lipids, alcohol-
soluble low molecular weight compounds and pigments. More ethanol is then used
for precipitation at a concentration of 70% with estimated volume of 20 mL per gram
of ulvan. Additional 99% ethanol (unspecified amount) is used for dehydration. Such
that for each gram of ulvan produced in the reported study, at least 30 mL of ethanol at
70–80% concentration is required for pretreatment and precipitation with more used
in dehydration post-extraction. As a greener alternative, bioethanol from renewable
source can be used. However, the sustainability of such extraction process will need
further consideration. The alcohol can also be cleaned and recovered through solvent
recovery processes such as adsorption, distillation and condensation.
8.6.4 Acetone
Following pretreatment with ethanol to remove lipids, pigments and low molecu-
lar compounds, the ulvan is washed with acetone (amount unspecified). Additional
acetone is used after the aqueous extraction in the final washing and dehydration
prior to drying of the crude extract. Acetone is an organic solvent sourced from
non-renewable sources. In the production of biopolymers such as ulvan, the goal is
to source polymers from renewable sources, and it is therefore important to seek
alternative solvents in the process of extraction.
In the extraction of the crude ulvan, energy consumed goes into heating of the system
to temperatures between 80 and 90 °C (Tabarsa et al. 2018). Heating 1 kg of water
by 1 K consumes 4.18 × 10−3 J of energy (Bird et al. 2005). Further energy is
consumed in the drying process. Further energy consumption takes place in the other
180 8 Ulvans
Table 8.1 Typical

Component Amount
consumption in ulvan
extraction process Green algae biomass 3–12.5 g per gram algaea
Water 30 ml/g ulva
Ammonium oxalate 0.02M
Ethanol 80% 10 ml
90% 10 ml
Acetone 98%
Energy
• Grinding 0.5 mm particle size
• Heating 65 °C 2 h
• Drying 60 °C overnight
• Centrifugation 8000 rpm 10 min
10,000 rpm 10 min
a Based on yield of ulvan ranging between 6 and 30%
unit operations which follow extraction such as separation and drying. Separation is
done using centrifugation at 10,000 rpm at 10 °C for 10 min. Centrifuges operate
from low rpm to medium rpm of about 2000 rpm to high rpm > 6000 rpm with
60,000 rpm being in the ultracentrifugation range. Therefore at 10,000 rpm, the ulvan
production separation process is making use of relatively high centrifugal force, thus
implying that the energy used up at this stage is considerably high. Manufacturers
also need to consider the energy consumed further down in the production process
for fractionating into different molecular weight ranges to obtain a less polydisperse
sample and for purification which is important in improving the value of the product.
As discussed within the chapter, molecular weight and purity have significant effect
on the bioactivity of ulvans.
Table 8.1 gives a summary of consumption in a typical ulvan extraction process.
These values are based on values reported in research studies (Tabarsa et al. 2018).
8.7 Applications
Bioactive properties of ulvans include antioxidant activity, cholesterol- and

triglyceride-lowering effects among others. We know that ulvans from different
species of green algae show species related variation in their chemical properties such
as molecular weight, sulfation, constituent monosaccharides and level of branching
(Kidgell et al. 2019). These variations in chemical properties result in variation in
the bioactive properties such that specific species have been identified with some
bioactive properties that are not seen in other species. In reviewing the applica-
tions of ulvan, we identify some of the species which can be used in the discussed
application.
8.7.1 Immune Modulation
The ability of certain substances to influence immune response is referred to as

immunomodulation and entities which are able to do these are referred to as
immunomodulators. This activity is primarily dependent on the activation of inflam-
matory process whereby the body releases chemicals to destroy pathogens from the
body. Ulvans aids in immunomodulatory process by activating the protein complexes
which control production of cytokines which are key to the inflammatory process.
Increased production of cytokines, enzymes and the products has been shown by
macrophage cells treated with ulvan from species of U. pertusa (Li et al. 2018a,
b), U. rigida (Leiro et al. 2007), U. intestinalis (Peasura et al. 2016), U. Ohnoi
(Fernández-Díaz et al. 2017) Enteromorpha prolifera (Kim et al. 2011a) and U. prolif-
era (Cho et al. 2010). Beyond macrophage cell studies, investigation of immunomod-
ulatory activity of ulvans has also been carried out in organisms such as fish (Cas-
tro et al. 2006), rats, mice and chickens. Ulvans aid in the bodies’ defense against
defective or pathogenes by enhancing macrophage functions such as phagocytosis
(Karnjanapratum et al. 2012).
The inflammatory cytokine production, hence immunomodulatory activity of
ulvan, is comparable to that of lipopolysaccharides albeit requiring a higher concen-
tration of ulvan to achieve the same effect. For instance in one study, a 500 µg mL
of ulvan was required to achieve the same immunomodulatory effect as 100 ng/mL
of lipopolysaccharides.
Molecular weight and degree of sulfation jointly affect the occurrence and potency
of the immunomodulatory activity of ulvans. Purity is also an important factor in
the effectiveness of the immunomodulatory activity of ulvans with purer samples
showing better immunomodulatory effect. This can be attributed to the reduction in
interference with the active functional groups as well as structural chemistry which
influences the bioactivity. In one study, ulvans extracted and purified from U. per-
tusa with molecular weight in the range 14,450–1690 kDa showed an increase in
immunomodulatory activity quantified as macrophage cell activation when com-
pared with ulvans with lower molecular weight of less than 365 kDa from the same
source (Tabarsa et al. 2012). However, this increased immunomodulatory activity
resulting from increased molecular weight is not replicated in other ulvan extracts
from other species in a different molecular weight range. For examples, ulvan from U.
intestinalis with a molecular weight of 28.7 kDa showed much higher immunomodu-
latory activity compared to ulvans from same source with higher molecular weight of
87.2 kDa (Tabarsa et al. 2018), thus indicating that the relationship between molecu-
lar weight and immunomodulatory activity is not the same either across all molecular
weight range or across species. Degree of sulfation also affects the immunomodula-
tory activity of ulvan. Most studies show that higher level of immunomodulation is
achieved with higher degree of sulfation (Leiro et al. 2007).
The immunomodulatory activity of ulvans makes them potentially applicable
as immune-stimulating agents if nutraceutical or pharmaceutical products. Species
of green algae not yet confirmed to possess or do not possess immunomodulatory
182 8 Ulvans
bioactivity include U. conglobata, U. compressa, U. flexuosa, U. gigantea and many

others (Kidgell et al. 2019).
8.7.2 Antioxidant
Ulvans like other sulfated polysaccharides act as exogenous antioxidants and enhance
the activities of the endogenous antioxidants. They do so through their radical scav-
enging activity and enhancement of the activities of antioxidant enzymes. The antiox-
idant activity of ulvans makes them applicable in addressing a broad range of human
defects which are associated with the presence of reactive oxygen and nitrogen
species. Such conditions include cancer, neurodegenerative diseases, cardiovascular
diseases and aging. Low molecular weight and high sulfate content have been shown
to correlate with the antioxidant activity of ulvans.
Although the mechanism by which ulvans boost endogenous antioxidant activity
is not yet fully established, it is thought that they do this by interfering in signaling
pathways for expression of antioxidant producing enzymes. This ability of ulvans
to stimulate antioxidant enzyme production has been demonstrated by ulvans from
species such as U. amoricana (Berri et al. 2017). Therefore, ulvan antioxidant activity
is both as a result of their chemical structure eliminating oxidative species and also
their ability to influence the gene expression for production of enzymes responsible
for endogenous antioxidant activity in the body.
8.7.3 Anticancer
Although no clinical trials have been reported yet, studies on animals such as mice
and rats and a range of human cancer cell lines such as colon cancer, breast cancer and
gastric cancer cell lines have provided evidence that ulvans could potentially have
anticancer properties. Ulvan from species such as fasciata, tubulosa, pertusa, latusa,
intestinalis and others (Kidgell et al. 2019; Kim et al. 2011b) has shown anticancer
properties.
Various approaches are used in the treatment of cancer: preventing the formation
of cancerous cells by removing factors such as oxidative processes which could lead
to mutation, prevention of growth of the cancer cells or inducing death of the cancer
cells. Thus, far evidence points to ulvans potentially play anticancer roles by either
preventing the proliferation of cancer cells or promoting apoptosis (programmed
cell death) in cancer cells. Example of studies indicates that the ability of ulvans to
promote cancer cell apoptosis is by Wang et al. (2014). In the aforementioned study,
the increase in pro-apoptotic tumor suppressor expression was observed in cancer cell
lines treated with ulvan from Enteromorpha intestinalis. The pro-apoptotic activity of
ulvan is also demonstrated by the reduction in expression of anti-apoptotic proteins.
The other anticancer approach ulvans is thought to use is the prevention of cancer
cell proliferation. This has been investigated by measuring the reduction in the level
of DNA replication activity in the cancer cells. This is quantified by the reduction
in the level of proliferating cell nuclear antigen (Hussein et al. 2015). The level of
DNA replication activity reduced to noticeable levels in rat hepatocytes when treated
with ulvan from Ulva lactuca. Anticancer activity of ulvans has also been measured
as a function or tumor mass and cytotoxicity where a reduction in tumor mass and
a high cytotoxicity are indicative of reduced cell proliferation and hence anticancer
activity.
Although a number of studies have presented anticancer properties of ulvans,
there are also studies which show no cytotoxicity of ulvan against cancer cells. An
example of such is studies on Ulvan intestinalis which show no cytotoxicity against
sarcoma tumor cells at a relatively high concentration of 50–800 µg per ml. However,
this study also further showed that ulvans from different species could take different
pathways to achieving anticancer activities. While no cytotoxicity was measured
in vitro, there was 61–7% (Jiao et al. 2009) reduction in the tumor cell mass in vivo
when applied to mice in vivo at a dose of 100–400 mg/kg.
The anticancer activity is very low compared to conventional chemotherapy
agents. With such a low level of effectiveness, ulvan can at best serve in adjunct roles
in cancer treatment. As a polymeric material with pH-dependent rheological prop-
erties, ulvan can serve roles in anticancer therapy delivery system as pH-responsive
polysaccharide systems (Yang et al. 2017) and nanoparticle delivery systems (Li
et al. 2018a, b). No conclusive data yet exists to relate the chemical structure such
as degree of sulfation or molecular weight to the anticancer activity of ulvan.
8.7.4 Anticoagulant Activity
Undesired blood clots could lead to severe health risks and could result from condi-
tions such as operated blood vessels. Anticoagulants have different mechanisms of
action with the general goal being to interfere with the pathways leading to blood
clotting. Anticoagulant activity of ulvan is affected by degree of sulfation at a par-
ticular molecular weight range. Most studies are in agreement that a higher degree
of sulfation leads to increased anticoagulant activity of ulvans. However, the effect
of molecular weight varies. For ulvans extracted from Enteromorpha prolifera, for
example, beyond a given molecular weight of 200 kDa, further increase in degree
of sulfation did not yield any anticoagulant property (Li et al. 2018a, b). Increasing
the degree of sulfation resulted in increased anticoagulant activity for ulvans of E.
prolifera with molecular weight less than 200 kDa. However, when the molecular
weight is increased beyond this value, the ulvan no longer had any anticoagulant
effect.
Anticoagulant effect of ulvans is much less than those of currently commer-
cially available anticoagulants such as heparin (Qi et al. 2013). It also varies from
species and source since the degree of sulfation and molecular weight is also species
184 8 Ulvans
and source dependent. Nonetheless, the low level of anticoagulant activity demon-
strated in various studies is potentially relevant in developing anticoagulant agent
with potential for interfering in the body’s intrinsic anticoagulant mechanism.
8.7.5 Antihyperlipidemic
When there is an imbalance in the way the body distributes and eliminates lipids and
cholesterol, this leads to metabolic syndrome often associated with hyperlipidemia.
This is often a cause of cardiovascular diseases. The components of macroalgae have
been linked with having antihyperlipidemic effects and a diet including macroalgae
has been shown to have preventive or curative effect on hyperlipidemia. To isolate
specific components in order to develop therapies for hyperlipidemia, the sulfated
polysaccharides of macroalgae have been investigated for their antihyperlipidemic
effect.
Ulvans from species which include U. pertusa, U. lactuca, U. prolifera and Ulva
fasciata have shown significant hyperlipidemic effect when measured as a function of
serum total cholesterol using rat and mice models. Evidence suggests that increased
degree of sulfation of ulvan may improve the antihyperlipidemic effect of the ulvan.
Oversulfated ulvan with up to 40.6% sulfation achieves a total cholesterol-lowering
effect of 44% compared to a 28% reduction measured for crude ulvan with a degree
of sulfation of 22.5% (Qi and Sheng 2015). Although it is obvious that the structure of
ulvans affects the mechanism and level of antihyperlipidemic effect, the relationship
between the chemical structure of ulvan and its antihyperlipidemic effect is unclear.
Possible mechanisms presented include gene regulatory effect and interference in
metabolism and biosynthesis (Ali and Agha 2009).
8.7.6 Antiviral Property
While ulvans alone show moderate to low antiviral properties, more important is their
potential impact on vaccination. Combination of ulvans with vaccination can improve
vaccination effectiveness by as much as 100% compared to when vaccination is done
alone (Song et al. 2016). This enhancement of vaccination is thought to be associated
with ulvan’s immunomodulatory activity. The impact of this could be need for less
quantities of vaccine in achieving the same level of vaccination, particularly in cases
when such vaccines are scarce.
Although some evidence points to improve antiviral activity with higher molecular
weight and higher degree of sulfation, the number of studies and species investigated
are not enough to make a conclusive relationship between the structural parameters
and the antiviral property of ulvan.
Antiviral activities have been investigated in ulvan from species of
U. clathrata, U. pertusa and U. compressa (Aguilar-Briseno et al. 2015; Song et al.
2016; Lopes et al. 2017) among others. These antiviral activities have been tested on
viruses such as influenza, herpes simplex, Japanese encephalitis, yellow fever, avian
influenza, dengue, Newcastle disease, dengue, West Nile and others (Kidgell et al.
2019). Thus far, much of the studies on the antiviral activity of ulvans have been
carried out in cell cultures and on mice models.
Starting in the early twenty-first century to date, commercial agal farming has gained
much attention from researchers, government and venture capitalists. The interest
in algae ranges from biofuel production from algae oil, bioethanol production from
the algal biomass, water remediation and extraction of the various bioactives for
pharmaceutical applications. All with the promise of largely profitable algae are
based on commercial venture.
Ulvan, being one of the polysaccharides produced by green algae, has potential
commercial application in food and pharmaceutics with the applications as discussed
in earlier sections within this chapter. However, commercial production of ulvan is
presently still at its infancy due to limitations in the commercially feasible extraction
methods to obtain uniform standard extracts.
Commercial ulvan production would benefit from the relatively high productivity
of the Ulva green algae, if well managed. One of such fast-growing species that has
high nutrient uptake rate is the U. prolifera (Zhang et al. 2019). Ulvan is the most
valuable polysaccharide of the Ulva green algae, and therefore, the main incentive
for commercial cultivation and processing of these green algae is either as food or
to extract ulvan. Presently, the use as food seems to gain precedence owing to it
being a less complex option in terms of finance and process technology. Consumed
as food, the Ulva already is known for improving gastrointestinal health and pre-
vention of certain chronic diseases (Kidgell et al. 2019). However, extraction of
the sulfated polysaccharide to which these health benefits are attributed results in
improved effectiveness and development of more effective derivatives. As processes
to extract purer and more uniform ulvan samples further develop this could encour-
age more commercialization of ulvan-based products for personal care and clinical
applications.
8.9 Conclusion
The ulvan producing green algae species are readily available and highly productive.
The cultivation of these species comes with the risk of propagating or contributing to
existing algae blooms. On the other hand, well-managed recovery of Ulva from the
wild or cultivation in well-managed systems can benefit from the rapid growth rate of
these species for ulvan production. Ulvans have the bioactive properties associated
186 8 Ulvans
with sulfated polysaccharides. In addition to the general sulfated polysaccharide

bioactivities, ulvans are particularly promising as immunomodulatory, antioxidant
and antihyperlipidemic agents. Much of the studies on ulvans bioactive properties
are at an early stage using cellular and animal test models. It is nonetheless important
to understand the potential application and the state of technology in the respective
applications. Such will be useful in advising further efforts in the use of ulvans.
References
Adrien A, Bonnet A, Dufour D, Baudouin S, Maugard T, Bridiau N (2017) Pilot production of

ulvans from Ulva sp. and their effects on hyaluronan and collagen production in cultured dermal
fibroblasts. Carbohyd Polym 157:1306–1314
Aguilar-Briseno JA, Cruz-Suarez LE, Sassi JF, Ricque-Marie D, Zapata-Benavides P, Gamboa EM,
Rodriguez-Padilla C, Trejo-Avila LM (2015) Sulphated polysaccharides from ulva clathrata and
cladosiphon okamuranus Seaweeds both inhibit viral attachment/entry and cell-cell fusion, in
NDV Infection. Marine Drugs 13(2):697–712
Ali MM, Agha FG (2009) Amelioration of streptozotocin-induced diabetes mellitus, oxidative stress
and dyslipidemia in rats by tomato extract lycopene. Scand J Clin Lab Inv 69:371–379
Berri M, Slugocki C, Olivier M, Helloin E, Jacques I, Salmon H, Demais H, Le Goff M, Nyvall P,
Collen (2016) Marine-sulfated polysaccharides extract of Ulva armoricana green algae exhibits
an antimicrobial activity and stimulates cytokine expression by intestinal epithelial cells. J Appl
Phycol 28:2999–3008
Berri M, Olivier M, Holbert S, Dupont J, Demais H, Le Goff M, Nyvall Collen P (2017) Ulvan from
Ulva armoricana (Chlorophyta) activates the P13K/Akt signalling pathway via TLR4 to induce
intestinal cytokine production. Algal Res 28:39–47
Bird RB, Stewart WE, Lightfoot EN (2005) Transport phenomena, 2nd edn. Wiley-India, New
Delhi, pp 270
Castro R, Piazzon MC, Zarra I, Leiro J, Noya M, Lamas J (2006) Stimulation of turbot phagocytes
by ulva rigida C. agardh polysaccharides. Aquaculture 254:9–20
Chen D, Wu XZ, Wen ZY (2008) Sulphated polysaccharides and immune response: promoters or
inhibitors. Panminerva Med 50:177–183
Cho M, Yang C, Kim SM, You S (2010) Molecular characterization and biological activities of water-
soluble sulfated polysaccharides from Enteromorpha prolifera. Food Sci Biotechnol 19:525–533
Fernández-Díaz C, Coste O, Malta EJ (2017) Polymer chitosan nanoparticles functionalized
with Ulva ohnoi extracts boost in vitro ulvan immunostimulant effect in Solea senegalensis
macrophages. Algal Res 26:135–142
Glasson CRK, Sims IM, Carnachan SM, de Nys R, Magnusson M (2017) A cascading biorefinery
process targeting sulfated polysaccharides (ulvan) from Ulva ohnoi. Algal Res 27:383–391
Hussein UK, Mahmoud HM, Farrag AG, Bishayee A (2015) Chemoprevention of
diethylnitrosamine-initiated and phenobarbital-promoted hepatocarcinogenesis in rats by sulfated
polysaccharides and aqueous extract of Ulva lactuca. Integr Cancer Ther 14:525–545
Jaswir I, Monsur HA (2011) Anti-inflammatory compounds of macro algae origin: a review. J Med
Plant Res 5:7146–7154
Jiao LL, Li X, Li TB, Jiang P, Zhang LX, Wu MJ, Zhang LP (2009) Characterization and anti-tumor
activity of alkali-extracted polysaccharide from Enteromorpha intestinalis. Int Immunopharmacol
9(3):324–329
Karnjanaprakorn S, Tabarsa M, Chou M, You SG (2012) Characterization and immunomodulatory
activities of sulfated polysaccharides from Capsosiphon fulvescens. Int J Biol Macromol 51:720–
729
References 187
Kidgell JT, Magnusson M, de Nys R, Glasson CRK (2019) Ulvan: a systematic review of extraction,
composition and function 39(101422):1–20
Kim JK, Cho MI, Karnjanaprakorn S, Shin IS, You SG (2011a) In vitro and in vivo immunomod-
ulatory activity of sulfated polysaccharides from Enteromorpha prolifera. Int J Biol Macromol
49:1051–1058
Kim S-K, Thomas NV, Li X (2011b) Anticancer compounds from marine macroalgae and their
application as medicinal foods. Adv Food Nutr Res 64:213–224
Lahaye M (1998) NMR spectroscopic characterisation of oligosaccharides from two Ulva rigida
ulvan samples (Ulvales, Chlorophyta) degraded by a lyase. Carbohydr Res 314:1–12
Lahaye M, Robic A (2007) Structure and functional properties of Ulvan, a polysaccharide from
green seaweeds. Biomacromol 8:1765–1774
Leiro JM, Castro R, Arranz JA, Lamas J (2007) Immunomodulating activities of acidic sulphated
polysaccharides obtained from the seaweed Ulva rigida C. agardh. Int Immunopharmacol 7:879–
888
Li J, Jiang F, Chi Z, Han D, Yu L, Liu C (2018a) Development of Enteromorpha prolifera
polysaccharide-based nanoparticles for delivery of curcumin to cancer cells. Int J Biol Macromol
112:413–421
Li W, Wang K, Jiang N, Liu X, Wan M, Chang X, Liu D, Qi H, Liu S (2018b) Antioxidant
and antihyperlipidemic activities of purified polysaccharides from Ulva pertusa. J Appl Phycol
30(4):2619–2627
Lopes N, Ray S, Espada SF, Bomfim WA, Ray B, Faccin-Galhardi LC, Linhares REC, Nozawa
C (2017) Green seaweed Enteromorpha compressa (Chlorophyta, Ulvaceae) derived sulphated
polysaccharides inhibit herpes simplex virus. Int J Biol Macromol 102:605–612
Michel G, Czjzek C (2014) Polysaccharide-degrading enzymes from marine bacteria. Marine
enzymes for biocatalysis: Sources, biocatalytic characteristics and bioprocesses of marine
enzymes. Woodhead Publishing Ser Biomed 5:429–464
Misurcova L, Skrivankova S, Samek D, Ambrozova J, Machu L (2012) Health benefits of algal
polysaccharides in human nutrition. Adv Food Nutr Res 66:75–145
Peasura N, Laohakunjit N, Kerdchoechuen O, Vongsawasdi P, Chao LK (2016) Assessment of
biochemical and immunomodulatory activity of sulphated polysaccharides from Ulva intestinalis.
Int J Biol Macromol 91:269–277
Pengzhan Y, Quanbin Z, Hong Z, Xizhen N, Zhien L (2004) Preparation of polysaccharides in
different molecular weights from Ulva pertusa Kjellman (Chlorophyta). Chin J Oceanol Limnol
22:381–385
Qi H, Sheng J (2015) The antihyperlipidemic mechanism of high sulfate content ulvan in rats. Mar
Drugs 13:3407–3421
Qi H, Zhao T, Zhang Q, Li Z, Zhao Z, Xing R (2005) Antioxidant activity of different molecu-
lar weight sulfated polysaccharides from Ulva pertusa Kjellman (Chlorophyta). J Appl Phycol
17(6):527–534
Qi H, Huang L, Liu X, Liu D, Zhang Q, Liu S (2012) Antihyperlipidemic activity of high sul-
fate content derivative of polysaccharide extracted from Ulva pertusa (Chlorophyta). Carbohydr
Polym 87:1637–1640
Qi XH, Mao WJ, Chen Y, Chen YL, Zhao CQ, Li N, Wang CY (2013) Chemical characteristics and
anticoagulant activities of two sulfated polysaccharides from Enteromorpha linza (Chlorophyta).
J Oceanogr Univ China 12:175–182
Reisky L, Stanetty C, Mihovilovic MD, Schweder T, Hehemann JH, Bornscheuer UT (2018) Bio-
chemical characterization of an ulvan lyase from the marine flavobacterium Formosa agariphila
KMM 3901T. Appl Microbiol Biotechnol 102(16):6987–6996
Rioux L, Turgeon SL (2015) Seaweed carbohydrates. In: Tiwari BK, Troy DJ (eds) Seaweed
sustainability: food and non-food application. Academic press, pp 141–192
Robic A, Rondeau-Mouro C, Sassi JF, Lerat Y, Lahaye M (2009) Structure and interactions of ulvan
in the cell wall of the marine green algae Ulva rotundata (Ulvales, Chlorophyceae). Carbohyd
Polym 77:206–216
188 8 Ulvans
Rybak A, Messyasz B, Laska R (2012) Freshwater Ulva (Chlorophyta) as a bioaccumulator of

selected heavy metals (Cd, Ni and Pb) and alkaline earth metals (Ca and Mg). Chemosphere
89(9):1066–1076
Scharnweber D, Hubner L, Rother S, Hempel U, Anderegg U, Samsonov SA, Pisabarro MT,
Hofbauer L, Schnabelrauch M, Franz S, Simon J, Hintze V (2015) Glycosaminoglycan deriva-
tives: promising candidates for the design of functional biomaterials. J Mater Sci Mater Med
26(9):232–249
Shao Q, He Y, Jiang, S (2011) Molecular dynamics simulation study of ion interactions with
zwitterions. J Phys Chem B 115:8358–8363
Song J, Chen X, Liu X, Zhang F, Hu L, Yue Y, Li K, Li P (2016) Characterization and comparison
of the structural features, immune-modulatory and anti-avian influenza virus activities conferred
by three algal sulfated polysaccharides. Mar Drugs 14(1):4
Tabarsa M, Han JH, Kim CY, You SG (2012) Molecular characteristics and immunomodulatory
activities of water-soluble sulfated polysaccharides from Ulva pertusa. J Med Food 15:135–144
Tabarsa M, You s, Dabaghian EH, Surayot U (2018) Water-soluble polysaccharides from Ulva
intestinalis: molecular properties, structural elucidation and immunomodulatory activities. J Food
Drug Anal 26:599–608
Varnier AL, Sanchez L, Vatsa P, Boudesocque L, Garcia-Brugger A, Rabenoelina F, Sorokin A,
Renault JH, Kauffmann S, Pugin A, Clement C, Baillieul F, Dorey S (2009) Bacterial rhamnolipids
are novel MAMPs conferring resistance to Botrytis cinerea in grapevine. Plant Cell Environ
32:178–193
Wang XX, Chen Y, Wang JJ, Liu ZX, Zhao SG (2014) Antitumor activity of sulfated polysaccha-
ride from Enteromorpha intestinalis targeted against hepatoma through mitochondrial pathway.
Tumor Biol 35:1641–1647
Yabe T, Ishii Y, Amano Y, Koga T, Hayashi S, Nohara S, Tatsumoto H (2009) Green tide formed
by free-floating Ulva spp. at Yatsu tidal flat, Japan. Limnology 10:239–245
Yang F, Fang X, Jiang W, Chen T (2017) Bioresponsive cancer-targeted polysaccharide nanosystem
to inhibit angiogenesis. Int J Nanomed 12:7419–7431
Young CS, Gobler CJ (2016) Ocean acidification accelerates the growth of two bloom-forming,
estuarine macroalgae. PLoS ONE 11(5):e0155152
Zhang Y, He P, Li H, Li G, Liu J, Jiao F, Zhang J, Huo Y, Shi X, Su R, Ye N, Liu D, Yu R, Wang
Z, Zhou M, Jiao N (2019) Ulva prolifera green-tide outbreaks and their environmental impact in
the Yellow Sea, China. Nat Sci Rev 0(0):1–14
Chapter 9
Laminarins
Abstract Laminarins are short-chain polysaccharides present in the cell wall of

brown algae. The simple structure of short-chain 1-3 linked β glucose unit that makes
it attractive in bioethanol production. Laminarin also has some bioactive properties
which are of research interests and potential commercial application. Extraction of
laminarin from brown algae can be carried out alongside the extraction of the other
polysaccharides in the brown algae cell wall, such that it contributes toward more
efficient utilization of the algal biomass. This chapter reviews the production process
and evaluates the environmental impacts of laminarin production, the commercial
potential in terms of availability of raw material as well as existing and potential appli-
cations. The occurrence of laminarin in nature and its chemistry are also presented
within the chapter.
Keywords Laminarins · Algae · Bioethanol · Aquatic · Biopolymer
9.1 Introduction
Laminarin is a type of sulfated polysaccharide found in brown algae. It serves as a

storage polysaccharide. Also referred to as laminarans, they are soluble polysaccha-
rides found in algae. The main repeating unit in laminarin structure is a 1-3 linked
β glucose unit. Some branching at the β 1-6 linkage is not uncommon in laminarin
chains, and other units such as mannitol and mannose have been reported to be present
along some laminarin polymer chains (Yvin et al. 1993). They are usually short-chain
polysaccharides with a degree of polymerization between 26 and 31 (Deville et al.
2007) or up to 60 repeating units (Yvin et al. 1993). Algae species such as Ecklonia
kurome, Laminaria japonica, Laminaria digitata and Eisenia bicyclis contain lami-
narin. They belong to the group of polysaccharides known as glucans, a diverse group
of polymers made up of glucose repeating units. These glucans vary in terms of the
orientation and linkages of these repeating units, which can be in forms such as alpha
or β, linear or branched, L or D forms (Caipang and Lazado 2015). Glucans also have
varying chain lengths from short-chain oligomers in tens of repeating units or less
to long-chain polymer structures with thousands of repeating units. Other examples
of glucans are cellulose and starch. Glucans exist in a variety of organisms such as

190 9 Laminarins
yeast, bacteria and algae. Laminarins can therefore be described as short-chain β

glucans with some degree of branching.
There is an increasing need for alternative sources of energy as the conventional
fossil fuels are being depleted exponentially as the population rises globally. While
crops such as corn and sugarcane are sources of carbohydrates which can be further
hydrolyzed and fermented into ethanol used as biofuel, the main issue with such
sources of alternative fuel is the competition with food, water and land for human
consumption and use for survival. Other crops which are sources of polymeric mate-
rials such as gum arabic have these similar issues. Laminarin is sourced from brown
algae, and this addresses half of the issues of other alternative fuels and biomaterials.
Laminarin is a glucan which can be hydrolyzed to glucose and other simple sugars
which can be fermented by microbes which in turn produce ethanol as a by-product.
As the world population grows, there is increasing demand to keep people alive
and disease-free. Furthermore, as disease resistance of bacteria to conventional drugs
increases, there is a need for more complex compounds for combating diseases and
improving immunity. For this reason, exploring naturally occurring polymers such
as laminarin for their bioactivities such as immunomodulatory and antimicrobial
activity becomes more important.
In this chapter, we explore another interesting biopolymer which can be obtained
from an aquatic organism, the brown algae, laminarin. The sources of laminarin,
chemical structure, applications and the environmental impacts of laminarin extrac-
tion process are all discussed with the reference to various recent studies from
different research studies around the world.
Glucans, in general, exist in yeast, bacteria, mushrooms, grains of cereals and algae
(Caipang and Lazado 2015). In brown algae, laminarin exists in the plastids where it
serves as a storage polysaccharide (Rioux and Turgeon 2015). Brown algae contain
~40% polysaccharides by dry weight (Deville et al. 2007), and one of these polysac-
charides could include laminarin. Brown algae which are sources of laminations are
referred to as Laminariales (Yvin et al. 1993). While alginate serves as a structural
polysaccharide in brown algae, laminarin serves as a storage polysaccharide (Konda
et al. 2015). The laminarin content could vary from 0 to 18% (Rioux and Turgeon
2015; Konda et al. 2015) in different species of brown algae, other studies report
higher laminarin content of up to 35% laminarin per dry mass of brown macroalgae
(Motone et al. 2016).
Storage polysaccharides generally have simpler structures such that they can eas-
ily be broken down to release energy and carbon when required by the organism.
Laminarin content in any given species varies in different growing seasons and growth
conditions. For instance, at low nitrate and nitrite content in the water, brown algae
proceed to produce laminarin as a stored carbon source; however, where nitrate and
nitrite are present, the brown algae directs its energy toward growth (Rioux and
Turgeon 2015). The brown algae species of Laminaria and Saccharina are the more
abundant and more common sources of Laminaria; nonetheless, laminarin is also
found in other brown algae such as Fucus, Undaria and Ascophyllum species. These
species also serve as sources for other algae polysaccharides such as alginates and
fucoidan. Laminaria varies in the chain length and level of branching depending on
different species and growth factors. These chemical structure variations also have
an effect on solubility (Kadam et al. 2015) and bioactivity (Liu et al. 2018) of the
specific laminarin.
Laminarin production is thought to be part of the brown algae coping strategy to
survive the winter season. It is produced at the end of the peak growth rate in spring,
such that laminarin serves as a storage reserve source of carbon during the winter
season (Misurcova et al. 2012). Water-soluble carbohydrates are a more accessible
source of energy storage compared to insoluble carbon sources such as cellulose
(Hildebrand et al. 2017). Laminarin is therefore one of the soluble polysaccharides
the brown algae uses as a source of carbon.
9.3 Chemistry of Laminarin
Laminarin is built up of β,1-3 linked glucan with a low level of branching at the 1-6
linkages. Some β,1-6 intrachain linkages may also be present (Kadam et al. 2015).
Laminarin has a similar structure to Lichenan found in moss and lentinan found in
mushrooms but differs in the degree of branching and nature of glycosidic linkages
(Ojima et al. 2018).
9.3.1 Repeating Units
Laminarin is composed of mostly neutral sugars with small amounts of uronic acid
(Misurcova et al. 2012). The composition of the sugars varies for different species. For
example, while Saccharina longicruris contains up to 99% neutral sugars which is the
highest composition of neutral sugars thus far observed in laminarins, Ascophyllum
nodosum has 89.6% neutral sugars and Fucus vesiculosus contains 84.1% neutral
sugars. Nonetheless, the neutral sugars are always significantly more abundant in
laminarin. The arrangements and conformation of the repeating units also vary for
different species. Figure 9.1 compares the structure of laminarin extracted from E.
bicyclis with that from L. digitata (Liu et al. 2018).
To understand the difference between the laminarin from the two different species
of brown algae, the laminarin forms the two sources that were hydrolyzed using
microorganism Coprinopsis cinerea which metabolizes the enzyme endo-β-1,3-
glucanase which breaks the β-1,3 glycosidic bond in laminarin. By analyzing the
residues from the hydrolysis of the different laminarin using high-performance anion
exchange, chromatography combined with mass spectrometry revealed the building
192 9 Laminarins
(a)
(b)
Fig. 9.1 Illustration of the secondary structure of laminarin extracted from two different species
of brown algae a E. bicyclis and b L. digitata, showing the arrangements of the repeating units
blocks of the laminarins. These building blocks are used to deduce the structure of
the different laminarins. This revealed a higher degree of branching in the E. bicyclis
which had a 25.99% branching while L. digitata had a 7.68% branching. This dif-
ference in the degree of branching was indicated by the presence of β-1,6 glycosidic
bonds in the residue which were not broken down by the glucanase whose activity
is specific to the β-1,3 glycosidic bond.
9.3.2 Molecular Weight
Laminarin is generally a low molecular weight polysaccharide with between 26 and

31 repeating units per strand (Deville et al. 2007). Some studies have reported degree
of polymerization of laminarin in different ranges of 31–40 while others report a
maximum degree of polymerization of 12 (Stone 2009). Laminarin extracted from
L. digitata is reported to have a degree of polymerization of ~25 (Hrmova and Fincher
2009). The molecular weight of laminarin extracted from a given species could also
vary with the stage of growth. For example, laminarin extracted from the mature
brown algae Fucus evanescens had a higher molecular weight than that extracted
when it was at an earlier growth stage (Misurcova et al. 2012). This must therefore
be considered when cultivating or harvesting brown algae for laminarin production.
Thus, far matured brown algae harvested in winter grown under reduced nitrate and
nitrite content seem to show the optimal composition of laminarin.
9.3 Chemistry of Laminarin 193
9.3.3 Solubility Branching
The degree of branching varies in different species; for instance, laminarin extracted
from the L. digitata brown algae has a degree of branching of about 12.5% while that
of E. bicyclis has a much higher degree of branching (Ojima et al. 2018). Laminarin
is generally referred to as a soluble sulphated polysaccharide. However, the solubility
of laminarin varies with structure and medium. Highly branched laminarin is more
readily soluble in cold or hot water while less branched laminarin will only dissolve
in hot water (Misurcova et al. 2012). This solubility variation also serves as a basis
for the extraction of laminarin where the degree of branching of isolated fraction can
be controlled by the temperature. The more branched can first be isolated at lower
temperature, and the more linear laminarin can be isolated at elevated temperature.
When dissolved in the aqueous solvent, laminarin, like other polymers, will result
in an increase in the viscosity of the solution. This increase in viscosity is related to
the molecular weight. As a low molecular weight polymer laminarin solutions will
not have the thickening effect of other higher molecular weight polysaccharides such
as carrageenan. However, the lower viscosity at higher solution concentration will
give less flow limitations during extraction and processing of liquid phases.
9.3.4 Methacrylated Laminarin
Modification of laminarin can be achieved by replacing some of the –OH groups

with methyl groups. These methacrylated laminarins are applied in tissue engineer-
ing for enhancing cell adhesion, for example (Martins et al. 2018; Wang et al. 2018).
Polysaccharides form hydrogels; however, they are limited in terms of maintaining
their mechanical properties when exposed to the physiological conditions in the body.
Although other materials exist for producing hydrogen with desirable mechanical
properties at physiological condition, biocompatibility and biodegradability limit the
application of such hydrogel-forming materials for biomedical applications. Modify-
ing laminarin with glycidyl methacrylate prior to cross-linking results in a hydrogel
with improved mechanical properties compared to unmodified laminarin hydrogels.
An example process for the production of methacrylated laminarin is as follows
(Wang et al. 2018): laminarin is dissolved in dimethyl sulfoxide (DMSO) in a mass (g)
of laminarin to volume (mL) of DMSO ratio of 1:10. This is carried out under nitrogen
atmosphere to prevent reaction with atmospheric air. This takes 1 h to dissolve and
is followed by the addition of 167 mg of 4-(N,N Dimethylamino) pyridine and then
dropwise addition of glycidyl methacrylate. The amount of glycidyl methacrylate
added determines the level of methacrylation. The methacrylation process takes 48 h
under room temperature. The reaction is terminated by bringing the pH to neutral
by adding hydrochloric acid. On completion, the methacrylated laminarin is then
separated from the reaction components to obtain purified methacrylated laminarin.
This purification can be achieved using dialysis for a period of 7 days. An example
194 9 Laminarins
dialysis condition for separation is dialysis against deionized water and a molecular
weight cutoff point of 2000 Da. Using this method, laminarin with a degree of
methacrylation of 5–10, 10–20 and 20–30% is obtained when 0.75, 1.5 and 3.0 mmol
of glycidyl methacrylate were used, respectively (Wang et al. 2018).
The methacrylated laminarin is then cross-linked to obtain hydrogels. The hydro-
gels obtained from methacrylated laminarin have a tensile strength of up to 0.63 MPa,
a compressive strength of 3.2 MPa, and will stretch by 650% of their original length
before breakage (Wang et al. 2018). Figure 9.2a shows the structure of laminarin,
and Fig. 9.2b shows the structure of methacrylated laminarin.
Fig. 9.2 Structure of

a laminarin and
b methacrylated laminarin
9.3 Chemistry of Laminarin 195
9.3.5 Biodegradation of Laminarin
Laminarin degrades into its repeating units which are glucose and other reducing
sugars such as mannose. Microbes which metabolize these laminarinase include C.
cinerea and Microbacterium oxydans (Kim et al. 2013). At an inoculum volume con-
centration of 20% (v/v), pH 6.0, and a temperature of 30 °C, a 10 g L−1 concentration
of laminarin substrate produced 5.11 g L−1 of reducing sugars and 2.88 g L−1 glu-
cose after a 6-day culture period using M. oxydans (Kim et al. 2013). The microbial
degradation of laminarin into simple sugars is important both for safe environmental
purpose and for commercialization of the sugars.
9.3.6 Chrysolaminarin
It is important to mention here, chrysolaminarin, also referred to as leucosin, another

biopolymer found in unicellular algae (Beattie et al. 1961). Diatoms are part of the
plankton and benthic algae which serve as a significant part of the aquatic food
chain. Diatoms have been used in art and are continuously studied for wide variety
of potential applications to advance human life such as use in photo-induced green
synthesis of nanoparticles (Chetia et al. 2017). Diatoms produce a polysaccharide
which is similar to laminarin known as chrysolaminarin. It is synthesized during the
day in the presence of light and used up at night to fuel heterotrophic metabolic activ-
ities (Caballero et al. 2016). The similarity in these two polysaccharides lies in the
fact that they are both made up of β,1-3 glucose and both water-soluble polysaccha-
rides. Chrysolaminarin contains up to 99.5% glucose repeating units while laminarin
has other units, mainly mannitol within the structure. There are no mannitol units
present in chrysolaminarin (Beattie et al. 1961). Chrysolaminarin is a linear poly-
mer and has a more crystalline secondary structure while laminarin could have low
or high level of branching. While laminarin serves as the storage polysaccharides
for the Phaeophyceae, chrysolaminarin serves as the storage polysaccharides for the
diatoms. Chrysolaminarin makes up between 12 and 33% dry weight of the diatom.
This could be up to 80% when grown at optimal conditions for chrysolaminarin
production (Hildebrand et al. 2017). Due to their simpler structure, other than the
potential bioactivities, chrysolaminarin has a potential to serve as a source of glucose
for ethanol production.
Laminarin can be sourced from a variety of brown algae which grow in different
marine areas. According to FAO in 2016, 31.2 million tonnes of algae are pro-
duced in 2016 (FAO 2018). Of this total, 34,000 tonnes of brown algae live weight
196 9 Laminarins
was cultivated in aquaculture globally. The live weight contains around 45% mois-
ture prior to natural drying in storage. After drying at room temperature, the algae
used as feedstock are generally around 25% moisture content (Konda et al. 2015).
This figure excluded L. japonica species, and this is one of the main Phaeophyceae
which acts as a major source of laminarin. Production of L. japonica in 2016 was
8,219,000 tonnes cultivated using aquaculture. China is the lead producers of algae
followed by Indonesia producing live weight of 14,387,000 and 11,631,000 tonnes,
respectively, in 2016. The figures from 2005 to 2016 show a continuous rise in the
production algae from different countries, with some few exceptional cases where a
drop in production is experienced as a result of isolated cases. For example, a drop
in production of Undaria pinnatifida species is reported from 2014 to 2015 to 2016,
where the cultivated amount was 2,359,000, 2,297,000 and 22,070,000, respectively.
Algae cultivated using aquaculture is given more attention here since 96.5% of the
global production of algae is from aquaculture. Although a small amount of algae
obtained from wild stock and some countries have implemented policies to conserve
the population of certain species of algae in the areas where they grow naturally
and contribute significantly to the aquatic ecosystem, aquaculture remains the main
method of cultivating algae for large-scale production for either direct consumption
as food or for extraction of biocompounds.
Although alginate is the most common biopolymer extracted from brown algae,
processes have been presented which allow the extraction of laminarin, fucoidan and
alginate from brown algae in a single process. Such developments make it possible
to optimize the utilization of the brown algae resource and minimize the component
which is discarded as waste. Furthermore, since the species of brown algae required
for alginate production are the same which are used for laminarin production, the
availability of brown algae for laminarin production is further maintained as it is a
raw material for multiple purposes.
9.5 Extraction of Laminarin
As a soluble polysaccharide, laminarin can be extracted by dissolving out in a suitable

solvent at specific conditions within which it is soluble. Laminarin is extracted from
the thallus of the brown algae. Extraction method commonly used for laminarin is
summarized in Fig. 9.3.
The highly branched laminarins are soluble in both hot water and cold water, while
the laminarins with less branching are only soluble in hot water. This difference in
solubility can be used as a basis for obtaining laminarin of desired level of branching.
Extraction is generally achieved by treatment with hot or cold acids or calcium
chloride. Acids commonly used are hydrochloric acid or sulfuric acid. Laminarin
is then precipitated with alcohol. The extract can then be redispersed in a suitable
solvent and then purified. Most common purification presented in many research
studies is the ultrafiltration and dialysis.
9.5 Extraction of Laminarin 197
Pulverization
Cultivation Washing of
biomass
<1mm particle
diameter
Extraction
Precipitation & in Water 2% CaCl
Separation
Ion
Ultrafiltration / exchange Lyophilization
dialysis
Packaging
Fig. 9.3 Flowchart summarizing laminarin production stages
Acid-based extraction is more commonly used. The process involves the treatment
of the brown algae biomass with acidic solution, and this could be hydrochloric
acid or sulfuric acid at a 0.1 M concentration at high temperature during which the
laminarin is released in the water (Kadam et al. 2015). This usually contains a mixture
of laminarin and other soluble polysaccharides. The laminarin is then precipitated out
with alcohol. Further, separation is required to obtain purified form of laminarin, and
this is done using methods such as ultrafiltration, gel permeation chromatography or
dialysis. Laminanrin’s lower molecular weight is used as a basis to separate it from
higher molecular weight polysaccharides which also dissolve in the same condition.
In a method presented for extraction of laminarin from Laminaria saccharina
(Yvin et al. 1993), 300 g of the algae biomass was ground in a cryogenic grinder
at −40 °C. This ground the biomass into smaller particle sizes with 50–100 μm
average diameter. This is then followed by treatment in 900 ml of sulfuric acid at a
0.3% concentration at a temperature of 80 °C for a duration of 1 h under continuous
stirring. The mass of algae used here is on wet basis with a solid content of 10–12%. In
the completion of the extraction, the system was neutralized by adding an alkali. This
is then followed by treatment with polyvinylpyrrolidone at a 1 g/ml for a duration
of 2 h. The mixture is separated using vacuum filtration, and further, purification is
achieved using ultrafiltration and dialysis. The final purified product is lyophilized
to form a dry powder. Using this process, 7 g of dry powder is obtained from 300 g
of the feedstock.
198 9 Laminarins
9.5.2 Calcium Chloride Extraction
In an another approach to the extraction of laminarin from brown algae, the alginate
can first be extracted and precipitated out followed by the extraction of laminarin. This
method as applied to the extraction of laminarin from the brown algae A. nodosum
is as follows (Yvin et al. 1993): The fresh algae biomass was ground using mill to
smaller particle size <1 mm. This gave a suspension with a 10–12% solid content.
The groundmass is then added to 2% aqueous solution of calcium chloride. For this,
300 g of the ground algae biomass (10–12% solid content) was added to 900 ml of
the calcium chloride solution. This addition of calcium chloride allowed the alginate
to precipitate. It is then required to separate the laminarin from the solid mass. This is
achieved by extraction in hot water at 60 °C since the laminarin is soluble in hot water.
The extraction is allowed a duration of 7 h under continuous stirring. After this period,
the laminarin is dissolved in the hot water, and this can then be separated by filtration.
Further, purification is achieved using ultrafiltration and dialysis. The residue from
the first extraction can then be taken through another extraction process to remove
residual laminarin. This repeated extraction is necessary since the solvent overtime
becomes saturated; a fresh solvent is then required to increase the concentration
gradient for further extraction.
Laminarin can also be extracted from brown algae through the use of enzymes.
These enzymes can either be enzymes which digest the non-polysaccharide com-
ponents of the brown algae biomass leaving behind laminarin in a mixture with
other polysaccharides. This mixture of polysaccharides can then be separated by
hydrothermal treatment, precipitation and/or molecular separation methods. Another
approach involves the partial degradation of the laminarin into even shorter-chain
laminarin oligosaccharides using laminarin degrading enzymes (Ojima et al. 2018).
This form of extraction is desirable where laminarin oligosaccharides are desired
for their bioactive properties. For example, laminarin oligosaccharides extracted
from the brown algae E. bicyclis showed immunomodulatory effects by promoting
the immune response of monocytes (Ojima et al. 2018). There is the β-1,3 glu-
canase which degrades glucans by breaking some specific forms of β-1,3-linkages.
The form of glucanase enzymes which are specific to laminarins is generally called
laminarinase. The different glucanases differ in their mode of actions and are bond-
specific. The endo-1,3;1,4-β-glucanase cleaves the β-1,3-glycosidic bond as well as
the β-1,4-linkage while another type of glucanase the lichenase splits only specific
terminal β-1,4-linkage and glucan endo-1,3-β-d-glucosidase would break the β-1,3-
linkage but only when at least two adjacent β-1,3-linkages are present (Ojima et al.
2018). These enzymes can be obtained from sources such as barley and baker’s yeast
(Hrmova and Fincher 2009).
9.5 Extraction of Laminarin 199
Enzyme extraction often results in the production of oligolaminarins and some

sugars. Enzyme extraction is carried out at temperatures of ~30 °C. The system is
then heated to 100 °C upon completion in order to deactivate the enzymes (Ojima
et al. 2018).
9.5.4 Combined Extraction of Laminarin and Other Brown

Algae Polymers
For optimal utilization of the aquatic resources such as brown algae for biopolymer
extraction, sustainable extraction methods, whereby extraction of one polymer does
not completely destroy or inhibit the extraction of the other useful polymers within
the brown algae, are very important.
The conventional approach to the extraction of biopolymers from algae is to
extract a single product by either dissolving away all the biopolymers from the fiber
or isolating the required biopolymer by methods such as precipitation in alcohol.
However, in an approach to optimize the use of the biomass, some research efforts
have been directed at combined extraction processes which allow the extraction of
multiple biopolymers in a single process from the same algae biomass. Higher com-
bined yield of polysaccharides can be achieved by combined extraction of laminarin,
fucoidan and alginate from brown algae; however, the molecular weights and other
properties such as viscosity and solubility differ from the conventional single extrac-
tion method (Abraham et al. 2019). Combined extraction of laminarin fucoidan and
alginate has been carried out of species such as the Durvillaea potatorum obtaining a
combined yield of 43.57% of polysaccharides compared to a single yield of 38.97%
of alginate. The combined extraction process required a modified biorefinery pro-
cess which could achieve the extraction of all three polysaccharides from a single
source where the extraction of one component does not hinder or destroy the other
products. The scale-up of such process in a large-scale continuous process requires
further modification to the entire production process. The cost of the additional unit
operations to extract multiple biopolymers should also be weighed against the value
of the biopolymers being extracted.
Here we look at some key environmental issues associated with laminarin extraction
process from the cultivation of the brown algae to the final product leaving the factory
gate.
200 9 Laminarins
9.6.1 Cultivation of Brown Algae
Brown algae have been harvested for centuries in different parts of the world where it
has been used in food and agriculture and other uses. Among the brown algae, Lam-
inaria has especially higher ability to extract minerals from polluted water and use
these as nutrients (Kim and Bhatnagar 2011). One main advantage of brown algae,
and algae in general, as a feedstock in biorefinery for the production of compounds
such as laminarin has the advantage of not requiring large land area for cultivation.
Algae also have a much higher productivity than land plants, and they can grow up to
83,000,000 tonnes per hectare annually, compared to land plants such as sugarcane
whose rate of growth are around 3–30,000,000 tonnes per hectare annually (Konda
et al. 2015). Therefore, the cultivation of brown algae is less demanding on the envi-
ronment than traditional crops such as corn and cotton which also have commercial
applications.
One of the advantages of laminarin in terms of the extraction process is that highly
branched laminarin can be obtained at low temperature in cold water extraction pro-
cess. Even the hot water extraction process required for the less branched laminarins
can be carried out at temperatures as low as 60 °C. Compared with other extraction
process where temperatures of ~90 °C are required.
When the algae needs to be cultivated in a closed system to maintain sterility,
artificial growth light and temperature and humidity control all required additional
energy consumption unlike where the brown algae is harvested from the wild. Where
laminarin is required for its bioactive properties such closed growth systems might
be required to meet regulatory standards.
The relatively lower viscosity of laminarin compared to other higher molecular

weight polymers such as alginate has the advantage of requiring less water for wash-
ing and separation. Unlike in alginate extraction, for example, where a large amount
of water is added to reduce the viscosity in order to allow flow in filtration. The
process of cultivating brown algae for laminarin production does not use water in
the same way as used in the cultivation of land plants. The water used in aquaculture
for algae is often recycled, and in some cases, the algae are grown in a symbiotic
relationship with fish. Table 9.1 only considers the water used in the extraction pro-
cess. This water is acidified with the addition of sulfuric acid, and it then needs to be
Table 9.1 Summary of

Consumption Amount
consumptions in laminarin
extraction process Brown algae 42 g per gram of laminarin powder
Energy >80 °C for 1 h
Water >170 mL per gram of laminarin
powder
Acid 129 mL at 3% w/v
Polyvinylpyrrolidone 1 g/mL
neutralized and treated before discharge or reuse. This water treatment process even
when recycled requires additional energy usage.
9.6.4 CO2 Emission
While the process of cultivation of brown algae is considered as carbon negative,

i.e., results in removal of CO2 for the environment, on the other hand, the process
of extraction results in the release of CO2 . These two processes are able to balance
each other out, particularly if the process of extraction is carried out in an optimally
sustainable manner such that the production of laminarin can be considered a carbon-
neutral process.
9.6.5 Acid Consumption
When compared to a process like extraction of chitin from mushrooms requires up to

2 M concentration of hydrochloric acid while extraction of laminarin requires a lower
acid concentration of 0.1 M. Therefore, laminarin extraction is a milder process on
the environment.
9.6.6 Solid Waste Generation
The brown algae biomass comprises of other compounds mainly alginate (32%),
mannitol (18%), little or no proteins, ash and salts (35%) (Konda et al. 2015). In the
process of extraction of laminarin, these other components are separated and either
go into further processing or are discarded and processed as waste. Brown algae
are particularly attractive for extraction of biopolymers as they have a lower lignin
content which makes the extraction process milder compared to biomass with higher
lignin content where more aggressive delignification processes are required. The
solid residue generated can be burnt to produce steam for heating in other processes
202 9 Laminarins
such as heating and powering turbines for the generation of electricity. This way the
waste produced is utilized in powering the process.
Laminarin is considered as the secondary biopolymer of brown algae where algi-
nate is the primary biopolymer with well-established commercial application. Extrac-
tion of laminarin alongside alginate and fucoidan in a coextraction process will allow
optimal utilization of the brown algae biomass according to reported studies. The
very important environmental impact of these polymers lies in their serving as alter-
natives to the non-renewable sourced polymers in similar applications. However, the
limitations lie in the cost and materials for production as well as high-cost additional
stages such as ultrafiltration. The unique bioactive properties of laminarin could boost
its commercial viability as a high-value product required in lower quantity. Table 9.1
gives a summary of typical consumptions in a laminarin extraction process.
9.7 Applications
Laminarin has been explored for a range of applications which includes the produc-
tion of bioethanol, animal feed and in food processing. The commercial value of
biopolymers is significantly increased when they possess bioactive properties. This
way they can be sold at low volume and high price. This is more profitable compared
to other non-bioactive applications where they are used as feedstock and required
in much larger amounts and at a lower cost. Brown algae as a whole have been
consumed for centuries in the Asian countries such as Japan, China and Korea as
food or medicinal preparations. The algae in general were believed to have several
health benefits. The goal of modern medicine is the isolation and purification of
the compounds within the algae which have these bioactive properties for optimal
effectiveness. Furthermore, separation of these bioactive compounds from the other
component of the algae allows optimal utilization of all the parts of the algae for
multiple potentially high-value products. For example, extracts of E. bicyclis have
anticancer, antiallergic and also have some benefits to the cardiovascular system, in
particular prevention of undesirable blood clot in the blood vessels through inhibit-
ing pathways which lead to platelet formation and thrombosis (Irfan et al. 2018). As
these extracts contain a mixture of compounds, separation of these combines could
identify what compounds have specific effects and contribute to optimal use of this
aquatic resource.
9.7.1 Bioethanol Production
The ability to produce ethanol from laminarin is significant in the sense that it provides
an alternative non-food source for ethanol production. Compared to other glucose
sources such as corn and sugarcane which are used more commonly for commercial
ethanol production, laminarin from algae source does not compete with staple foods
and does not compete with land. Although several species of algae are used as food
in many Asian countries, in many other parts of the world they are not consumed as
food and therefore not likely to pose a threat to food security.
The limitation to the use of polysaccharides such as cellulose for ethanol produc-
tion is that there are limited microbes which can break down cellulose into glucose.
Microbes such as Saccharomyces cerevisiae can break down sucrose, a much simpler
structure into glucose and metabolize this and produce ethanol as a by-product in a
single process. Inclusion of an additional stage in the production process for breaking
down the feedstock into glucose or simple sugars will significantly add to the cost
of production.
There are microbes which are capable of fermenting laminarin to produce ethanol
such as Pichia angophorae (Horn et al. 2000) yeast S. cerevisiae and bacterium Sac-
charophagus degradans (Motone et al. 2016). In an age, where fossil fuel depletion
is a major concern, new alternative ways to produce fuel are vital to the survival
of the modern world which is heavily dependent on fuel consumption. Production
of bioethanol from biomass depends on a carbon source and a microbe which syn-
thesizes the enzyme to break down the carbon source for its metabolism and in
the process produces ethanol as a by-product. Laminarinase enzyme extracted from
organisms such as the baker’s yeast S. cerevisiae and bacterium S. degradans can
degrade laminarin into its sugar units. In a coculture of these two organisms with a
medium containing 20 g/L of laminarin, 5.2 g/L of ethanol is obtainable (Motone
et al. 2016). In a separate study in a batch fermentation process, a yield of 0.43 g
ethanol per g substrate was achieved at a pH of 4.5 and oxygen level of 5.8 mmol L−1
h−1 (Horn et al. 2000).
One of the limitations of bioethanol production from laminarin is the relatively
lower yield and preference of the microbe for batch systems (Horn et al. 2000). For
large scale and lower cost of production, scaled up continuous process is required by
the biorefineries. For the successful production of bioethanol from laminarin, the cost
of extraction of laminarin from brown algae needs to be minimized as bioethanol
is a high volume and low-cost commodity and feedstock production needs to be
as cheap as possible. Compared to, for example, sugar extraction from sugarcane
which mainly requires mechanical processes and thus less cost of chemicals for
extraction, extraction of laminarin requires additional cost of chemicals such as
sodium hydroxide or calcium carbonate for extraction. Additional unit operations
such as ultrafiltration also add to the cost of laminarin as a feedstock for ethanol
production. Therefore, although a one-step fermentation process to obtain ethanol
from laminarin is possible, the cost implications presently limit the commercial
application.
204 9 Laminarins
9.7.2 Gut Health
Laminarin has potential to serve as a dietary fiber in human nutrition with some
bioactive benefits. The possibility of laminarin acting in the modulation of the com-
position and function of the gut is one which has been investigated by researchers in
a number of reported in vitro and in vivo studies. Although they are indigestible by
any of the known digestive enzymes of the human body and cannot be broken down
into smaller units for metabolism, their passage through the alimentary canal can
potentially pose some health benefits. In an in vitro study which modeled the human
digestive system based on an anaerobic batch fermenter system which replicates the
microflora and biochemical composition of the human gut system, the variation in
the composition with the inclusion of laminarin was investigated. The study revealed
that laminarin is digested by the bacteria in the human gut by >90%. In further stud-
ies on rats, it was shown that laminarin affected the mucus content which in turn
affects bacterial attachment to the walls of the intestine. Laminarin also affects the
pH and biochemical composition of the gut, particularly the short-chain fatty acid
content (Deville et al. 2007). These parameters, which can be affected by laminarin,
contribute to the metabolism in the intestine. Laminarin has a pH lowering effect in
the intestine, and this lower pH is beneficial to metabolism, it promotes beneficial
bacterial growth, enhances the absorption of minerals and has been associated with
cancer prevention.
One of the attractions of dietary fibers is the possibilities of having probiotic
effects, that is, enhancing the growth of beneficial bacteria in the human digestive
system, mainly Bifidobacterium and Lactobacillus (Deville et al. 2007). There are
conflicting reports as to whether laminarin has prebiotic effects or not (Michel et al.
1999; Deville et al. 2007).
The studies on the effect of laminarin on gut health have advanced to in vivo test on
mammals such as piglets. For example, studies on piglets following weaning stage of
growth showed that laminarin improved their resistance to the pathogenic microbes,
Salmonella typhimurium (Bouwhuis et al. 2017). This can be achieved either by
administering laminarin directly to the piglet or to the show during weaning and
then transferred to the piglet in the breast milk. This was achieved with a dosage
of 300 ppm laminarin per tonne of feed; therefore, this improvement in gut health
can be achieved with a relatively low amount of laminarin. The improvement in gut
health is due to a combination of factors; the laminarin either induced the expression
of genes which gave the piglet improved defense against the pathogen or the direct
antimicrobial effect of laminarin fed in the diet on S. typhimurium in the gut or it
improved the architecture of the gut, making it more effective at eliminating the
pathogenic microbe.
9.7.3 Anticancer Activity
There have been several studies which investigate the anticancer effect of laminarin
and the evidences presented show that laminarin has promising use as a functional
food with anticancer properties. Cancer prevention and treatment occur by many
routes; anticancer drugs and compounds can act by either inducing cell death or
enhancing the body’s own defense against the cancer cells. The anticancer and anti-
tumor bioactivities of laminarin have been demonstrated in human melanoma cells,
colon cancer cells and adenocarcinoma cells of rats among others. For example,
apoptosis (programmed cell death) of LoVo human metastatic colon cells has been
activated by laminarin, and laminarin hydrolysates have shown ability to inhibit
growth of some human melanoma and colon cancer cells (Ji and Ji 2014). In other
studies, laminarin has been shown to prevent the spreading of cancer cells beyond
the point of origin to other tissue (metastasis) by inhibiting the cancer cells’ ability
to disrupt the extracellular matrix, hence preventing invasion (Deville et al. 2004). It
should be noted that the studies mentioned here and much of the existing studies on
the anticancer effects of laminarin have only been carried out at the cellular level and
on laboratory rats. The findings point to some potential of laminarin as a functional
dietary fiber which the intake could yield these anticancer effect. This is a long way
from drug development where the route by which the active compound gets to the
cell in a more complex organism, such as mammals and humans, introduces several
challenges.
9.7.4 Immunomodulatory Effect
Disease resistance is important in animal farming. Spreading of disease could result

in devastating financial loss of livestock and could also reduce the growth rate and
hence the yield of the animal. Low-cost disease management through boosting the
immunity of animals is an effective means of disease management and prevention.
Laminarin has a low molecular weight glucan that has been reported to have immune-
modulatory effect in grouper fish (Yin et al. 2014). Laminarin immunological effect
is also dose-dependent; fish fed with a concentration of laminarin of 0.5, 1 and
1.5% over a period of 48 days. Fish fed with a diet-containing laminarin showed an
increased specific growth rate, and they were able to more effectively utilize nutrients.
Furthermore, an increase in the number of immune response cells were observed in
fish fed with laminarin compared to those with laminarin-free diets. Boosting immune
response in fish through the nutrient present in the feed adds economic value to the
fish feed and also presents the fish farmer with lower risk of fish developing diseases
and improved yield.
Laminarin has been explored extensively for its effect on the growth and develop-
ment of pigs at different developmental stages, from development of young piglets
206 9 Laminarins
to the maternal health of the sow. Laminarin has even been shown to improve the
development of porcine embryo (Jiang et al. 2018).
Other species have also shown improved growth rate in response to laminarin
administration. These include young chicks and piglets. For example in post-hatch
chicks, it is important that they have a moderate growth rate and attain sufficient
immunity to survive the early life stage as lots of the livestock could be lost during this
fragile stage. It is possible to achieve this required immunity through diet-containing
immune modulatory agents. Laminarin tested on such post-hatchery chicks have
shown that chicks could grow faster and develop intestine with more efficient nutri-
ent uptake and immune response (Sweeney et al. 2017). However, the immunomod-
ulatory effect of laminarin seems to be limited as it did not improve resistance to
Campylobacter jejuni, a major pathogen. Therefore, while laminarin might provide
some immunomodulatory effect, it does not provide complete immunity against
pathogens.
The immunomodulatory bioactivity of laminarin can be partly attributed to the
ability of laminarin like other algae polysaccharides to bind with certain proteins
called the lipopolysaccharide and beta-1,3-glucan-binding protein (LGBP). These
proteins are able to recognize specific polymer chain patterns of these algae polysac-
charides such as laminarin. These patterns are referred to as the pathogen-associated
molecular patterns (PAMPs). It is deduced that the LGBPs bind with laminarins to
form complexes which then aid in the ability to attach to and destroy pathogens.
This protein binding ability of laminarin has been investigated in shrimp, resulting
in the activation of the prophenoloxidase system which boosts the shrimp’s innate
immunity (Chen et al. 2016). Most of the studies on laminarin’s immunomodula-
tory bioactivity are toward its use as a functional food for optimizing productivity in
farmed animals.
9.7.5 Plant Growth Agent
While the use of fertilizers and manure provides nutrients to support plant growth,
the growth of the plant also depends on the ability of the plant to effectively take up
and utilize the nutrients, water and sunlight. This is down to the intrinsic functioning
of the plant. This intrinsic growth factor can be enhanced by using compounds which
can interact with the plant’s cellular function and enhance its metabolic and hence
growth process as well as defense against pathogenes. Laminarin has been shown to
accelerate the process of seed germination through interfering with pathways which
lead to the formation of alpha-amylase (Yvin et al. 1993). Laminarin is thought
to do this by acting as an elicitor: a compound which induces particular responses
in an organism which enables its ability to survive its environment. This occurs by
difference means such as by triggering the production of certain enzymes or initiating
or inhibiting certain biochemical processes. Laminarin can be used in powdered or
liquid form either mixed with additives or dispersed in water and applied to the
plant. Concentrations of 0.005–100 g per liter have been presented for plants such
as tomatoes, potatoes and carrots in a patented formulation (Yvin et al. 1993)
The use of laminarin to accelerate seed germination and plant growth has signifi-
cant economic benefits. Plants which germinate faster would reduce the time required
to be spent in the nursery thereby reducing the amount of intensive care required for
the plants. It also allows for optimal utilization of nutrients and fertilizers. The use of
laminarin which is from a natural source could also be used as a means to accelerate
growth in organic farming, depending on the regulation of the region.
9.7.6 Food Processing and Preservation
The shelf life of food products plays a major role in their marketing and distribution
as well as the use. Fresh meat products are required to retain their organoleptic
properties such as taste and texture throughout the period of storage and consumption.
The organoleptic properties of these foods are affected by the chemical and microbial
composition. It has been observed that the food fed to the animals during rearing
significantly affects the organoleptic properties of the meat after slaughter. One of
the main factors which lead to loss of organoleptic properties of meat is the lipid
content. Before the action of spoilage microbes the loss of quality of the meat occurs
due to the deterioration of other food quality index. The oxidation of the lipid within
the meat leads to rancidity. Furthermore, as consumers are becoming increasingly
health conscious, meat products with lower saturated fats are increasing in demand.
In one study, it is reported that pigs fed with a diet including 450 mg of l per kg of
feed laminarin for a period of 6 weeks have lower saturated fat content and lower
lipid oxidation in storage (Moroney et al. 2015). Although the reduced lipid oxidation
from laminarin feed in the pork did not occur in cooked minced pork, cooked minced
pork from pigs fed with a diet-including laminarin showed reduced saturated fatty
acid content.
9.7.7 Antimicrobial Activity
Laminarin extracted from E. bicyclis and L. digitata shows some antimicrobial activ-
ity against gram-positive and gram-negative bacteria. E. bicyclis with a higher degree
of branching showed a stronger antimicrobial activity against gram-negative bacte-
ria. There are results indicating that a higher degree of branching could support the
antimicrobial activity of laminarins (Liu et al. 2018). However, a weaker activity
was observed against gram-negative bacteria. Antimicrobial activity of laminarin is
based on early stage studies. It is mentioned here as a potential future application of
laminarin.
Other bioactive properties of laminarin include anticoagulant, hypocholes-
terolemia. However, although studies in the late 50s found that laminarin, when
208 9 Laminarins
administered intravenously, has anticoagulant properties comparable to that of hep-

arin, it was also extremely toxic to the rabbits’ tested on at the pharmacokinetically
relevant doses (Thorpe and Adams 1957). Consequently, no further studies have been
carried out on the anticoagulant application of laminarin. Oral administration of lam-
inarin for food and other bioactive properties do not pose the toxic effect as when
delivered directly to the blood intravenously. The first-pass metabolism through the
alimentary canal abates this.
Macroalgae cost contributes to about 24% of the total annual running cost of
macroalgae-based biochemicals refinery for bioethanol and biochemical production
(Konda et al. 2015). The rest of the annual running cost goes into the purchase of
consumables, facilities, labor and utilities. A megatone of brown algae is estimated
to be priced at 100 USD, if laminarin is processed into sugars, the minimum selling
price for the sugars produced from a corefinery of laminarin alongside alginate would
be 21–47 cents per pound. If fermented to ethanol, the minimum selling price for the
ethanol will be 3.6–8.5 USD per gallon (Konda et al. 2015). This costs much higher
than fossil fuels or ethanol from conventional process. The commercial application
of laminarin is therefore of more economic relevance in the bioactive applications
where the high value can compensate for the relatively higher production cost.
As research interest increases in the numerous benefits of laminarin as a potential
bioactive compound, so does demand for analytical grade laminarin which is used
to study the structure, properties, develop new applications and better understanding
of the structure property relations of laminarin. Companies such as Sigma-Aldrich,
Sigma Chimie SARL and Seikagaku Kogyo supply laminarins of different specifi-
cations. These have been used in various studies on laminarins (e.g., Ojima et al.
2018; Yvin et al. 1993). These supply companies therefore play a significant role
in the advancement of the research in laminarin through ensuring their availability.
In studies focused on the structure and applications of laminarin, several hours and
resources could be saved in repeated extractions by buying already prepared samples.
9.9 Conclusion
Laminarin, another biopolymer obtained from the brown algae, has several bioac-
tive properties which makes it a valuable biopolymer in the food and animal feed
industry. Its lower molecular weight makes separation by ultrafiltration—an useful
method in its extraction. It can be extracted either as a sole product or alongside
other polysaccharides in the brown algae such as alginate and fucoidan. It seems
more attention and has been placed on the application of laminarin in animal feed
as a feasible commercial application in the near future. Application for human use
9.9 Conclusion 209
includes food preservation and in the production of bioethanol. The environmental

impact of the extraction process lies in the consumption of fossil energy to power the
processes, the land-use change and CO2 emissions in the transportation and pack-
aging processes. This is weighed against the CO2 consumed during the process of
cultivation of the algae, the use of algae to clean polluted water and the potential for
use in the replacement of fossil-based fuel if the process of bioethanol production
from laminarin is commercialized.
References
Abraham RE, Su P, Puri M, Raston CL, Zhang W (2019) Optimization of biorefinery of alginate,
fucoidan and laminarin from brown seaweed Durvillaea potatorum. Algal Res 38. Article 101389
Beattie A, Hirst EL, Percival E (1961) Studies on the metabolism of the Chrysophyceae. Compara-
tive structural investigations on leucosin (chrysolaminarin) separated from diatoms and laminarin
from brown algae. Biochem J 79:531–537
Bouwhuis MA, Sweeney T, Mukhopadhyay A, McDonnell MJ, O’Doherty JV (2017) Maternal
laminarin supplementation decreases Salmonella typhimurium shedding and intestinal health in
piglets following an experimental challenge with S. typhimurium post-weaning. Anim Feed Sci
Technol 223:156–168
Caballero MA, Jallet D, Shi L, Rithner C, Zhang Y, Peers G (2016) Quantification of chrysolaminarin
from the model diatom Phaeodactylum tricornutum. Algal Res 20:180–188
Caipang CMA, Lazado CC (2015) Nutritional impacts on fish mucosa: immunostimulants, pre- and
probiotics. In: Beck BH, Peatman E (eds) Mucosal health and aquaculture, pp 211–272
Chen Y, Chen J, Kuo Y, Lin Y, Huang C (2016) Lipopolysaccharide and β-1,3-glucan-binding
protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in
white shrimp Litopenaeus vannamei. Dev Comp Immunol 55:144–151
Chetia L, Kalita D, Ahmed GA (2017) Synthesis of Ag nanoparticles using diatom cells for ammonia
sensing. Sens Bio-Sensing Res 16:55–61
Deville C, Damas J, Forget P, Dandrifosse G, Peulen O (2004) Laminarin in the dietary fibre concept.
J Sci Food Agric 84:1030–1038
Deville C, Gharbi M, Dandrifosse G, Peulen O (2007) Study on the effects of laminarin, a
polysaccharide from seaweed, on gut characteristics. J Sci Food Agric 87:1717–1725
FAO (2018) The state of world fisheries and aquaculture 2018: meeting the sustainable development
goals. Rome. Licence: CC BY-NC-SA 3.0 IGO. ISBN 978-92-5-130562-1
Hildebrand M, Manandhar-Shrestha MK, Abbriano R (2017) Effects of chrysolaminarin syn-
thase knockdown in the diatom Thalassiosira pseudonana: implications of reduced carbohydrate
storage relative to green algae. Algal Res 23:66–77
Horn SJ, Aasen IM, Ostgaard K (2000) Ethanol production from seaweed extract. J Ind Microbiol
Hrmova M, Fincher GB (2009) Plant and microbial enzymes involved in the depolymerization
of (1,3)-β-D-glucans and related polysaccharides. In: Bacic A, Fincher GB, Stone BA (eds)
Chemistry, biochemistry and biology of 1-3 beta glucans and related polysaccharides. Academic
Press, pp 119–170
Irfan M, Kwon TH, Yun BS, Park NH, Rhee MH (2018) Eisenia bicyclis (brown alga) modulates
platelet function and inhibits thrombus formation via impaired P2Y12 receptor signaling pathway.
Phytomedicine 1(4):79–87
Ji CF, Ji YB (2014) Laminarin-induced apoptosis in human colon cancer LoVo cells. Oncol Lett
7(5):1728–1732
210 9 Laminarins
Jiang H, Liang S, Yao XR, Jin YX, Kim NH (2018) Laminarin improves developmental competence
of porcine early stage embryos by inhibiting oxidative stress. Theriogenology 115:38–44
Kadam SU, Alvarez C, Tiwari BK, O’Donnell CP (2015) Extraction of biomolecules from seaweeds.
In: Tiwari BK, Troy DJ (eds) Seaweed sustainability. Academic Press, pp 243–269
Kim SK, Bhatnagar I (2011) Physical, chemical and biological properties of wonder kelp—lami-
naria. In: Kim SK (ed) Marine medicinal foods. Advances in food and nutrition research, vol 64,
pp 85–96
Kim EJ, Fathoni A, Jeong G, Jeong HD, Kim JK (2013) Microbacterium oxydans, a novel alginate-
and laminarin-degrading bacterium for the reutilization of brown-seaweed waste. J Environ
Manage 130:153–159
feasibility of macroalgae as a potential feedstock for biorefineries. Bioenergy Res 8:1046–1056
Liu Z, Xiong Y, Yi L, Dai R, Yuan S (2018) Endo-β-1,3-glucanase digestion combined with the
HPAEC-PAD-MS/MS analysis reveals the structural differences between two laminarins with
different bioactivities. Carbohyd Polym 194:339–349
Martins CR, Custodio CA, Mano JF (2018) Multifunctional laminarin microparticles for cell
adhesion and expansion. Carbohyd Polym 202:91–98
Michel C, Bernard C, Lahaye M, Formaglio D, Kaefer B, Quemener B (1999) Algal oligosaccharides
as functional foods: in vitro study of their cellular and fermentative effects. Sci Ailm 19:311–332
Misurcova L, Skrivankova S, Samek D, Ambrozova J, Machu L (2012) Health benefits of algal
polysaccharides in human nutrition. Adv Food Nutr Res 66:75–145
Moroney NC, O’Grady MN, Robertson RC, Stanton C, Kerry JP (2015) Influence of level and dura-
tion of feeding polysaccharide (laminarin and fucoidan) extracts from brown seaweed (Laminaria
digitata) on quality indices of fresh pork. Meat Sci 99:132–141
Motone K, Takagi T, Sasaki Y, Kuroda K, Ueda M (2016) Direct ethanol fermentation of the
algal storage polysaccharide laminarin with an optimized combination of engineered yeasts. J
Biotechnol 231:129–135
Ojima T, Rahman MM, Kumagai Y, Nishiyama R, Narciso J, Inoue A (2018) Polysaccharide-
degrading enzymes from marine gastropods. Methods Enzymol 605:457–497
Rioux LE, Turgeon SL (2015) Seaweed carbohydrates. In: Tiwari BK, Troy DJ (eds) Seaweed
sustainability. Academic Press, pp 141–192
Stone BA (2009) Chemistry of β-glucans. In: Bacic A, Fincher GB, Stone BA (eds) Chemistry,
biochemistry and biology of 1-3 beta glucans and related polysaccharides. Academic Press, pp
5–46
Sweeney T, Meredith H, Vigors S, Mcdonnell MJ, Ryan M, Thornton K, O’Doherty JV (2017)
Extracts of laminarin and laminarin/fucoidan from the marine macroalgae species Laminaria
digitata improved growth rate and intestinal structure in young chicks, but does not influence
Campylobacter jejuni colonisation. Anim Feed Sci Technol 232:71–79
Thorpe HM, Adams SS (1957) The anticoagulant activity and toxicity of laminarin sulphate K. J
Pharm Pharmacol 9(1):459–463
Wang H, Xu Z, Wu Y, Li H, Liu W (2018) A high strength semi-degradable polysaccharide-based
hybrid hydrogel for promoting cell adhesion and proliferation. J Mater Sci 53:6302–6312
Yin G, Li W, Lin Q, Lin X, Lin J, Zhu Q, Jiang H, Huang Z (2014) Dietary administration of
laminarin improves the growth performance and immune responses in Epinephelus coioides.
Fish Shellfish Immunol 41(2):402–406
Yvin JC, Levasseur F, Amin-Gendy DCP, Tran TK, Patier P, Rochas C, Lienart Y, Cloarec B (1993)
Laminarin as a seed germination and plant growth accelerator. EP0649279A1
Chapter 10
Aquatic Plants and Algae Proteins
Abstract Proteins are quite diverse group of polymers. For this reason, this book
divides proteins into three chapters—the present chapter on proteins from algae and
aquatic plants, a chapter on collagen and a third chapter on enzymes. Proteins from
aquatic plants and algae are of particular importance since they serve as a source of
plant protein which do not require land space for cultivation. As the demand for pro-
tein for nutrition rises alongside global population and arable land spaces decrease
due to desertification and demand for living space, an alternative way to grow pro-
tein is in the aquatic environment. To this end, this chapter reviews the process of
extracting proteins from aquatic plants and algae and discusses the environmental and
economic significance. In the process, chemistry and applications are also discussed.
Keywords Proteins · Algae · Aquatic · Phycobiliproteins · Biopolymer
10.1 Introduction
Proteins are rather ubiquitous; they are found in all living things. Described most
simply as the building blocks of life, proteins are biopolymers that can be found
in a wide range of organisms across the five kingdoms—animals, plants, protista,
fungi and prokaryotes. Aquatic-sourced proteins are applicable in various industries
from food to cosmetics to biomedical and many others. They have been sourced for
their nutritional value in food, skin enhancing effects in cosmetics, productions of
scaffolds in tissue engineering, catalyzing chemical processes, in the production of
biofuel, in cancer treatment and much more. Within this book, proteins are covered
in three chapters—the present chapter which covers proteins from aquatic plants and
algae, a chapter on collagen and another chapter covering enzymes.
Aquatic plants such as duckweed and water hyacinth have been investigated for
their protein components and how these can be used in applications such as ani-
mal and human nutrition as a source of protein is also investigated (Adeyemi and
Osubor 2016; Aguilera-Morales et al. 2018). Algae, which include both microalgae
and macroalgae, have so far gained more attention as aquatic source of proteins.
The microalgae include the diatoms, the blue-green algae, the golden algae and the

212 10 Aquatic Plants and Algae Proteins
microscopic green algae (Bacillariophyta, Cyanophyta, Chrysophyta and Chloro-

phyta, respectively). All of these algae are a diverse group with thousands of species.
This gives the possibility for a largely diverse range of proteins.
Their ability to grow in the aquatic environment means these aquatic organisms
have adapted some features which are distinct from terrestrial plants. Every different
feature means these plants and algae have different proteins which are different from
those of animals, since these proteins are important in developing these specific
features. The aquatic environment therefore serves as a promising source of new
proteins and peptides or at the very least a more productive source, considering the
higher biomass accumulation rate of aquatic plants and algae compared to those of
terrestrial plants.
Aquatic plants and algae continue to play an increasingly important role in the
livelihoods of individuals, they can serve as a source of food, means of livelihood and
their economic value can be significantly increased through exploring their protein
content. These proteins can be further processed into even higher value commodities
such as functional foods and bioactive agents in supplements and drugs. This chapter
explores some of the aquatic plants and algae, their protein components and existing
and potential commercial applications of these proteins. Within this chapter, aquatic
plants and algae proteins are reviewed as the total protein content in the biomass. Sub-
sequent chapters will then explore specific proteins, namely enzymes and collagen
in separate chapters.
It should be noted that while some of these plants and algae could serve as a
source of beneficial proteins, their mismanagement or the mismanagement of the
aquatic environment within which they grow could lead to adverse commercial,
health and environmental impact. A particularly common one of such is the algae
bloom which results in release of toxins into the environment which are harmful
to human beings (Pulz and Gross 2004). This has become a serious environmental
concern with increasing frequency in aquatic habitats such as along the coast of the
Yellow Sea in the Jiangsu Province and Shandong Province in China (Zhang et al.
2019).
As the world population continues to increase so does the demand for food. Protein
forms a large part of the daily nutritional requirement, and much of these come from
terrestrial plants and animals. Aquatic source of protein is mainly fish and aquatic
animals. Algae and aquatic plants can provide an alternative or additional protein
source which do not require land for cultivation and grow at a much faster rate as
well as offer some additional health impacts.
Aquatic plants and algae contain considerable amounts of protein which make them
commercially relevant sources of protein. For example, the blue-green cyanobac-
teria Spirulina contain between 60 and 70% protein by dry weight of its biomass
(Yucetepe et al. 2018). Many aquatic organisms produce more than one biopolymer
Table 10.1 Protein yields

Organism Protein yield (%) References
from some aquatic plants and
algae Kappaphycus 12.69–23.61 Kumar et al. (2015)
alvarezii
Undaria pinnatifida 15 Suetsuna and
Nakano (2000)
Azolla (water fern) 28 Brouwer et al.
(2019)
Lemna gibba 21.5 Aguilera-Morales
duckweed et al. (2018)
Ulva lactuca 17.2 Aguilera-Morales
et al. (2018)
P. tenera (red 47 Fleurence (1999)
algae)
Nannochloropsis 17.4 and 24.8 Safi et al. (2017)
gaditana
(microalgae)
Spirulina platensis 29.05 Yucetepe et al.
(blue-green algae) (2018)
of commercial value. The rate of accumulation of each polymer varies; furthermore,

the optimal conditions for maximum accumulation of one polymer are most likely
not the optimal condition for the others. For example, the red marine alga Porphyrid-
ium marinum contains up 46% protein by dry weight when grown in medium with
17.6 mM potassium nitrate. However, when grown with 5.9 mM potassium nitrate,
the protein content is 35% and when grown with 3.5% mM potassium nitrate, the
protein content is even lower at 25% dry weight (Li et al. 2019). This red microal-
gae accumulates maximum starch at light intensity of 100 µmol photons m−2 s−1 ,
sodium nitrate (NaNO3 ) concentration of 1 g/L and a sodium chloride concentration
of 20 g/L (Hlima et al. 2019). However, this condition is not the optimal condition
for protein accumulation. Furthermore, while the starch content increases the light
intensity and salinity of growth environment increases the starch content in these
algae, it does the opposite for the protein content. This is explained to be as a result
of the conditions favoring biochemical processes which lead to starch formation that
does not favor those biochemical reactions leading to protein formation. Table 10.1
gives yields of protein obtained from different species based on values reported from
different studies in the literature.
10.3 Chemistry of Aquatic Proteins
Proteins are polymeric structures made up of polypeptides which are chains made
up of several amino acids which are repeating units joined together by amide links
that links the amine group with a carboxylic acid group. They are heteropolymers
whose repeating units can be trillions of variations of arrangements of 20 different

amino units. Unlike other aquatic polymers that have been explored in this book
such as fucoidans and carrageenans, proteins are monodisperse; they have uniform
molecular weight, hence number of repeating units within their polymer chains.
Proteins differ from one another by the sequences of amino acids in their primary
structure, the interaction of the polypeptide chain in the secondary structure and the
folding of these chains in the tertiary structure. Some proteins further have a quater-
nary structure defined by the combination of different folded polypeptide structures
by hydrogen bonds. One such protein with a quaternary structure is hemoglobin.
Some researchers have explored how to develop other monodisperse polyamides
from proteins as a route to achieving monodispersity in polymerization reactions
(Yang et al. 2003).
Some proteins from aquatic organisms share the same general chemistry as pro-
teins from terrestrial organisms. Proteins such as collagen, luciferase and amylase
are examples of proteins found in land as well as aquatic organisms. However, some
of these proteins have some distinct features in terms of their chemical structure that
differs from one another. The rest of this section will highlight some of the features
specific to aquatic proteins.
Despite some of the adverse effects associated with the consumption of high
quantity of meat, animal protein has the highest amount of all essential amino acids
required for healthy nutrition for humans. To counter the risks associated with meat
consumption such as high fat content leading to cardiovascular disease, it is recom-
mended to consume a diet consisting of mainly plant-based proteins and achieve
the required essential amino acids through a combination of a range of plant protein
sources such as legumes, grains, fruits and vegetables (FAO, WHO 1991).
Despite being considered as highly nutritious consisting of proteins, vitamins and
minerals, amino acid composition of algae is rather limited. Some of the essential
proteins, which are not synthesized by humans and required by the body, are not
present in algae. This is the general case with plant protein, whereby some essential
amino acids are missing in plants of both terrestrial and aquatic. These essential
amino acids missing in plants include leucine, histidine, tryptophan, lysine, valine,
threonine, methionine and phenylalanine (Young and Pellett 1994). The amino acid
missing in particular plant or algae varies from species to species. For example, in
red algae, leucine and isoleucine are usually present in low amounts in brown algae
cysteine, while lysine and methionine are the missing or limited amino acids in brown
algae. Tryptophan and lysine are generally in limited amounts in all algae (Bleakley
and Hayes 2017). Amino acids which are more abundant in algae include aspartic
acid and glutamic acid which could be up to 22–44% in species Fucus sp. and up to
32% in Ulva sp. (Fleurence 1999).
While algae and aquatic plants cannot serve as a main source of proteins due
to the absence of some essential amino acids, they nonetheless can serve as an
additional more efficient protein source. Polypeptides from plant sources tend to be
cyclic peptides, and these are known to have more bioactive properties than the linear
structures. The cyclic structures tend to be more stable than the linear structures (Tan
and Zhou 2006). This stability could ensure they retain their secondary structure
10.3 Chemistry of Aquatic Proteins 215
until they reach the bloodstream, thus allowing them a better chance of eliciting their
bioactivities.
Algae and aquatic plants, therefore, can contribute to a more diversified protein
source for nutrition and other applications. Due to the increased rate of productivity
of aquatic plants and algae, they could contribute a more reliable source of plant type
proteins which can be used for nutrition and as bioactive agents.
In 2016, 89,000 tonnes of microalgae was farmed across 11 countries of the world—
88,600 tonnes of this was from China. These include species such as Haematococcus
pluvialis, Nannochloropsis spp., Chlorella spp. and Spirulina spp. All are being
farmed in large, medium and small scales (FAO et al. 2018). While macroalgae
get a larger revenue from their food sales, microalgae are mostly sold as high-value
functional products. Therefore, despite the lower annual tonnes produced, microalgae
are valued at around a billion USD annually, compared to that of macroalgae at
6 billion USD (Bleakley and Hayes 2017).
The protein content in macroalgae is comparable to those found in animal-based
proteins and is higher than those found in land plants such as soybean, wheat and
legumes. Algae yield around 2.5–7.5 tonnes per hectare annually, while microalgae
yield 4–15 tonnes per hectare annually. These yields are rather high compared to
the conventional plant-based proteins such as wheat, soybeans and legumes which
yield 1.1, 0.6–1.2 and 1–2 tonnes per hectare annually (Van Krimpen et al. 2013).
Macroalgae and microalgae could contain similar or even more protein that terrestrial
plants typically used as protein source. Spirulina, a microalgae which have gained
much popularity as a nutrient source, could contain up to 63% protein per dry weight
(Tokuşoglu and Üunal 2003). The red algae species Porphyra tenera contains up to
47% protein per dry weight (Fleurence 1999).
Protein content in any particular algae varies with factors such as growth season,
temperature, harvest period, region and nutrient content of water. The types of pro-
teins present also vary. How different species react to particular changes in growth
condition in turn affects the types of proteins they metabolize. The algae make use of
these proteins to survive and function within its environment; therefore, the stimulus
it gets from these environments determines what protein it is prompted to produce.
These factors can be used to manipulate particular algae to produce desired type
of protein by controlling the growth environment. This requires an in-depth under-
standing of the correlation between environmental factors and the metabolism of the
specific species. For example, highest protein yield is obtained from the algae Kap-
paphycus alvarezii in August, November and February when studied over 12 months
from September 2004 to April 2006 in Northwestern India (Kumar et al. 2015).
Aquatic plants such as water fern, duckweed and water hyacinth contain relatively
moderate-to-high amounts of proteins. 28% protein content by dry weight has been
reported for water fern (Brouwer et al. 2019) and 21.5% for duckweed (Aguilera-
Morales et al. 2018). While there are up to hundreds of thousands of species of algae,
some of which belong to the plant kingdom, there are just about a hundred species
of non-algae aquatic plants. The recent rise in algae bloom incidents in different
parts of the world further eludes to the fast rate of growth of algae. Water hyacinth,
ferns and duckweed are also fast-growing plants which can be explored as abundant
sources of aquatic proteins. Despite the fact that aquatic plants and algae could serve
as an abundant source of proteins, well managed and controlled systems for the
cultivation of aquatic plants and algae is important in order to avoid undesirable
population blooms which could result in devastating environmental impacts.
10.5 Extraction of Proteins from Algae and Aquatic Plants
Protein extraction from aquatic plant could be with different goals. It could be carried
out with the aim of processing the proteins from algae into more digestible or more
appealing forms. Example of such is incorporation of algae proteins into snacks
(Lucas et al. 2018). The proteins could be extracted for their bioactive properties
for use in non-food applications such as cosmetics, pharmaceutical or biotechnology
applications. The latter section of this book will explore some of the applications of
such proteins from aquatic plants and algae.
Conventional extraction processes can be carried out using water, acids or alkali.
Other methods also exist which make use of more advanced techniques, some in
combination with the conventional techniques, to improve ease and effectiveness of
extraction. One of the factors which limit full commercial exploitation of proteins
from algae for processed food is the cost and environmental implications of the
extraction process to prepare them into edible forms. However, when considering
high-value applications such as for use as therapeutic agents, these added costs could
then be compensated.
In algae, proteins are usually present alongside polysaccharides such as cellulose,
alginate, ulvans and carrageenans. The extraction strategy could therefore be to either
break down these polysaccharides in order to free up the proteins, or to break down the
protein into a more soluble form which is then dissolved out of the fibrous structure.
This is then followed by other unit operations such as separation and drying.
10.5.1 Physical Extraction
This process involves the application of mechanical force to the biomass which then
frees up the water-soluble protein from the fiber. This can be achieved by applying
shear through grinding, homogenizing or pressing (Barbarino and Lourenço 2005).
It can also be achieved by applying osmotic pressure through immersion in water
for an extended period of time (Marrion et al. 2003). Ultrapure water is used to have
10.5 Extraction of Proteins from Algae and Aquatic Plants 217
minimal concentration of minerals in the water and optimize concentration gradient

and consequently osmotic pressure. Using this method to extract both water-soluble
and insoluble proteins from Palmaria palmata, one study reports a yields of 0.0677%
for the osmotic method and a yield of 0.0692% (mass protein per dry weight of algae)
using shear force (Harnedy and FitzGerald 2013). Extraction yield of up to 40% has
been reported using the physical method (Harnedy and FitzGerald 2013).
10.5.2 Chemical Extraction
This method requires the use of an alkali or acidic solution for extraction of the
protein. The protein becomes soluble at altered pH and in some cases elevated tem-
peratures. This is likely to obtain the protein in a denatured form. Yields of up to
59% can be obtained using this method (Barbarino and Lourenço 2005). The alkali
or acid could also be used to disrupt the cell wall structure, allowing the protein
to be released more easily. Sodium hydroxide and hydrochloric acid are commonly
used; others include polyethylene glycol, potassium carbonate and N-acetyl-cysteine
(Bleakley and Hayes 2017).
In some cases, the protein can be extracted alongside other components which can
be used alongside the protein, for example, as animal feed. In this case, the processor
can eliminate the cost of separating the protein from these components and simply
needs to obtain the protein from the biomass. An example of this is extraction of leaf
protein concentrate from the water hyacinth. Here the extraction of the leaf protein
concentrate was carried out by first blanching the washed water hyacinth leaves for
5 min with acetic acid at 5% concentration. The blanched leaves where then rinsed
with deionized water to get rid of the acids on the surface after which the biomass was
dried. The next step is to extract the fats, and this was done by soaking in 95% ethanol
within which the fat is soluble. This process was allowed 6 h to allow the alcohol
penetrate into the walls and dissolve the fats and other ethanol-soluble compounds.
This leaves behind the carbohydrates, proteins, minerals and other compounds such
as alkaloids and phenolic compounds. The residual fat in the leap protein concentrate
depends on the yield of extraction. This concentrate can then be used as a protein
source in applications such as animal feed.
Another way to extract proteins from the algal biomass is through the use of enzymes.
The role of the enzyme is to breakdown the polysaccharides to sugar units which are
soluble and separating the protein. The water-soluble proteins can be separated from
the water-soluble sugars by separation method such as precipitation of the protein
or crystallization of sugars. The enzymes used depend on the type of algae and its
components. Enzymes such as carrageenase, xylanase and cellulase are used for
extraction of protein from red algae (Joubert and Fleurence 2008; Fleurence et al.
1995). One limitation of the enzyme extraction method is the high concentration of
enzymes required to achieve commercially feasible yields. This could be around (48.0
× 103 units/100 g) (Bleakley and Hayes 2017). Extraction yields of the enzymatic
method are higher compared to the other conventional methods. Extraction yields of
up to 87% (weight of protein extracted per weight of protein present in the biomass)
have been reported (Harnedy and FitzGerald 2013).
10.5.4 Ultrafiltration and Diafiltration
These processes can be used in combination to optimize the yield and/or quality of
protein extract. For example, in the extraction of water-soluble protein from Nan-
nochloropsis gaditana microalgae, the cell disruption in order to free the proteins
prior to ultrafiltration or diafiltration was carried out with either a combination of
high-pressure homogenization and ultrafiltration/diafiltration or a combination of
enzyme treatment with protease enzyme and ultrafiltration/diafiltration (Safi et al.
2017). This method is presented as a mild biorefinery process which has less environ-
mental impact in terms of minimal use of solvents and acids or alkali. High-pressure
homogenization at 150 GPa permeating at 9 L h−1 was sufficient to break the cell
walls of the microalgae and release the proteins in the aqueous system. The ultracen-
trifugation followed by diafiltration was then required to separate the polysaccharides
from the proteins. Although the system was maintained at room temperature with
no additional heat input, due to the frictional forces resulting in heat generation dur-
ing homogenization, a cooling system was necessary to maintain the temperature at
30 °C. When cell disruption was achieved using enzymatic treatment, this was car-
ried out at a temperature of 50 °C and a pH of 8. The enzyme treatment was allowed
a period of 4 h using alcalase, a protease enzyme.
The combination of enzyme treatment with ultrafiltration and diafiltration resulted
in a higher yield than when high-pressure homogenization was combined with ultra-
filtration/diafiltration in the particular study where a yield of 24.8% was obtained for
the former and 17.4% for the latter. This does not necessarily mean that the enzyme
method would always result in a higher yield for all algae; this needs to be carried
out for a range of species and a correlation drawn between yield and species for both
methods before such conclusions can be made. Figure 10.1 shows a flowchart of the
extraction process involving cell disruption, ultrafiltration and diafiltration.
10.5.5 Assisted Extraction
The conventional extraction methods can be modified by using additional techniques

to assist the extraction process. An example is the use of ultrasound. This could be
used to create tiny bubbles within the extraction medium, weather neutral, alkaline
10.5 Extraction of Proteins from Algae and Aquatic Plants 219
Biomass Cell disruption Centrifugation Solid residue
Supernatant
Ultrafiltration Filtrate
Concentrate
Diafiltration Filtrate
Protein
Fig. 10.1 Schematic summary of protein extraction from algae using cell disruption, ultrafiltration
and diafiltration
or acidic. These acoustic cavities exert pressure and generate heat within the cell
wall causing disruption of the cell wall (Mason et al. 1996). This makes it easier for
the water, alkali, acid or enzyme to penetrate the cell wall and act toward releasing
the protein. In one example, when applied prior to acid extraction of protein from
the alga Ascophyllum nodosum, ultrasound increased the protein yield by 540%. The
required extraction time was also reduced by 50 min (Kadam et al. 2017). Such
reduction in processing time and increase in yield are significant toward a more fea-
sible commercial-scale extraction of algal protein. An integrated extraction process
combined the ultrasound with other methods to extract protein from microalgae. The
ultrasound method combined with the sugaring out method and liquid biphasic flota-
tion achieved a yield of up to 93.33% (protein extracted as a percentage of protein
present in the biomass) at optimum conditions (Sankaran et al. 2018). The process
at large scale had an efficiency of 85.25%. While costly equipments and technical
expertise are required to achieve a less fossil-sourced energy-demanding process, it
is important to have high efficiency to compensate for the cost incurred in equipment
and technical manpower for production.
Another way to improve the conventional extraction method is through the use of
pulsed electric field. This method is used for other applications such as drug delivery
where the electric field is used to open up the skin or other tissue, to enhance the
transport of drugs which will otherwise not permeate the tissue or cell membrane and
to permeate faster. This involves application of high voltage to the biomass for a short
period of time, usually a fraction of a second. When pulsed electric field is applied
to a membrane, this opens up temporary channels through which larger molecules
can permeate the membrane (Silva and Sulaiman 2019). This principle has also been
applied for extraction of protein from algal biomass. For example, protein yield from
Spirulina was increased by 13%, while that from chlorella was increased by 27%
using the pulsed electric field-assisted extraction (Garcia et al. 2018). These are two
of the common microalgae which have been commercially explored for food and
non-food applications. Therefore, a commercially feasible method which increases
yield is bound to have significant commercial implications with a ready-to-adapt
market.
Other methods include microwave-assisted method (Barba et al. 2015), use of
subcritical water or supercritical CO2 (Herrero et al. 2006; Dumay and Morancais
2016) and membrane technology where a semipermeable membrane is used to sep-
arate a component against a concentration gradient. This method is often preceded
by cell disruption by methods such as homogenization or enzyme treatment to make
the protein available in solution (Safi et al. 2017). These processes have the common
goal of developing means of disrupting the cell wall using extreme fluid properties
at specific temperature and pressure in order to aid extraction of the protein. All of
these processes are mostly limited by financial requirement of necessary facilities
and scale up of the process.
Extraction using more novel methods such as electrophoresis without additional

chemicals or heat input can be considered since it significantly results in significantly
higher yields and the electric current can be generated using green energy sources.
As the technology for extraction of protein from algae and plant biomass advances,
particularly in an age where more attention is being paid to environmental impact,
the research in this area is bound to move toward developing more environmentally
sustainable extraction processes which are financially feasible.
10.6.1 CO2 Emission
Use of supercritical CO2 could be a way of not only minimizing emissions but
also removal of CO2 . This will, however, incur more cost in production which is
not commercially feasible. A tonne of microalgae requires to produce an estimated
1.8 tonnes of CO2 (Kliphuis et al. 2010). Since the process of growing the biomass
removes CO2 from the environment, it can be generally regarded as a CO2 -negative
process. This is then weighed against the carbon emitted in the other processes
involved in the extraction of protein and going further down the value chain to the
packaging and transportation to final consumer.
10.6.2 Solid Waste Generation
Compared to lignocellulosic land plants such as corn and sugarcane, algae biomass
has simpler composition. Brown algae, for example, comprise 15% laminarin, 18%
mannitol, 32% alginate and 35% ash and salts. Lignocellulosic plants, for exam-
ple, corn stover, contain 35% cellulose, 19% hemicellulose, 16% lignin and other
compounds up to 27% (Konda et al. 2015). This means the solid residue generated
from the extraction of protein from algae results in less diverse range of compounds
which make the utilization of this waste less complex compared to residue from
lignocellulosic biomass.
In extraction of proteins, water is used as the main extraction medium. Water is

also consumed in the washing of the biomass prior to extraction. In the cultivation
process, aquatic plants and algae have one major advantage over terrestrial plants of
not requiring freshwater. They can, in fact, be used to clean up water by extracting
the nutrients in the water for their metabolism. However, careful control of the levels
of the different nutrients is required for optimal growth and control of the yield of
the desired biopolymer from the aquatic plant or algae. In the extraction of protein
from Spirulina platensis, for example, 1 g of biomass required 15 ml of distilled
water as extraction medium (Yucetepe et al. 2018). Much of the water used in the
extraction process is then evaporated in the drying process, and this requires some
energy input. The water used in washing is either sent to external water treatment
plants or recycled internally. Where acids or alkalies are used, the water needs to
be neutralized and this requires the use of either more acid or alkali in the water
treatment.
10.6.4 Acids and Alkali
Although the enzyme extraction method relies on enzymes to break down the cell
walls with the aim of eliminating or at least minimizing the need for additional
chemicals, in some cases it is necessary to use acids or base to achieve the required
pH for enzymes to function optimally. Enzymes are proteins, and the activity of
these proteins is determined by their configuration as well as conformation (O’Brien
et al. 2012). The conformation is dependent on the pH of the environment within
which they are acting, therefore for specific enzymes, a specific pH is required. For
example, while alcalase is used, a pH 8 was required (Safi et al. 2017). This will
require the addition of an alkali to achieve this pH. However, this is relatively mild
as it is close to a neutral pH.
Energy consumed depends on the method being used and the efficiency of the oper-
ating system. While ultrasound is being used, one study reports 0.4 kWh required per
liter of the biomass suspension being processed during the disruption of microalgal
cell (Keris-Sen et al. 2014). While physical method such as high-pressure homoge-
nization is being used, energy to generate the amount of shear to disrupt the cell is
required.
Amount of heat required for extraction varies with the method being used. For
example, in extraction of water-soluble proteins from microalga N. gaditana where
cell disruption is carried out by high-pressure homogenization, the process of homog-
enization results in generation of heat from the friction forces and the exothermic
processes of cell disruption. This process thereby requires cooling to prevent over-
heating of the system (Safi et al. 2017). Proteins are prone to denaturation at elevated
temperature; therefore, the temperature should be kept as close to ambient as possible.
To achieve optimal cell disruption, a pressure of 150 GPa was sufficient. This resulted
in over 90% of the cells being disrupted in a single pass. This process required an
energy input of 0.44 kWh per kg of biomass. Repeated passes and increasing homog-
enization pressure did not result in higher percentage of cell disruption. Such studies
are necessary in terms of saving cost and time. Indeed, two passages would have
required an energy input of 7.5 kWh per kg resulting in only 3% increase in yield.
Energy also used in the additional unit operations is required to separate and purify.
Centrifugation of the suspension following cell disruption by physical, chemical or
enzyme treatment requires around 10,000 g for 10 min (Safi et al. 2017). Use of
enzymes reduces the heat energy required in the energy-demanding methods.
10.7 Applications
A variety of proteins can be found in algae, and some of these have distinct functions.
Some edible algae and aquatic plants are consumed in their whole form as food,
skin care, aquaculture or animal feed. Here we look at applications which involve
extracting protein from aquatic plants and algae for protein specific applications
which range from food to clinical applications. Some of the proteins described here
are not only limited to aquatic plants and algae and can be found in other terrestrial
plants and animals.
10.7.1 HIV Microbicides
A type of protein which is currently receiving attention in HIV prevention research

is lectins. Lectins are present in animals, microorganisms and terrestrial. They are
also present in algae such as cyanobacterium (Singh et al. 2017). They are a diverse
group of proteins which include scytovirin, griffithsin and cyanovirin. Lectins have
the ability to bind to polysaccharides, glycans and glycolipids in a reversible manner
(Harnedy and FitzGerald 2011; Singh et al. 2015). This binding ability allows them
to cause cell agglutination and precipitation of glycoconjugates. This makes them
applicable in diverse applications such as antiviral, antitumor and antiinflammatory
agents and well as development of protein expression systems and nutraceuticals. The
use of lectins as microbicides to prevent the transmission of HIV has been explored
in several research studies (Janahi et al. 2018; Hopper et al. 2017; Alexandre et al.
2010). The lectins can selectively bind to the HIV cells by binding to the glycans
and polysaccharides on the surface of the virus which shields it from attack by the
antimicrobial agents or from being recognized by the body’s immune system. Lectins
also act by preventing the virus cells from binding to uninfected cells. This has been
demonstrated in in vitro studies on vaginal mucosa using scytovirin an algal lectin
(Janahi et al. 2018).
10.7.2 Phycobiliproteins
Phycobiliproteins are another form of proteins present in algae. These are water-
soluble proteins that serve a role in capturing of light in photosynthesizing organ-
isms. They are common in red algae and cyanobacteria (Dumay and Morancais
2016). Phycobiliproteins find commercial applications as natural dyes for food and
cosmetics (Spolaore et al. 2006). They also have been explored for other applications
such as fluorescent imaging and flow cytometry (Aneiros and Garateix 2004). More
recently, phycobiliproteins have been associated with some bioactive properties such
as antiviral, antiinflammatory and antioxidant activities (Sekar and Chandramohan
2008). Algae are also a source of certain bioactive polypeptides (Fan et al. 2014).
These have 2–30 repeating units of amino acids which have some bioactive properties
depending on the types and sequence of amino acids within these short chains. These
bioactive proteins are usually derived from the long-chain proteins and polypeptides
through hydrolysis or fermentation to break down the chains into shorter ones.
10.7.3 Food
Aquatic plant and algae play an increasingly important role in the future food security.
The world population is expected to reach 9 million by 2050, and the rate of food
production is being threatened due to several factors such as dangerous weather
conditions due to climate change and social problems leading to violence which has
resulted in reduced farming activities in these conflict regions (FAO et al. 2018).
There is an urgent need to develop alternative ways to produce food and produce it
much faster than conventional farming methods. With a much faster rate of nutrient
accumulation and growth, algae and aquatic plants are much more productive than
land plants. Furthermore, they do not require land for growth which means they do
not compete with space for living and can be grown away from disputed land areas.
Proteins are an important food content and are necessary in maintaining a balanced
diet. FAO estimates that 10.9% of people in the world are undernourished as of 2017
and that figure is said to have been growing in the past two years. The number of
malnourished people in the world rose from 804 million as of 2016 to 821 million
in 2017 (FAO et al. 2018). Alongside carbohydrates and fats, protein forms one of
the macronutrients required by humans for energy and growth.
In many parts of the world, fish serves as a major source of protein. With recent
algal blooms which result in the release of toxins into the water, fish in some parts is
at threat of being unsafe for human consumption. Aquatic plants and algae play two
roles in this aspect. The possible use to clean up water and as alternative sources of
proteins; either directly consumed as food, nutraceuticals or used as ingredients in
food products (Sinha et al. 2019).
An aquatic plant can be regarded as edible if it contains some nutrients which can
be broken down by the enzymes in the body and if it does not contain any toxins.
Recent studies present a protein digestibility of 99.36% for protein extracted from
the blue-green algae Spirulina. This is relatively high compared to that of soybean
which is 85% (Yucetepe et al. 2018). Although this value was derived from in vitro
studies, it indicates that such protein is suitable for food applications and indeed
some Spirulina-based food products are available in the market today. Thousands of
species of macroalgae and microalgae exists, such that the different proteins within
them also largely vary. The digestibility and types of amino acids which make up
the proteins vary for different algae (Boisen and Eggum 1991; Bleakley and Hayes
2017).
When consumed directly as food, algae have relatively low bioavailability. This
is thought to be due to the fact that proteins are usually present alongside other
polysaccharides within the cell wall of the algae. These fibers with which the proteins
are attached are indigestible; for the protein to be available as a nutrient, it must be
released from the fiber and then broken down into forms which can be absorbed
into the body along the alimentary canal. The digestibility therefore depends on the
enzymes present within the body which can break down the protein and free it from
the fibrous structure. Most often, it is therefore necessary to extract these proteins
and then make them into edible preparations as processed foods before they can have
relevant digestibility or bioavailability.
While egg and casein protein have digestibility coefficients of 94.2 and 95.1%,
respectively, microalgae Chlorella sp., Scenedesmus obliquus and Spirulina sp. have
digestibility coefficients of 76.6, 88.0 and 77.6%, respectively (Becker 2007). The
digestibility varies widely among algae species. For example, Undaria pinnatifida
has a bioaccessibility of 87%, Ulva lactuca has a digestibility of about 85.7%, while
P. tenera has a bioavailability of 78%. Some studies report red algae have to be more
digestible than brown algae, digestibility of red algae studied ranged between 83 and
87% while that of the brown algae ranged between 78.7 and 82% (Tibbetts et al.
2016). Much of the digestibilities referred to herein are based on in vitro studies.
Algae have been made into plant protein-based snacks of various forms. These
have been shown to have similar sensory appeal to conventional snacks. Lucas et al.
(2018) reported a sensory acceptability index of 82% for snacks which have been
enriched with Spirulina as a protein and nutrient enrichment. Algae has been part of
human diet for centuries; therefore, this could serve as a basis for using proteins from
algae as protein enrichment in foods. This is important in, for example, producing
protein supplements where a high protein content is required. Isolation of specific
component such as protein also allows for standardization and quality control of
products through identification of the specific compounds present and their quantity.
People of different regions have used freshwater plants for different applications
for centuries. Example of such freshwater plants includes wild rice (Zizania spp.),
Chinese water chestnut (Eleocharis dulcis), Indian lotus (Nelumbo nucifera), water-
cress (Rorippa nasturtium-aquaticum), water spinach (Ipomoea aquatica), water
caltrop (Trapa natans), water mimosa (Neptunia oleracea), wild taro (Colocasia
esculenta) and cattails (Typha spp.). Some have been consumed as food and used
as animal feed or in construction of structures, while some are simply valued for
their aesthetic appeal and revered as traditional/religious symbols—an example of
this is the Indian lotus. There are some interests in extracting proteins from some
of the aquatic plants which are popularly known to cause some environmental nui-
sance which often also lead to economic losses through the destruction of aquatic
wildlife. For example, aqueous extract of water hyacinth was shown to contain over
56.38% protein and had 17 out of the 20 essential amino acids for humans. The water
hyacinth leaf protein concentrate was also reported to have metal composition within
acceptable limits which could make it a potential source of protein for human use
(Adeyemi and Osubor 2016). The protein content in the water hyacinth leaf protein
concentrate was much higher than the carbohydrate (33.61%), fat (4.11), ash (4.88%)
and crude fiber (1.02%). Such that, over half of the leaf protein concentrate of the
water hyacinth is protein; this makes it potentially suitable as a high-protein food or
supplement.
10.7.4 Animal Feed
The protein in algae is used in animal, poultry and fish feed with the aim of improving
yield and quality since protein forms an important part of the diet. However, much
of the work in this area has looked at using whole or semi-processed macro- and
microalgae. Few studies have reported on exclusively extracting the proteins from
algae and incorporating in animal feed. This is due to the fact that animal feed is
required to be sufficiently low cost; therefore, expensive protein isolation prior to
incorporation in the feed would not be commercially feasible. A number of commer-
cially available animal feeds incorporate algae as a rich source of protein alongside
the other nutrients present in algae. Ruminants are particularly good candidates for
algae-derived protein-based feed as they can better digest the fiber and have a better
bioavailability of the protein within. This biological process is quite similar to the
extraction of algal protein using the enzymes which break down the polysaccharides
within the fiber, thereby making the protein available for extraction.
Animals fed with diet which included algae as a protein source have shown
improvement in immune system, increased productivity and improved quality of
meat and milk. This has been observed in a variety of commercially bred animals
such as steers used for beef and pigs used for pork (Allen et al. 2001; Saker et al. 2001;
Montgomery et al. 2001; Braden et al. 2004; Gatrell et al. 2014). In poultry, chicken
broilers fed with microalgae Spirulina show improved fertility and a more aestheti-
cally pleasing egg yolk color (Zahroojian et al. 2013). However, these attributes are
not directly linked with the protein content. The improvement in egg yolk color is
thought to be due to the presence of beta carotene (Anderson et al. 1991). In aquacul-
ture, both microalgae and macroalgae are used as feed for aquaculture animals such
as mollusks, sea bream and shrimps (Müller-Feuga 2013). They also act to regulate
the nutrient and mineral content in the water (Chuntapa et al. 2003).
Aquatic plants such as water fern, duckweed and American pondweed have been
used as animal feed as a protein source as well as other nutrients. Some aquatic plants
such as duckweed species, Lemna gibba, have relatively high protein content. For
example, a crude protein content of 21.5% was measured for L. gibba, while green
algae species, U. lactuca, was 17.2% in the same study (Aguilera-Morales et al.
2018). Both the duckweed and U. lactuca contained eight of the essential amino
acids necessary for fish feed.
10.7.5 Antihypertensive
The antihypertensive properties of compounds can be measured by their ability to

inhibit the activity of the angiotensin-converting enzyme (ACE I). This enzyme
converts the inactive angiotensin into an active ACE I which is then converted to ACE
II which caused constriction of the cardiac blood vessels leading to hypertension.
Bioactive peptides act as antihypertensive agents by preventing the conversion of
inactive ACE to the active ACE I, which in turn prevents the formation of ACE II
(Seca and Pinto 2018; Daskaya-Dikmen et al. 2017).
Another therapeutic approach to hypertension is the enhancement of vasodilation
to reduce blood pressure. Like flow in a channel, as the vessel is widened, the pressure
in the vessel is reduced. Vasodilation is facilitated by the enzyme bradykinin through
a series of processes. The body’s natural control system releases the appropriate
enzyme in response to the pressure in the blood vessels. A normal blood pressure
is therefore dependent on this control system, where the vasoconstricting enzymes
and substrates are working in conjunction with the vasodilating enzymes to maintain
homeostasis. Angiotensin I is also thought to be responsible for degradation of the
bradykinin, thus preventing vasodilation (Daskaya-Dikmen et al. 2017). Therefore,
by inhibiting the angiotensin enzyme, vasodilation is also promoted.
The antihypertensive properties shown by these peptides are absent in the parent
proteins. These parent proteins need to be broken down into the peptide forms before
they can manifest the antihypertensive bioactivity. This is usually achieved artificially
through enzymatic hydrolysis, heat treatment or fermentation. This could possibly
happen naturally in the body and perhaps why the seaweeds have been associated
with such properties when consumed as foods (Sabirin et al. 2016).
Proteins and peptides are susceptible to enzymatic breakdown in the gastroin-
testinal tract. Orally consumed peptides may therefore not have sufficient bioactivity
to elicit their antihypertensive effect when taken into the body through this route.
The peptides’ bioactivity is highly dependent on the sequence of amino acid and
the primary and secondary structures of the short chain (usually 2–20 amino acid
residues). When taken orally, these are susceptible to being digested as other food
proteins and broken down and absorbed into the body as amino acids. Therefore, for
these peptides to be able to elicit antihypertensive effect, they need to either have
resistance to the enzymes within the gastrointestinal tract, completely bypass the
gastrointestinal tract by using other routes of delivery into the bloodstream or be
prepared in forms which protects the active ingredient (in this case the polypeptide)
until it gets to the target.
On the other hand, the intestinal enzyme activity could act on these peptides and
convert them to more active antihypertensive short-chain peptides. This has been
demonstrated in peptides extracted from Ulva rigida, where the peptides containing
three amino acid residues produced from the hydrolysis of the parent protein were
further broken down by the intestinal mucosa peptidase into a smaller peptide con-
taining two amino acid residues (Paiva et al. 2016). In this case, the intestinal enzyme
activity was beneficial in improving the antihypertensive effect of the peptides by
partial digestion.
Indeed, the studies on the antihypertensive effect of these algae-derived peptides
are relatively advanced and have been carried out on animal and human test subjects
(Saito et al. 2002; Saito and Hagino 2005). Algae species from which peptides have
been shown to have antihypertensive activity have been extracted that include U.
pinnatifida, Porphyra yezoensis and U. rigida (Seca and Pinto 2018).
The presence of particular groups such as D-amino acids thiophene oxazole and
some alpha and beta amino acids within the polypeptide structures of some marine
plants gives them some excellent bioactive properties. Some products also exist in the
market as functional foods with antihypertensive effects. Examples are Evolus® and
Ameal-S 120® (Seca and Pinto 2018). The potency of the different peptides varies,
and the hypertensive effect is also dose dependent. Example prescribed dosage is
1.8 g daily of Pyropia yezoensis-derived peptides to human patients (Saito et al.
2002) and 10 mg per day per kg of body weight to hypertensive rats through oral
administration (Suetsuna and Nakano 2000).
10.7.6 Antioxidant
Compounds which inhibit the activities of reactive oxygen species or remove them for
the body are referred to as antioxidants (Yucetepe et al. 2018). Algae and other aquatic
plants have been investigated widely for their antioxidant properties. The antioxidant
properties of proteins from algae can be attributed to their bioactive proteins. Some
of the antioxidant properties of some polysaccharides such as fucoidans which show
antioxidant activity have been thought to be due to the presence of proteins attached
to them.
Proteins extracted from aquatic plants and algae have been explored for a range
of other applications which include antiinflammatory, anticancer, antiviral, lipid and
glucose-lowering effect, anticancer, immunomodulatory effect, appetite suppression,
antimicrobial, cytomodulatory and hypocholesterolemic.
Protein is an important component of the human diet. It is one of the three main
macronutrients which are vital for energy production, the others being fats and car-
bohydrates. For much of the world, sea-sourced food is the primary source of protein,
largely fish. With the growing demand for increased food production rate and protein
being a very important part of this, algae have a large potential present and future
market to serve as a rich protein source. Extraction of the protein from the algae and
aquatic plants improved digestibility and also reduces the cost of transportation of
the whole algae or aquatic plant biomass, which usually has high water content.
A non-essential amino acid, glutamic acid, has a sodium salt, monosodium glu-
tamate, which naturally occurs in the red sea weed Saccharina japonica, commonly
known as MSG, which is commercially used as a flavor enhancer. Although not
a protein, it is an amino acid. It is more commercially produced from fermenta-
tion of glutamic acid by corynebacterium species (Kakogawa et al. 1972) and made
one of its earliest commercial appearance in the form of the flavor enhancer brand
named “Aji-no-moto” (Choudhury and Sarkar 2017). One of the particular appeals
of an algae-sourced amino-acid-based flavor is that it serves as an option to meat- or
fish-based seasoning to vegans and vegetarians.
The extraction of proteins can be carried out alongside extraction of polysac-
charides and lipids as a way to make optimal use of all the commercially valuable
compounds within the algae. Presently, companies and researchers who are focused
on extraction of particular compound from algae tend to discard the other parts
of the algae which include the proteins. This is particularly due to the additional
costs required for further processing and the complexity of developing a multiprod-
uct business model. Companies who are presently involved in production of algae
protein-based products include Nova Scotia in Canada, Calpis Co Ltd in Japan and
Valio Ltd in Finland.
10.9 Conclusion 229
10.9 Conclusion
Aquatic plants and algae are a rich source of proteins. While they are not a total source
of proteins, they can be used to augment dietary protein requirement and serve as
a relatively cheap and abundant source of protein for direct and indirect human
consumption. The higher biomass accumulation rate and higher quality of protein of
aquatic plants and algae compared to terrestrial crops give them an advantage. Beyond
food, these aquatic polymers also have important bioactive properties such antiviral
and antihypertensive activities which make them applicable as nutraceuticals and in
the potential future clinical applications such as HIV prevention.
References
Adeyemi O, Osubor CC (2016) Assessment of nutritional quality of water hyacinth protein

concentrate. Eqypt J Aquat Res 42:269–272
Aguilera-Morales ME, Canales-Martinez MM, Avila-Gonzalez E, Flores-Ortiz CM (2018) Nutri-
ents and bioactive compounds of Lemna gibba and Ulva lactuca as possible ingredients to
functional foods. Latin Am J Aquat Res 46(4):709–716
Alexandre KB, Gray ES, Lambson BE, Moore PL, Choge IA, Mlisana K, Abdool Karim SS,
McMahon J, O’Keefe B, Chikwamba R, Morris L (2010) Mannose-rich glycosylation patterns
on HIV-1 subtype C gp120 and sensitivity to the lectins, griffithsin, cyanovirin-N and scytovirin.
Virology 402(1):187–196
Allen V, Pond K, Saker K, Fontenot J, Bagley C, Ivy R, Evans R, Brown C, Miller M, Montgomery
J (2001) Tasco-Forage: III. Influence of a seaweed extract on performance, monocyte immune
cell response, and carcass characteristics in feedlot-finished steers. J Anim Sci 79:1032–1040
Anderson DW, Tang CS, Ross E (1991) The xanthophylls of Spirulina and their effect on egg-yolk
pigmentation. Poult Sci 70:115–119
Aneiros A, Garateix A (2004) Bioactive peptides from marine sources: pharmacological properties
and isolation procedures. J Chromatogr B 803:41–53
Barba FJ, Grimi N, Vorobiev E (2015) New approaches for the use of non-conventional cell dis-
ruption technologies to extract potential food additives and nutraceuticals from microalgae. Food
Eng Rev 7:45–62
Barbarino E, Lourenço SO (2005) An evaluation of methods for extraction and quantification of
protein from marine macro- and microalgae. J Appl Phycol 17:447–460
Becker E (2007) Micro-algae as a source of protein. Biotechnol Adv 25:207–210
Bleakley S, Hayes M (2017) Algal proteins: extraction, application, and challenges concerning
production. Foods 6:33 (1–34). https://doi.org/10.3390/foods6050033
Boisen S, Eggum B (1991) Critical evaluation of in vitro methods for estimating digestibility in
simple-stomach animals. Nutr Res Rev 4:141–162
Braden K, Blanton J, Allen V, Pond K, Miller M (2004) Ascophyllum nodosum supplementation:
a preharvest intervention for reducing Escherichia coli O157:H7 and Salmonella spp. in feedlot
steers. J Food Proteins 67:1824–1828
Brouwer P, Nierop KGJ, Huijgen WJJ, Schluepmann H (2019) Aquatic weeds as novel protein
sources: alkaline extraction of tannin-rich Azolla. Biotechnol Rep E00368 (in press)
Choudhury S, Sarkar NS (2017) Algae as a source of natural flavour enhancers—a mini review.
Plant Sci Today 4(40):172–176
Chuntapa B, Powtongsook S, Menasveta P (2003) Water quality control using Spirulina platensis
in shrimp culture tanks. Aquaculture 220:355–366
Daskaya-Dikmen C, Yucetepe A, Karbancioglu-Guler F, Daskaya H, Ozcelik B (2017) Angiotensin-

converting enzyme (ACE) inhibitory peptides from plants. Nutrients 9(316):1–19
Dumay J, Morancais A (2016) Proteins and pigments. In: Seaweed in health and disease prevention,
pp 275–318
Fan X, Bai L, Zhu L, Yang L, Zhang X (2014) Marine algae-derived bioactive peptides for human
nutrition and health. J Agric Food Chem 62:9211–9222
FAO, WHO (1991) Protein quality evaluation—report of joint FAO/WHO expert consultation. FAO,
Rome, Italy
FAO, IFAD, UNICEF, WFP, WHO (2018) The state of food security and nutrition in the world 2018.
Building climate resilience for food security and nutrition. Rome, FAO. Licence: CC BY-NC-SA
3.0 IGO. ISBN 978-92-5-130571-3
Fleurence J (1999) Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends
Food Sci Technol 10:25–28
Fleurence J, Massiani L, Guyader O, Mabeau S (1995) Use of enzymatic cell wall degradation for
improvement of protein extraction from Chondrus crispus, Gracilaria verrucosa and Palmaria
palmata. J Appl Phycol 7:393–397
Garcia SE, van Leeuwen J, Safi C, Sijtsma L, Eppink MHM, Wijffels RH, van den Berg C
(2018) Selective and energy efficient extraction of functional proteins from microalgae for food
applications. Bioresour Technol 268:197–203
Gatrell S, Lum K, Kim J, Lei X (2014) Nonruminant nutrition symposium: potential of defatted
microalgae from the biofuel industry as an ingredient to replace corn and soybean meal in swine
and poultry diets. J Anim Sci 92:1306–1314
Harnedy PA, FitzGerald RJ (2011) Bioactive proteins, peptides, and amino acids from macroalgae.
J Phycol 47:218–232
Harnedy PA, FitzGerald RJ (2013) Extraction of protein from the macroalga Palmaria palmata.
LWT Food Sci Technol 51:375–382
Herrero M, Cifuentes A, Ibanez E (2006) Sub- and supercritical fluid extraction of functional
ingredients from different natural sources: plants, food-by-products, algae and microalgae: a
review. Food Chem 98:136–148
Hlima HB, Dammak M, Karkouch N, Hentati F, Laroche C, Michaud P, Fendri I, Abdelkafi S (2019)
Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium
marinum. Int J Biol Macromol 15(129):152–161
Hopper TSJ, Ambrose S, Grant CO, Krumm SA, Allison TM, Degiacomi MT, Tully MD, Pritchard
LK, Ozorowski G, Ward AB, Crispin M, Doores KJ, Woods RJ, Benesch JLP, Robinson CV,
Struwe WB (2017) The tetrameric plant lectin BanLec neutralizes HIV through bidentate binding
to specific viral glycans. Structure 25(5):773–782
Janahi EMA, Haque S, Akhter N, Wahid M, Jawed A, Mandal RK, Lohani M, Areeshi MY, Almaki
S, Das S, Dar SA (2018) Bioengineered intravaginal isolate of Lactobacillus plantarum expresses
algal lectin scytovirin demonstrating anti-HIV-1 activity. Microb Pathog 122:1–60
Joubert Y, Fleurence J (2008) Simultaneous extraction of proteins and DNA by an enzymatic
treatment of the cell wall of Palmaria palmata (Rhodophyta). J Appl Phycol 20:55–61
Kadam SU, Álvarez C, Tiwari BK, O’Donnell CP (2017) Extraction and characterization of protein
from Irish brown seaweed Ascophyllum nodosum. Food Res Int 99(3):1021–1027
Kakogawa TS, Takasago UJ, Kakogawa TK, Takasago YT (1972) Process for producing
monosodium glutamate. Patent 3655746
Keris-Sen UD, Sen U, Soydemir G, Gurol MD (2014) An investigation of ultrasound effect on
microalgal cell integrity and lipid extraction efficiency. Bioresour Technol 152:407–413
Kliphuis AM, de Winter L, Vejrazka C, Martens DE, Janssen M, Wijiffels RH (2010) Photosyn-
thetic efficiency of Chlorella sorokiniana in a turbulently mixed short light-path photobioreactor.
Biotechnol Prog 26:687–696
feasibility of macroalgae as a potential feedstock for biorefineries. BioEnergy Res 8(3):1–11
References 231
Kumar SK, Ganesan K, Rao SVP (2015) Seasonal variation in nutritional composition of
Kappaphycus alvarezii (Doty) Doty—an edible seaweed. J Food Sci Technol 52(5):2751–2760
Li T, Xu J, Wu H, Jiang P, Chen Z, Xiang W (2019) Growth and biochemical composition of
Porphyridium purpureum SCS-02 under different nitrogen concentrations. Mar Drugs 17(124):1–
16
Lucas BF, de Morais MG, Santos TD, Costa JAV (2018) Spirulina for snack enrichment: nutritional,
physical and sensory evaluations. LWT Food Sci Technol 90:270–276
Marrion O, Schwertz A, Fleurence J, Gueant JL, Villaume C (2003) Improvement of the digestibility
of the proteins of the red alga Palmaria palmata by physical processes and fermentation. Mol
Nutr Food Res 47:339–344
Mason T, Paniwnyk L, Lorimer J (1996) The uses of ultrasound in food technology. Ultrason
Sonochem 3:S253–S260
Montgomery J, Allen V, Pond K, Miller M, Wester D, Brown C, Evans R, Bagley C, Ivy R, Fontenot
J (2001) Tasco-Forage: IV. Influence of a seaweed extract applied to tall fescue pastures on sensory
characteristics, shelf-life, and vitamin E status in feedlot-finished steers. J Anim Sci 79:884–894
Müller-Feuga A (2013) Microalgae for aquaculture: the current global situation and future trends. In:
Richmond A, Hu Q (eds) Handbook of microalgal culture: applied phycology and biotechnology,
2nd edn. Wiley, Oxford, UK, pp 352–364
O’Brien E, Brooks BR, Thirumalai D (2012) Effects of pH on proteins: predictions for ensemble
and single molecule pulling experiments. J Am Chem Soc 134(2):979–987
Paiva LS, Lima EMC, Neto AI, Baptista J (2016) Isolation and characterization of angiotensin
I-converting enzyme (ACE) inhibitory peptides from Ulva rigida C. Agardh protein hydrolysate.
J Funct Foods 26:65–76
Pulz O, Gross W (2004) Valuable products from biotechnology of microalgae. Appl Microbiol
Biotechnol 65:635–648
Sabirin F, Soo KK, Ziau HS, Kuen LS (2016) Antihypertensive effects of edible brown seaweeds
in rats. Int J Adv Appl Sci 3:103–109
Safi C, Olivieri G, Campos RP, Engelen-Smit N, Mulder WJ, van den Broek LAM, Sijtsma L (2017)
Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration.
Bioresour Technol 225:151–158
Saito M, Hagino H (2005) Antihypertensive effect of oligopeptides derived from nori (Porphyra
yezoensis) and Ala-Lys-Tyr-Ser-Tyr in rats. J Jpn Soc Nutr Food Sci 58:177–184
Saito M, Kawai M, Hagino H, Okada J, Yamamoto K, Hayashida M, Ikeda T (2002) Antihyperten-
sive effect of Nori-peptides derived from red alga Porphyra yezoensis in hypertensive patients.
Am J Hypertens 15:210A
Saker K, Allen V, Fontenot J, Bagley C, Ivy R, Evans R, Wester D (2001) Tasco-Forage: II. Monocyte
immune cell response and performance of beef steers grazing tall fescue treated with a seaweed
extract. J Anim Sci 79:1022–1031
Sankaran R, Manickam S, Yap YJ, Ling TC, Chang JS, Show PL (2018) Extraction of proteins from
microalgae using integrated method of sugaring-out assisted liquid biphasic flotation (LBF) and
ultrasound. Ultrason Sonochem 48:231–239
Seca AML, Pinto DCGA (2018) Overview on the antihypertensive and anti obesity effects of
secondary metabolites from seaweeds. Mar Drugs 16(237):1–18
Sekar S, Chandramohan M (2008) Phycobiliproteins as a commodity: trends in applied research,
patents and commercialization. J Appl Phycol 20:113–136
Silva FVM, Sulaiman A (2019) Polyphenoloxidase in fruit and vegetables: inactivation by ther-
mal and non-thermal processes. In: Encyclopedia of food chemistry. Reference module in food
science, pp 287–301. https://doi.org/10.1016/B978-0-08-100596-5.21636-3
Singh RS, Thakur SR, Bansal P (2015) Algal lectins as promising biomolecules for biomedical
research. Crit Rev Microbiol 1(1):77–88
Singh RS, Walia AK, Khattar JS, Singh DP, Kennedy JF (2017) Cyanobacterial lectins characteristics
and their role as antiviral agents. Int J Biol Macromol 102:475–496
Sinha S, Patro N, Patro IK (2019) Amelioration of neurobehavioral and cognitive abilities of F1

progeny following dietary supplementation with Spirulina to protein malnourished mothers. Brain
Behav immun (in press)
Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae.
J Biosci Bioeng 101:87–96
Suetsuna K, Nakano T (2000) Identification of an antihypertensive peptide from peptic digest
of wakame Undaria pinnatifida. J Nutr Biochem 11:450–454. https://doi.org/10.1016/S0955-
2863(00)00110-8
Tan NH, Zhou J (2006) Plant cyclopeptides. Chem Rev 6:840–895
Tibbetts SM, Milley JE, Lall SP (2016) Nutritional quality of some wild and cultivated seaweeds:
nutrient composition, total phenolic content and in vitro digestibility. J Appl Phycol 28:3575–3585
Tokuşoglu Ö, Üunal M (2003) Biomass nutrient profiles of three microalgae: Spirulina platensis,
Chlorella vulgaris, and Isochrisis galbana. J Food Sci 68:1144–1148
Van Krimpen M, Bikker P, van der Meer I, van der Peet-Schwering C, Vereijken J (2013) Cultivation,
processing and nutritional aspects for pigs and poultry of European protein sources as alternatives
for imported soybean products. Wageningen UR Livestock Research, Lelystad, The Netherlands,
p 48
Yang J, Gittlin I, Krishnamurthy VM, Vazquez JA, Castello CE, Whitesides GM (2003) Synthesis
of monodisperse polymers from proteins. J Am Chem Soc 125(41):12392–12393
Young VR, Pellett PL (1994) Plant proteins in relation to human protein and amino acid nutrition.
Am J Clin Nutr 59:1203S–1212S
Yucetepe A, Saroglu O, Daskaya-Dikmen C, Bildik F, Ozcelik B (2018) Optimisation of ultrasound-
assisted extraction of protein from Spirulina platensis using RSM. Food Technol Econ Czech J
Food Sci 36(1):98–108
Zahroojian N, Moravej H, Shivazad M (2013) Effects of dietary marine algae (Spirulina platensis)
on egg quality and production performance of laying hens. J Agric Sci Technol 15:1353–1360
Zhang Y, He P, Li H, Li G, Liu J, Jiao F, Zhang J, Huo Y, Shi X, Su R, Ye N, Liu D, Yu R, Wang
Z, Zhou M, Jiao N (2019) Ulva prolifera green-tide outbreaks and their environmental impact in
the Yellow Sea, China. Natl Sci Rev 1–14
Chapter 11
Enzymes
Abstract Enzymes play important roles in catalyzing biological processes such

as breaking down complex compounds in food into simpler forms which can be
utilized to produce energy and aid metabolism and growth. These enzymes are also
used commercially in products such as biological cleaning agents, biofuel, food and
medicine. Aquatic sources of enzymes include internal organs of fish, carnivorous
plants and microorganisms. Organisms which inhabit extreme aquatic environments
are also sources of enzymes which perform better than other enzymes in industrial
processes. Some aquatic enzymes also provide the option of more environmentally
friendly processes for biofuel production.
Keywords Enzymes · Aquatic · Proteins · Extremozymes · Biopolymers
11.1 Introduction
In other chapters of this book, different forms of proteins such as collagen, aquatic
plants and algae proteins have been covered. Enzymes are discussed in a separate
chapter as they are indeed a diverse group of compounds, and their source, processing
and commercialization can be differentiated from other proteins. While the majority
of enzymes are sourced from microorganisms, plants and terrestrial animals, the
aquatic environment is a great source of a wide variety of enzymes. Since 70% of the
earth is made up of water and life itself is thought to have begun in water, the aquatic
environment is therefore expected to be an even more abundant source of enzymes
compared to land.
Enzymes play numerous roles in a range of food, pharmaceutical, chemicals and
cleaning products. Their use also extends to biofuel production where enzymatic
transesterification of fats can be used in biodiesel production as an alternative to
the alkali-catalyzed transesterification. A number of processes would either occur
too slowly to be commercially feasible or not at all without the use of enzymes.
In food processing, enzymes are used in the production of products such as fish
sauce, they are also employed in more effective alternative processes for product
preparation such as fish descaling. Within the chapter, some of the different industrial
applications of different enzymes are discussed. The origins of these enzymes, the

234 11 Enzymes
environmental implications of their production process, biochemistry, as well as the

demand and commercial production are also discussed with the aim of giving the
reader an understanding of the integral role of these classes of aquatic polymers
within the natural world and in industry.
The diversity of organisms present in the aquatic environment and the intricate
food chain makes it a promising source of enzyme-producing organisms. The large
variety of organisms present in the aquatic environment, some of which feed on
other organisms and non-living matter means these organisms in the aquatic envi-
ronment produce the enzymes necessary to catalyze a wide variety of biological
processes. Organisms including fish which produce proteolytic enzymes for break-
ing down proteins in the other sea life they feed on, marine bacteria which produce
chitinolytic enzymes for breaking down chitins in the shells of crustaceans, and
amphipods inhabiting the Challenger Deep of the Mariana Trench, the deepest part
of the ocean, produce cellulases and amylases that allow them to digest wood from
shipwreck and fallen plants as a means of surviving in the deep dark part of the sea
where food is extremely scarce at extreme temperature and pressure (Kobayashi et al.
2012). Other examples of enzymes obtained from aquatic sources include trypsin,
pepsin, lipase, collagenase and chymotrypsin.
Fish, particularly the internal organs of fish, fish viscera, is one of the most widely
explored aquatic sources of enzymes as an alternative to the conventional sources
(microorganisms, terrestrial plants and animals). Enzymes found in fish include
hydrolases, proteases and carbohydrases, listed in order of their abundance (Kim
et al. 2002). Algae are also a promising aquatic-sourced alternative for enzyme pro-
duction. Algae have the ability to break down a wide range of compounds in the
water and use for energy and carbon production. They can take up nutrients from
water and break down carbon compounds. Photosynthesizing algae also make use of
light to power their growth and energy production. They also have biochemical pro-
cesses in place to protect from pathogens and environment. To do all these, they make
use of enzymes which facilitate their synthesis. It is therefore expected that when
they are harvested some of these enzymes remain within their biomass. Enzymes
present in algae include carboxylase, oxygenase, dehydrogenase, amylase, cellulase,
lipase, sucrose, phosphate, sulfatase, glycogen synthase, phosphatase, starch syn-
thase, glycolate oxidase and peroxidases. All of these serve specific roles in the algae
(Mogharabi and Faramarzi 2016). For example, superperoxide dismutase which is
found in red algae as well as blue-green algae catalyzes the process which defends
the algae from oxygen toxicity. The amount and composition of the enzymes present
vary for different species of algae.
A wide variety of microorganisms exist in different zones of the aquatic envi-
ronment. These microorganisms have adapted to survive in a range of conditions.
Examples are microorganisms which exist in the underwater volcanoes of the deep
sea where temperatures could be over 100 °C, while other bacteria are found in higher
depths in waters where the temperatures can be as low as −2 °C in, for example, the
Antarctic waters. The relatively less complex structure of the microorganisms makes
them an attractive option for commercial enzyme production. This also allows the
possibility of genetically engineering microorganisms to produce desired enzymes,
11.1 Introduction 235
where the gene coding for enzyme production from an aquatic organism can be
placed in the nucleus of that of another organism which grows more effectively at
the industrial scale. Microorganisms can be cultured in bioreactors which mimic the
conditions in the part of the aquatic environment they inhabit for large-scale enzyme
production. Within this chapter, we discuss an example process for production of
enzymes from microorganisms obtained from the aquatic habitat.
Thus far within this text, we have discussed the main polymers extracted from
algae for commercial applications. These include the common ones such as alginates,
carrageenan and agar and those produced in lesser amounts like fucoidan, laminarin
and ulvan. In the process of extraction of these biopolymers, the enzymes are mainly
discarded or destroyed alongside other residues. However, algae are a source of a
variety of enzymes of commercial value. It is therefore worth exploring the potential
for commercial enzyme production from algae.
This chapter therefore explores the diversity of enzymes which are obtained from
the aquatic environment, and how the aquatic-sourced enzymes differ from those
of the terrestrial origin and in doing so the economic and environmental impact of
aquatic-derived enzymes.
The aquatic environment is host to a diverse range of organisms. These organisms

have adapted the means to survive a range of conditions which range from mild
to extreme temperature, light, salinity and pressure. The complexity of life in the
aquatic environment has resulted in the organisms inhabiting the aquatic environment
evolving a range of biochemical processes for survival. These biochemical processes
are catalyzed by the diverse range of enzymes, thus making the aquatic environment
a formidable source of enzymes. Microorganisms, plants and animals within the
aquatic environment metabolize enzymes which have several industrial applications.
Some of these such as lipase and proteases are well established commercially, while
some are still at infancy in terms of commercial application.
11.2.1 Microorganisms
The ease of genetic manipulation and mass culture for commercial production make
microorganisms attractive source of commercial enzymes. Moraxella from Antarctic
seawater and marine streptomycete are examples of microorganisms which produce
the enzyme lipase (Zhang and Kim 2010). Vibrio fluvialis, Listonella anguillarum and
Vibrio mimicus are examples of marine bacteria which produce the enzyme chitinase
which hydrolyzes chitin. Microorganisms play a significant role in the breakdown
of the dead tissue, and organisms in the aquatic environment, for example, the shed
scales of marine zooplankton, are converted to carbon and energy by microorganisms
236 11 Enzymes
Table 11.1 Examples of some enzymes produced by different organisms

Enzyme Example source Substrate References
Cellulase Cladosporium Cellulose Trivedi et al. (2015)
sphaerospermum (isolated
from Ulva algae)
Chitinase Pseudoalteromonas piscicida Chitin Paulsen et al. (2016)
Lipase Pseudomonas putida Triglyceride Sivasubramani et al. (2013)
Protease B. mojavensis (from Protein Haddar et al. (2009)
seawater)
Agarase Acinetobacter sp. PS12B Agar Leema and Sachindra (2018)
(marine bacteria)
Alginate lyase Vibrio splendidus Alginate Zhuang et al. (2018)
Alginate lyase Mollusk (Lambis sp.) Alginate Sil’chenko et al. (2013)
Carrageenase Cellulophaga lytica Carrageenan Yao et al. (2013)
Amylase Hirondellea gigas (giant Amylose Kobayashi et al. (2012)
amphipod)
Fucoidanase Bacterium (Formosa algae) Fucoidan Silchenko et al. (2013)
Cutinase Thermobifida fusca Cutin Hong et al. (2019)
which degrade chitin, and this serves as a source of food for these microorganisms.
Humans have for long developed methods to breakdown polymers for different pur-
poses such as production of fuel and processing of food, using chemical catalysts.
Due to limitations and some adverse effects of the use of such chemicals, there
is a shift toward the use of biological catalysts to replace the chemical catalysts.
Table 11.1 gives some examples of catalysts and their sources.
The enzymes obtained from microorganisms have shown superior stability and
activity compared to enzymes from plants and animals. However, the need to optimize
the aquatic resource and the existing need to minimize the waste from the processing
of resources such as seafood make the production of enzymes from aquatic plants
and animals just as important.
11.2.2 Symbiotic Microorganisms
Aquatic organisms often have symbiotic relationships which aid their individual
survival. Some of the microorganisms present in larger organisms produce enzymes
which have industrial applications. For example, symbiotic bacteria present in the
marine shipworm produce protease (Greene et al. 1996). These microorganisms
harbored by the fish can then be removed from the respective organ, while the rest of
the fish is used for food and other products. Microorganisms existing in the seawater
are also a source of enzymes. These microorganisms can be collected and harvested
in bioreactors for the production of desired enzymes. One such enzyme-producing
microorganism found in seawater is the Bacillus mojavensis A21 (Haddar et al. 2009)
which produces protease.
11.2.2.1 Biofilms
Some microorganisms form biofilms which protect them from harsh conditions and
substances. The biofilm also supports their adhesion to wet surfaces in the natural
aquatic environment and in wet surfaces in everyday life. These biofilms are com-
posed of polymeric materials which are present in the exoskeletal matrix of microor-
ganisms such as polysaccharides. Alginate forms a large amount of the biofilm mate-
rial. At different points in the developmental stage, the biofilms need to be broken
down to allow spreading of the film and/or the bacteria. This breakdown is achieved
by alginolytic enzymes produced by the microorganisms. Recently, the possibility of
using the alginate lyase enzyme produced by these biofilm-forming microorganisms
to destroy biofilm-forming bacteria is being explored (Daboor et al. 2019). Biofilms
aid resistance of bacteria to antibiotics in humans and antiseptic chemical in clean-
ing of surfaces. Extraction of alginolytic enzymes from biofilms to then attack the
biofilms of these resistant bacteria provides a promising way to address antibiotic
resistance.
11.2.3 Fish Viscera
Some of the common commercially available enzymes are sourced from the internal
organs (viscera) and muscles of fish (Shahidi and Kamil 2001). These parts are
generally not eaten; therefore, their production does not compete with food. It is
also in line with the sustainable development goal of optimizing the utilization of
fisheries resources since these parts are otherwise discarded. Examples of enzymes
from aquatic source are the pepsin and collagenase from crab hepatopancreas (Kim
et al. 2002). Pepsin is also obtained from fish. Extracts from squid offal contain
collagen-degrading enzymes. The occurrence of enzymes in an aquatic animal can
be affected by the type of diet the animal consumed. For example, juvenile green
abalone showed a variation in the digestive enzymes they produced when fed with a
diet of either algae or sea grasses (Garcia-Carreno et al. 2003). This can be attributed
to the fact that the body will produce the type of enzyme required to digest the food it
consumes. The activity of the enzyme produced by an organism at certain conditions
is dependent on the habitat of the organisms.
238 11 Enzymes
11.2.4 Carnivorous Plants
While some plants depend on the nutrient supply from the soil or water they are
grown in for their growth and survival, there are a special group of plants which have
adapted features that allow then to capture prey, digest them and use their biomass as
nutrients. Examples of species of aquatic carnivorous plants are Aldrovanda vesicu-
losa, Utricularia vulgaris, U. reflexa Oliver, U. stygia Thor and U. intermedia Hayne
(Adamec 2010). However, there are over 700 species of known carnivorous plants.
These plants mainly feed on insects which they lure into the trap using sweet
nectar they secrete. Once the insect is trapped, the enzymes within the plants digest
the insect to produce the nutrients needed by the plants. The enzymes produced by
the plants must therefore include all the essential enzymes to break down protein,
lipids, chitin, glycogen and other compounds which make up the insect’s body that
can be converted to produce the carbon, nitrogen, sulfate and phosphate needed for
plant growth. These carnivorous plants have been confirmed to have different enzyme
compositions for digesting prey (Schulze et al. 2012). The minerals present in the
insect body are also taken up by the plant. These enzymes are expected to have
quite significantly high activity as some of these plants have been known to digest
relatively large rodents.
Other than the enzymes they produce, carnivorous plants also make use of diges-
tive enzymes of the symbiotic bacteria present within them to digest prey. An example
of a carnivorous plant which has been confirmed to do this is the Utricularia brevis-
capa (Lima et al. 2018). This carnivorous plant grows in the floodplains in Brazil and
can therefore be classified as aquatic. It floats on water by means of its parenchyma
which is filled with air and traps insects with the aid of its segmented leaf structure
with utricles which capture prey by creating a hydrostatic pressure. Bacteria species
which are present in these carnivorous aquatic plants include Bacillus, Aquitalea,
Sphingomonas, Azospirillum, Chromobacterium, Novosphingobium and Acidobac-
teria (Lima et al. 2018). Therefore, a carnivorous aquatic plant can have a host of
bacterium in its trap which aids in the digestion of the prey. The types of bacteria
and population distribution vary depending on the habitat.
Understanding the mechanisms of action of such plants could contribute toward
the development of plant-based biological pest control and enzyme-based pesticides
for aquaculture.
11.2.5 Extremophiles
Some organisms have adapted to survive in extreme conditions which require them to
produce enzymes not commonly found in organisms of their kind. An example of such
is the deep-sea giant amphipod, Hirondellea gigas, which has developed enzymes
for digesting wood (Kobayashi et al. 2012). These organisms exist in the Mariana
Trench, which is the deepest part of the ocean where the most extreme conditions exist
and little life or organic matter exists to feed on. Other wood-digesting organisms
which live in the higher zones of the water like shipworms and piddocks also exist.
The activity of the enzymes and optimal conditions for activity vary as a result of
the difference in the conditions under which these organisms exist.
This adaptation to extreme conditions gives enzymes sourced from these
extremophiles the particular advantage of having relatively higher activities at
extreme conditions (Fernandes 2016). Since the aquatic organisms which are the
source of these enzymes are adapted to processing food and metabolic activities
using these enzymes at such extreme conditions, the enzymes they produce for these
activities are therefore conformed to retain their activity at such conditions. Such
property becomes commercially useful where products such as food and pharma-
ceutics need to be processed at extreme conditions while retaining the activity of the
enzymes.
Enzymes are therefore abundant in the aquatic environment and are present in a
diverse range of organisms. Their occurrence in a diverse range of conditions such
as temperature, salinity, pH, pressure and light intensity gives rise to the availability
of enzymes that are optimally active in more varied conditions compared to enzymes
sourced from terrestrial organisms. The fact that the aquatic environment makes up a
larger portion of the earth and hosts more diversity of organism also results in more
naturally occurring enzymes in the aquatic environment.
11.3 Chemistry of Some Aquatic Enzymes
Enzymes are mostly proteins (some enzymes exist that are not proteins), chains of
amino acids linked together by peptide bonds. Amino acids being the repeating units
of these proteins are a diverse class of compounds which are characterized by the
presence of an amine and a carboxylic acid group attached to an alkyl. Enzymes can
have molecular weights in the thousands to tens of thousands range. Proteinase A,
for example, has a molecular mass of 50 kDa, while proteinase B has a molecular
mass of 95 kDa (Komori and Nikai 2013).
The functioning of an enzyme depends on the primary, secondary and tertiary
structure of the enzyme. Most enzymes are globular proteins which fold up in spe-
cific patterns in their tertiary structure. This unique tertiary structure allows them
to combine with specific substrates in a unique manner and by so doing catalyzes
specific processes. The activity of enzymes varies at different conditions. For exam-
ple, alkaline proteinase will act at pH above 7, while acid proteinase will act at the
low end of pH and lipases will only act at the interface between water and oil since
lipase is hydrophilic and the triglyceride it breaks down is hydrophobic (Bele et al.
2014a, b). Therefore, unlike nutritional protein which is required to be broken down
to produce amino acids which are then utilized in this form, enzymes are required to
retain their tertiary structures in order to serve their purpose.
Some enzymes are specific to particular bonds, while some can catalyze a wide
range of processes. For example, lipases catalyze the breakdown of fats to produce
240 11 Enzymes
fatty acids, glycerol and monoglycerides, while proteases cleave amide bonds to
produce shorter chain peptides. Figure 11.1 illustrates the cleavage of amide bond to
form a more stable cyclic peptide obtained from Ulva rigida (Seca and Pinto 2018),
and Fig. 11.2 illustrates the breakdown of triglyceride into fatty acids and glycerol.
These are examples of two different processes which are catalyzed by proteolytic
enzymes and lipase enzymes, respectively.
Lipases also catalyze a wide range of reactions which include hydrolysis,
acidolysis, esterification, alcoholysis, aminolysis and interesterification (Bele et al.
2014a, b). In catalyzing the range of reactions which break down fat, lipases can also
be used to catalyze the synthesis of other lipids. Examples of such are the acidolysis
to release the hydroxyl from the COOH group from the fatty acid and alcoholysis
which releases the alcohol group of the glycerol allowing the formation of triglyc-
eride. Therefore, lipases are not just able to catalyze the breakdown of triglycerides
but also formation of other similar compounds. An example of lipase-catalyzed lipid
production is the formation of ergosterol ester (He et al. 2019) from ergosterol, a
cholesterol equivalent produced in fungi (Bell 2007). Lipase-catalyzed formation of
Fig. 11.1 Cleavage of amide linkage to form amino acid

11.3 Chemistry of Some Aquatic Enzymes 241
Fig. 11.2 Transesterification reaction
ergosterol ester from ergosterol is an important process because the ergosterol ester
has more diverse application than ergosterol. The ester form has a lower melting
point, higher solubility in oil and improved stability.
The presence of enzymes in a sample can be confirmed by their level of activity.
For example, to confirm the presence and activity of proteolytic enzymes, the amount
of protein degraded into amino acid can be measured using the Bradford method and
the amount of lipase can be measured by introducing into a lipid sample such as
castor oil and quantifying the amount of fats converted to fatty acids in a given time
by titrating with an alkali and using an indicator to identify the quantity of alkali
required to neutralize the fatty acids formed. The formation of clear white crystals
upon introduction of a lipase containing sample onto agar plates containing 1% (v/v)
Tween 20 or Tween 80 is also used as an indication of lipase activity (Bele et al.
2014a, b).
Understanding the chemical structure of different enzymes is important in iden-
tifying their activities and hence applications. This provides a better understanding
of the aquatic world. For example, the ability of salmon sharks to maintain relatively
high body temperature in the cold Alaskan waters which could be as low as −2 °C
(Bernal et al. 2005) is partly attributed to the difference in the enzyme activity in
red and white muscles and the arrangement of these muscles within the organism’s
internal organs (Glancy and Balaban 2011).
11.3.1 Enzyme Stability in the Deep Sea
The tertiary protein structure of enzymes needs to be retained in order to ensure

they remain active and serve their intended function. The tertiary structure can be
destroyed by factors such as temperature, pH, salinity and pressure. The functioning
of these enzymes is crucial to the survival of the organism; therefore, for organ-
isms living in extreme conditions, certain mechanisms are put in place to retain the
enzyme activity. Understanding the way in which the enzyme structure is retained at
such extreme conditions can be adapted to commercial applications of enzymes, for
example, in developing enzyme-catalyzed reaction at high pressure.
At high pressures in the sea, marine organisms have developed a special com-
pound which allows them to retain their protein structure at such pressures. This
242 11 Enzymes
compound is referred to as trimethylamine oxide (TMAO) (Seibel and Walsh 2002).

Although not a polymer or an enzyme in itself, its unique interaction with proteins
is of much significance to better understanding, conserving and utilizing the aquatic
environment and the resources therein. Water molecules are much smaller than pro-
tein molecules such that they can penetrate into the protein structure at high pressure
and destabilize the protein (Yancey et al. 2014). In the absence of TMAO, the small
molecules will be pushed into the protein structure and cause disruption of the pro-
tein tertiary and secondary structure thus preventing it from functioning, eventually
resulting in the death of the organism.
Although the precise mechanism of action is yet to be uncovered, it is seen in
higher concentration in the organisms which live in these deep waters compared to
those in the lower depths, and hence, it is attributed to their ability to survive at
the high pressures which exist in the deep sea. TMAO is also attributed with the
resistance of the enzymes in the fish to urea and also preventing the freezing of the
fish’s bodily fluid at the low temperatures in the deep sea (Seibel and Walsh 2002).
The compound which helps retain the enzyme structure and hence activity in fish
against the high pressure in the deep sea may, however, pose a health risk for humans.
TMAO has been associated with adverse cardiovascular events in humans (Velasquez
et al. 2016). However, more studies are required to confirm the mechanism by which
this occurs and other associated factors.
In 2016, 89,000 tonnes of microalgae was farmed across 11 countries of the world,
88,600 tonnes of which was from China. These include species such as Haemato-
coccus pluvialis, Nannochloropsis spp., Chlorella spp. and Spirulina spp., all being
farmed in large, medium and small scales. While macroalgae get a larger revenue
from their food sales, microalgae are mostly sold as high-value functional products.
Therefore, despite the lower annual tonnes produced, microalgae are valued at around
a billion USD annually, compared to that of macroalgae at 6 billion USD.
The protein content in macroalgae is comparable to those found in animal-based
proteins and is higher than those found in land plants such as soybean, wheat and
legumes. Algae yield around 2.5–7.5 tonnes per hectare annually, while microalgae
yield 4–15 tonnes per hectare annually. These yields are rather high compared to
the conventional plant-based proteins such as wheat, soybeans and legumes which
yield 1.1, 0.6–1.2 and 1–2 tonnes per hectare annually (van Krimpen et al. 2013).
Macroalgae and microalgae could contain similar or even more protein than terrestrial
plants typically used as protein source. Spirulina, microalga which have gained much
popularity as a nutrient source, could contain up to 63% protein per dry weight
(Tokusoglu and Unal 2003). The red algae species Porphyra tenera contains up to
47% protein per dry weight (Fleurence 1999).
Protein content in any particular algae varies with factors such as growth season,
temperature, harvest period, region and nutrient content of water. The types of pro-
teins present also vary. How different species react to particular changes in growth
condition in turn affects the types of proteins they metabolize. The algae make use of
these proteins to survive and function within its environment; therefore, the stimulus
it gets from these environments determines what protein it is prompted to produce.
These factors can be used to manipulate a particular alga to produce desired type
of protein by controlling the growth environment. This requires an in-depth under-
standing of the correlation between environmental factors and the metabolism of
the specific species. For example, highest protein yield is obtained from the alga
K. alvarezii in August, November and February when studied over 12 months from
September 2004 to April 2006 in northwestern India (Kumar et al. 2014).
Aquatic plants such as water fern, duckweed and water hyacinth contain relatively
moderate to high amount of proteins, 28% protein content by dry weight has been
reported for water fern (Brouwer et al. 2019) and 21.5% for duckweed (Aguilera-
Morales et al. 2018). While there are up to hundreds of thousands of species of algae,
some of which belong to the plant kingdom, there are just about a hundred species
of non-algae aquatic plants. The recent rise in algae bloom incidents in different
parts of the world further eludes to the fast rate of growth of algae. Water hyacinth,
ferns and duckweed are also fast-growing plants which can be explored as abundant
sources of aquatic proteins. While they could serve as an abundant source of proteins,
well managed and controlled systems for the cultivation of aquatic plants and algae
are important in order to avoid undesirable population blooms which could result in
devastating environmental impacts.
11.5 Extraction of Enzymes from Aquatic Organisms
The processes involved in the isolation of enzymes depend on the source from which
it is being extracted. Here we take examples of methods reported for the extraction
of enzymes from fish viscera, aquatic microorganisms and algae.
11.5.1 Enzyme Extraction from Fish Viscera
The fish viscera can be obtained as a by-product of fish processing factories, for
example, in the production of sushi, canned fish, dried fish and packed fish fillets.
In all the aforementioned processes, the internal organs of the fish are removed as
waste. The internal organs can also be collected from the fishmongers in markets.
Here the method used by Jayapriya et al. (2014) is presented as an example for the
extraction of digestive enzyme from fish viscera.
The collected organs are washed with distilled water followed by grinding. The
pH is then adjusted to 8 by adding Tris-HCl (0.02 M). Crude extract is obtained
244 11 Enzymes
as the supernatant after centrifugation at 6000 rpm for 15 min. This crude extract
contains the enzymes mixed with other compounds. Ammonium sulfate at 80%
saturation is then added to precipitate the crude enzyme from the liquid supernatant.
The precipitate is then separated from the rest of the liquid by centrifugation at
10,000 rpm for 15 min. To further purify the enzyme, the solid precipitate is dissolved
in 0.2 M Tris-HCl buffer at pH 8. This solution is then purified by dialysis. This
method can be applied for the extraction of enzymes from waste of fish such as seer
fish sardines, great barracuda, red snapper and milk shark. The protease extracted
using this method has optimum enzyme activity at a pH of 10.
11.5.2 Extraction of Enzyme from Algae
The process of isolating enzymes from algae requires first freeing up the enzymes
from the solid biomass. Like most enzymes, the enzymes present in algae are water-
soluble globular proteins. Homogenization of the algae biomass followed by sep-
aration of the liquid and solid by centrifugation will yield a liquid supernatant
which comprises a mixture of some soluble polysaccharides and other proteins. The
enzymes can then be precipitated out of the solution. This is then redissolved and
further purified using methods like gel filtration (Mogharabi and Faramarzi 2016).
In one example extraction process reported by Bele et al. (2014a, b), the algae
biomass is collected and washed with water to remove debris. It is then dried and
ground into powdered form. The powdered algae biomass is mixed with distilled
water to allow all water-soluble components to dissolve. The liquid is then separated
from the solid mass. The protein is precipitated out of the aqueous phase using
ammonium sulfate added at a mass-to-volume ratio of 0.0663 g:1 ml. The solid
protein precipitate is then recovered by centrifugation at 10,000 g for 15 min at 4 °C.
The solid fraction contains the enzyme. This is then redissolved in trichloroacetic acid
(TCA) buffer and further purified by gel filtration using Sephadex grade 100 (Bele
et al. 2014a, b). This method was used to extract enzymes from Ulva lactuca, Ulva
fasciata, Chaetomorpha antenna, Gelidium pusillum and Enteromorpha compressa.
Magnesium sulfate has been conventionally used for precipitation of globular
proteins for several decades (Howe 1921). Other salts such as sodium sulfate and
magnesium sulfate can also be used for similar purposes. The choice of salt depends
on the requirements of the processor. For example, protein precipitates formed using
magnesium sulfate are gelatinous in nature and this results in relatively slow filtra-
tion; sodium sulfate is preferred when working at temperatures above 34 °C, and
ammonium sulfate can be used where the nitrogen element in the salt does not pose
a challenge in processing or analysis.
Enzyme extraction from marine algae can also be carried out using two-phase
extraction with PEG-4000 (Bele et al. 2014a). In this method, sodium sulfate is
added to the supernatant of the centrifuged sample 0.75 g in 10 ml mass of sample per
volume of salt solution. To this, ~3 ml of PEG-4000 is added at a 50% concentration.
11.5 Extraction of Enzymes from Aquatic Organisms 245
This results in the solution separating into two phases (indicating the presence of
globular proteins). The phases can be separated by pipetting out each phase.
In a third method, the protein can be separated using acetone–ether precipitation.
An oil–water emulsion is first formed using castor oil and sunflower oil in a bile salt
solution at a neutral pH and gum acacia as emulsifier. The ground algae biomass
is homogenized with acetone at ice-cold temperature. This is followed by filtration
with the solid residue retained and washed with acetone, an equal volume of acetone
and ether and then with ether. This process removes the lipids, polysaccharides and
other compounds which dissolve in acetone and ether from the algae leaving behind
the protein as the solid mass. The water-soluble protein is then obtained from the
dry mass by dissolving in cold water followed by centrifugation at 15,000 rpm for
10 min. The supernatant is obtained as the enzyme containing fraction (Bele et al.
2014a, b).
The salts used in precipitation can be removed by methods such as ion-exchange
resin or gel filtration using Sephadex G-25. The method of precipitation significantly
affects the yield of protein and the enzyme activity. For example, using the ammonium
sulfate precipitation method, protein concentration in the extract from the alga E.
intestinalis was 117 μg/ml and the enzyme activity was 0.123 meq/min/g using
castor oil as the fat substrate. Protein concentration from U. lactuca was lowest of
all the algae tested in the said study. It had a protein concentration of 87 μg/ml and
an enzyme activity of 0.109 meq/min/g (Bele et al. 2014a, b).
Upon purification using methods like gel filtration, the purity of the enzyme is
confirmed by SDS-PAGE (Jayapriya et al. 2014). This confirms the molecular weight
of the polypeptide extracted from the sample. A single band confirms that the pro-
tein extracted has a single molecular weight value. For example, purified protease
extracted from great barracuda viscera shows a single band at 34 kDa (Jayapriya
et al. 2014), while crude lipase extracted from algae shows a band ranging between
45 and 60 kDa (Bele et al. 2014a, b), indicating a less pure sample.
Since in both cases the enzyme is purified mainly of the bases of molecular
weight, the extract contains all proteins within a specified molecular weight range.
Some of these might not be enzymes and will also contain a combination of different
enzymes. The distinction is made by introducing the enzyme into a reaction it cat-
alyzes following incubation at the appropriate conditions. For example, the extract
from algae is mixed with castor oil formulation following incubation at 37 °C and
shaking at 200 rpm (Bele et al. 2014a, b). The amount of fatty acid produced is then
determined by titration against alkali (sodium hydroxide) to obtain a measure for the
lipase activity present within the sample.
11.5.3 Enzyme from Marine Microorganisms
Several marine organisms isolated from the waters and from aquatic organisms have
proven to be reliable sources of enzymes such as chitinase, cellulase, lipase and
246 11 Enzymes
agarase. Here we look at the process of extraction of lipase from marine microorgan-
isms. The general approach to commercial production of enzymes from microorgan-
isms is to cultivate the microorganism in a medium which promotes the metabolism
of the desired enzyme. For this purpose, it is important to study the growth kinetics
of the microorganism in order to identify the growth phase during which it produces
the desired enzyme at optimal rate. Once the desired amount of enzyme is produced
by the microorganism, the rest of the process is then directed at recovering and
purifying the enzyme free of the microorganism. One of the challenges of sourcing
enzymes from microorganisms is the risk of contamination of the final product with
the microorganism which could be pathogenic, especially where the enzyme is used
in food or pharmaceutical products.
Enzyme production from microorganisms is illustrated here using the method
presented in the literature for the extraction of lipase from marine bacteria as a case
study. In the study by Sivasubramani et al. (2013), marine bacterium Pseudomonas
putida isolated from the Vellar estuary in the Cuddalore district, Tamil Nadu, South-
ern India, is used as the source of the lipase enzyme. Other lipase-producing marine
bacteria strain includes Bacillus spp. (Valsa et al. 2003) and marine Streptomyces
sp. (Yuan et al. 2016).
The temperature, pH, salinity and nutrient composition are critical parameters to
be controlled when culturing microorganisms for enzyme production. In this partic-
ular example, the optimal values for these parameters were a neutral pH, 35 °C and
a sodium hydroxide concentration of 0.6%. The nutrient in the medium consisted
of 1% dextrose and 0.2% yeast extract. Companies such as Sigma-Aldrich supply
nutrient media which are optimal for required microorganism culture. The culture
was incubated for a duration of 48 h.
First, the microorganisms are isolated from the water of the estuary. The sample
is then incubated in Tween 80 agar plates to isolate the lipase-producing microbes.
The optimal growth conditions of the most productive strain are then identified by
growing at different growth conditions and finding the optimum pH, temperature,
salinity, nutrient composition and duration. The bacteria strain is then grown in a
shake flask using the optimal parameters determined. For a particular microorganism
and desired enzyme, the optimal growth conditions will vary. The optimal conditions
will also vary for different strains; therefore, the processor must determine the optimal
growth conditions for a specific strain and target enzyme.
The inoculum is then introduced into a bioreactor in a ratio of 1:100 (volume of
inoculum to volume of bioreactor medium). The culture was grown over a duration of
48 h at the optimal conditions. At the end of the process, the medium was filtered. The
liquid filtrate was precipitated with ammonium sulfate followed by centrifugation
at 3000 rpm for 30 min at 4 °C. The solid precipitate is then dissolved in 0.05 M
Tris-HCl and then purified by dialysis. The enzyme activity of the purified protein
was 9.474 U/mg at the highest when tributyrin is used as the triglyceride substrate.
SDS-PAGE analysis showed that the partially purified protein sample containing
the enzyme derived using the method described had molecular weights of 34, 45
and 52 kDa, which are within the range of molecular weight of lipase. The actual
molecular weight of lipase has been reported to be lower, and others have reported
11.5 Extraction of Enzymes from Aquatic Organisms 247
higher molecular weights of lipase from SDS-PAGE analysis. For example, Yuan
et al. (2016) reported lipase extracted from marine bacteria with a molecular weight
of 29 kDa.
The enzyme activity varies depending on the organism it is sourced from.
Table 11.2 compares the enzyme activity of some enzymes from different species of
fish, algae, microorganisms and the giant deep-sea amphipod H. gigas. The different
units are given as presented in the different studies to measure enzyme activities. In
the study carried out of H. gigas (Kobayashi et al. 2012), the enzyme activity of the
different enzymes present in the deep-sea residing amphipod varied significantly for
each enzyme. The amylase activity 112.1 mU/μg is much higher than the cellulase
activity. The activity of the other enzymes, protease, xylanase and mannanase is even
much lower. This is indicative of the types of nutrients available to the organisms for
energy and growth.
Table 11.2 Enzyme activity of protease and lipase from different sources
Enzyme Source Activity References
Lipase Ulva lactuca (Puducherry) 0.057 meq/min/g Bele et al. (2014a, b)
Lipase Enteromorpha compressa 0.062 meq/min/g Bele et al. (2014a, b)
(Puducherry)
Lipase Gelidium pusillum 0.086 meq/min/g Bele et al. (2014a, b)
(Kovalam)
Lipase Chaetomorpha antenna 0.068 Bele et al. (2014a, b)
(Puducherry)
Lipase Marine bacteria 1.763 U/mg Sivasubramani et al.
Pseudomonas (2013)
Lipase Bacteria from Antarctic 7.894 U/mg Rashidah et al. (2006)
water Arthrobacter
gangotriensis
Protease Red snapper Crude: 3.79 U/mg Jayapriya et al. (2014)
Purified: 58.80 U/mg
Protease Great barracuda Crude: 1.51 U/mg Jayapriya et al. (2014)
Protease Sardines Crude: 2.32 U/mg Jayapriya et al. (2014)
Protease Milk shark Crude: 1.53 U/mg Jayapriya et al. (2014)
Protease Aureobasidium pullulans Purified: 623.1 U/mg Zhang and Kim (2010)
(marine yeast)
Amylase H. gigas 112.1 mU/μg Kobayashi et al. (2012)
Mannanase H. gigas 8.22 μU/μg Kobayashi et al. (2012)
Cellulase H. gigas 3.42 mU/μg Kobayashi et al. (2012)
Protease H. gigas 0.26 mU/μg Kobayashi et al. (2012)
248 11 Enzymes
It is noted that the process of recovery of the enzymes from the solid mass fol-
lowing cultivation of the microbes or homogenization of plant and animal biomass
is quite similar. The common goal is therefore to free up the proteins and then isolate
the desired enzymes.
11.6 Immobilization of Enzymes
Since enzymes are biological catalysts, they do not take part in the actual reaction
or biochemical process, they serve as surfaces for substrates to attach, and once
the process is complete, the enzymes are released and become available again. In
industrial processes, the enzymes need to be recovered and stored in a suitable form
for reuse. This is achieved by immobilizing the enzyme. Enzymes are mostly pro-
teins which are soluble in water. This makes their recovery quite challenging after
a process; evaporation or crystallization at high temperature is not an option since
most enzymes are denatured or completely destroyed at temperatures about ~45 °C.
Immobilization of enzymes therefore entails converting them into insoluble forms
which can be more effectively collected, stored and reused. This can be achieved by
chemical or physical immobilization process (Dutta 2008). Other than being a source
of enzymes, aquatic organisms are also used in the immobilization of enzymes and
these are also discussed within the section.
Whole cells of enzyme-producing organisms can also be immobilized with the
activity of the enzyme and organisms remaining active. Although the use of immo-
bilized enzymes and biological catalysts is at industrial scale which is still at infancy
(Moreno-Garcia et al. 2018), immobilized enzymes offer benefits such as avoiding
the live organism or enzyme from getting into the final product and cost savings.
Whole algae cells of N. muscorum, for example, can be immobilized in sodium algi-
nate. When immobilized in 2% alginate at 30 °C, a concentration of 0.5 g/L and a
stirring rate of 100 rpm, the cells retained their activity even after five cycles of use.
The algal cells also showed higher yield in the immobilized form compared to when
used in their free form to catalyze the bioconversion of androst-4-ene-3,17-dione
to testosterone (Arabi et al. 2010). The rest of this section discusses the different
enzyme immobilization techniques, and in the process, some biopolymer used in
enzyme immobilization is also discussed.
11.6.1 Chemical Immobilization
The chemical immobilization process can be done by covalently bonding the vacant
functional groups on the enzymes to insoluble supports. These supports could be
monomers which are then copolymerized with the enzymes. Chemical immobiliza-
tion can also be achieved through cross-linking the enzyme with multifunctional
11.6 Immobilization of Enzymes 249
reagents such as glutaraldehyde. In any form of chemical immobilization, it is impor-

tant that the active site of the enzymes is not altered; otherwise, the enzyme is
destroyed. Therefore, the functional groups involved are those which do not affect
the enzyme’s activity.
Natural polymers are also used in chemical immobilization of enzymes. Agarose
is commonly used for such application. Polymers generally have minimal reactivity
with enzymes; therefore, when used for immobilization of enzyme, polymers are
first activated with reagents prior to contacting with the enzymes. This pretreatment
is targeted at freeing up specific functional groups on the polymer to allow covalent
reaction with the inactive sites on the enzyme.
11.6.2 Physical Immobilization
In the physical method, immobilization can be achieved by entrapment, microen-

capsulation or adsorption of the enzyme within or unto a support. The adsorption
method makes use of surface-active compounds unto which the enzymes are phys-
ically adsorbed. Cellulose and collagen are examples of natural polymers used as
adsorbents for physical enzyme immobilization. Other adsorbents are calcium car-
bonate, alumina, clays and glass plates. Hydroxyapatite, although not a polymer,
is also an interesting material used as physical enzyme immobilization adsorbent
which occurs naturally in the aquatic environment. Hydroxyapatite is present in the
scales of fish where it makes up part of the extracellular matrix. Adsorption method is
particularly preferred as a method of enzyme immobilization as it is relatively simple
process, it is reversible and there is little chance of the enzyme being deactivated in
the process of immobilization. It, however, has the disadvantage of the weak bond
between the enzyme and the support which are mainly due to secondary molecular
interactions unlike the chemical method which are formed with covalent bonds.
The entrapment method makes use of cross-linked polymers within which the
enzymes are trapped. The polymer is mixed with the enzyme prior to cross-linking.
The enzyme dispersed within the polymer is then trapped within the network upon
cross-linking. In the microencapsulated method, the enzymes are placed in the
semipermeable membrane microcapsules. The microcapsules are formed using inter-
facial polymerization where the enzymes are encapsulated within the droplets of
polymers formed. This method yields an immobilized enzyme system with high
surface area.
Table 11.3 gives a summary of consumption in the process of a typical enzyme

extraction process using the method presented by Bele et al. (2014a, b) as an example
case study. The reader should note that the processes vary for different feedstocks.
250 11 Enzymes
Table 11.3 Estimated

Consumption Amount
consumption in enzyme
extraction from algae Water ~20 ml per gram algae
Energy Drying (atmospheric)
Grinding
Cooling (4 °C)
Centrifugation: 10,000 g at 4 °C
Filtration and purification
Biomass ~3.7 g algae biomass required per
gram protein
Salts and buffers Ammonium sulfate ~0.663 g per
gram of algae
TCA 10 ml per gram enzyme extract
Solid waste generated 82.8%
11.7.1 Cultivation of Algae
A wide variety of enzymes can be obtained from different algae species. Currently,
thousands of species of algae are not cultivated commercially (Arabi et al. 2010).
Production of enzymes from algae could increase the diversity of algae species being
cultivated for different purposes with enzyme production included.
11.7.2 Replacement of Alkali or Acid Catalyst in Biodiesel

Production
Production of biodiesel can be made even more environmentally friendly by elim-

inating the use of acids and alkali and replacing with lipase, a biological catalyst.
The current limitation with this approach to biodiesel production is that the enzyme-
catalyzed transesterification reaction is slower compared to the chemically catalyzed
one. The issue of reusability of the enzyme can be addressed by immobilization of
lipase on a support surface such that the enzyme can be used to catalyze several
cycles (Narwal and Gupta 2013).
The consumption of water begins with the first washing of the algae biomass to get
rid of debris. More water is then used to dissolve the proteins prior to centrifugation.
The exact amount of water used is not specified within the test referenced for the
extraction of enzyme from algae. However, taking a common mass-to-volume ratio
of 1:10 as a minimal used in similar extraction processes, we can estimate that a

minimum of 20 ml of water is used in the extraction of enzyme from 1 g of algae.
11.7.4 Use of Salts and Buffers
Where ammonium sulfate is used, this eventually breaks down into nitrogen, hydro-
gen and sulfate which can be taken up by plants and other organisms if adequately
disposed of. Unlike other more intensive processes such as the extraction of chitin
from crustacean shells or extraction of collagen from animal tissue which makes use
of strong acids and alkali, enzyme extraction is relatively milder and requires use of
milder chemicals.
The homogenization of the algae biomass into powdered form achieves improved
release of the proteins into water as a result of reduced particle size and mechan-
ical disruption, and this process requires significant amounts of energy. Drying of
the samples prior to homogenizing reduces the amount of energy consumed in this
process. The quantity of energy consumed at this stage varies depending on the
machinery used and the efficiency.
The drying process is done at ambient conditions; high-temperature oven-drying
is not an option here as this will result in the destruction of the tertiary structure of
the enzyme which is a key to their activity as catalysts.
Energy is also consumed in the centrifugation at different stages at ~10,000 to
15,000 g for periods of 10 and 15 min at low temperatures ~4 °C. Therefore, energy
is required for cooling of the system. Cooling is usually achieved using electricity
in refrigerated centrifuges. The environmental impact of both centrifugation and
cooling energy consumption depends on the source of electricity. This could be
hydroelectric, nuclear, gas, solar, etc.
While up to 60% of fish is eaten as food, the rest of it either goes to waste or
used in lower value applications such as fish meal, pet food, fertilizers and fish oil.
Production of enzymes from the solid waste generated from fish significantly boosts
the value chain. Enzymes have a much broader application in industry compared to
the former, and adding this to the value chain of the fish food industry can contribute
revenue which could compensate for the cost of production of other products from
the fish waste such as production of chitin from the fish scales (chitin is discussed
252 11 Enzymes
in a separate chapter in this book). Proteins make up around 17.2% of U. lactuca

(Aguilera-Morales et al. 2018) for example. This means that approximately 82.8%
of the rest of the algae form the solid waste generated. This can then be further
processed to extract other biopolymers such as ulva and carrageenan.
11.8 Applications
Enzymes from aquatic sources find applications ranging from food processing to
cleaning agents. In various chapters of this book, reference has been made to some
extraction processes which make use of enzymes such as proteases to extract biopoly-
mers. Therefore, enzymes play a role in the process of production of other valuable
products.
11.8.1 Biological Cleaning Agents
Much of the stains in everyday life are food stains, blood stains, ink stains or other
stains which are likely to have polymeric components such as protein in blood and
food. Enzymes which can break down these compounds are therefore ideal cleaning
agents as they can break down the large molecules of the stain into smaller molecules
which wash off more easily. An example of enzymes used for such application is
protease. Protease has been long known for its cleaning properties. Protease extracted
from the marine shipworm’s gland of Deshayes has superior cleaning power when
compared to the conventional phosphate detergents (Greene et al. 1996). The said
protease remains active at temperatures up to 50 °C and is completely inactivated at
70 °C. This is ideal as this is the general temperature range for eco-friendly washing
at room temperature. Protease also has potential application as a cleaning agent for
contact lenses as it retained stability and cleansing activity in the chemicals such as
hydrogen peroxide and sodium hypochlorite used in the cleaning and storage of such
product (Greene et al. 1996). Another example of protease from microorganism
which shows good cleansing properties is the protease obtained from a culture of the
bacterium B. mojavensis which is obtained from seawater (Haddar et al. 2009).
Protease from the viscera of fish has been shown to effectively remove blood
stain from cloth (Jayapriya et al. 2014). The effectiveness of the enzyme depends on
the species it is extracted from. For example, enzyme from great barracuda showed
a stronger stain removal activity compared to that from sardines, milk shark, seer
fish and red snapper. Other enzymes such as lipase which breaks down triglycerides
into fatty acids and glycerol can also be used to remove fat-based stains. Lipase is
obtained from marine algae (Bele et al. 2014a, b).
The enzymes are combined with regular detergents, solutions and chemicals to
serve as biological cleaning agents. It is therefore important that these enzymes are
stable at the pH and temperatures and maintain their activity and stability when in
contact with the different chemicals which are used in the cleaning products.
11.8.2 Biodiesel Production
The production of biodiesel is a transesterification reaction between fatty acid and

methanol in the presence of a catalyst. This yields glycerol and soap as a by-product
alongside the biodiesel. Lipase produced from algae has potential application in
biofuel production. As mentioned earlier, one of the reactions catalyzed by lipase
is the transesterification reaction. In fact, lipase-catalyzed transesterification has
up to sevenfold higher yield than sodium hydroxide-catalyzed transesterification.
This has been observed when the transesterification of lipids extracted from green
microalga Tetraselmis sp. using both catalysts was compared (Teo et al. 2014). The
aforementioned study made use of immobilized lipase.
11.8.3 Antifungal Agents and Pesticides
The key activity of enzymes is in catalyzing the breakdown of compounds such as

chitin, cellulose and lipids. This breakdown of compounds can be associated with
antimicrobial and pesticide activity. Fungi and insects, for example, are made up of
chitin which provides them with a level of resistance to environment or chemical
damage. Therefore, enzymes which can break down chitin present in the fungal
cell walls and insect exoskeletons can act as antifungal and antimicrobial agents.
The marine bacterium Pseudoalteromonas piscicida, for example, shows antifungal
activity against seven different fungi strains which includes P. galatheae, V. neptunius,
P. piscicida, P. rubra, P. fuliginea and V. fluvialis (Paulsen et al. 2016). This form of
antifungal and pesticides provides an alternative to the chemical-based ones which
are often less selective and could have adverse effects on whole crop and product.
11.8.4 Bioactive Oligomers
Some biopolymers have improved bioactivity when they are partially hydrolyzed into
smaller molecular weight oligomers. These are short-chain polymers with a degree
of polymerization less than 100. The bioactivity of some polymers such as alginate
and fucoidan can be tuned by breaking specific bonds on the polymer chain. For
example, alginate which is made up of a combination of block and alternating chains
of mannuronic and guluronic acid can be broken down into smaller chains of either
purely mannuronic or guluronic acid oligomers or alternating chains of mannuronic
and guluronic acid units using enzymes which are specific to the respective sites.
254 11 Enzymes
For example, a poly-1 → 4-alpha-l-guluronate lyase enzyme cleaves the 1 → 4

glycosidic bond of alpha-l-guluronic acids (Sil’chenko et al. 2013). This enzyme
was extracted from the mollusk Lambis sp.
11.8.5 Fish Processing: Skin and Scale Removal
Proteolytic enzymes are used in removal of skin and scales in processing of fish
food. The use of enzymes for skin removal in fish has been known for a few decades
(Amano 1962; Kim et al. 2002). Manually descaling and deskinning fish are often
time-consuming and require more man power. This process could be made faster by
treating the fish with enzymes where a batch of fish can be processed at once.
The goal of enzyme-assisted skin removal is to remove the fish skin without
damaging the muscles which are the part required for the fish food product. The
fish skin is often removed as it contains fat and in particular food preparations such
as fish fillets, the texture is undesirable. In other seafood such as squid, the removal of
the skin is necessary to improve the organoleptic properties and also improve storage.
In removal of fish skin, a combination of pepsin and carbohydrase is often used to
aid efficient removal. Lipase can be applied in the defatting of fish during processing
(Zhang and Kim 2010). This results in a less physically demanding process compared
to manual fat expression and is also milder than use of alkaline for defatting.
Proteolytic enzymes from fish are also used in the process of extracting other com-
pounds from aquatic waste. For example, in the extraction of glycosaminoglycans
from the viscera of Nile tilapia, proteolytic enzymes are used in the deproteinization
of the tissue (Nogueira et al. 2019). GAG is also a polymer which is valued for its
anticoagulant properties and other pharmacological applications. The enzymes from
aquatic waste, either endogenous or exogenous, are also useful in obtaining other
valuable by-products from aquatic waste.
11.8.6 Fish Sauce and Fish Protein Hydrolysate Production
Fish sauce is produced from the fermentation of fish such as sardines and anchovy;
generally, fish growing in the pelagic zone of the water are less expensive catch. The
process occurs at room temperature, pH between 5.5 and 6.5 and high salinity which
is achieved by adding 20–30 weight % of salt. A mixture of endogenous enzymes is
allowed to ferment the fish and produce the sauce as the fermented by-product. The
high salinity eliminates much of the bacteria which could also ferment the fish to
produce undesirable by-products; therefore, the salinity ensures the right conditions
for the endogenous enzymes and microorganisms which will result in the production
of fish sauce. The process of fermentation takes place between 6 and 12 months at
the end of which an ambered colored solution of fish sauce and salt is obtained from
the bottom drain.
Although much of the enzymes which act on the fish protein to produce fish sauce
are the endogenous enzymes, some bacterial activity, particularly in the first 3 weeks,
does result in the production of fatty acids and other metabolites which contribute
to the distinct flavor of fish sauce (Kim et al. 2002). These are likely the halophiles
which can survive the high salinity. The activity of trypsin and chymotrypsin is
significantly reduced during the process due to the high salinity; however, the activ-
ity of these enzymes is still important to the production of fish sauce. Proteolytic
enzymes remain at maximal activity throughout the process. The level of activity
of the microorganisms or enzymes is also dependent on the species of fish and the
conditions.
The activities of these enzymes can be accelerated by either increasing the pH to
alkaline conditions, reducing the pH to higher acidity which accelerates hydrolysis
or increasing the temperature to 45 °C which is high enough to accelerate the process
but without denaturing the enzyme. At the varied pH, certain enzymes which promote
the breakdown of the fish proteins will be enhanced. For example, at higher pH and
lower salinity trypsin, chymotrypsin and alkaline proteinase are more active. These
methods could reduce the fermentation time by about 80%; however, taste of the
resulting sauce is often inferior to that at longer period.
Fish protein hydrolysate is produced by treating the fish with only proteolytic
enzymes. The proteolytic enzymes specifically act on the peptide bonds. In the pro-
cess of production of fish protein hydrolysate, other enzymes are inactivated in order
to ensure only the proteolytic enzymes act on the fish protein. This results in the
production of soluble protein which is then separated, concentrated and dried.
Enzymes can also be used to process the wastewater from cooking or process-
ing of fish and marine invertebrates into fish products such as sauces and protein
hydrolysate. This further aids the optimal utilization of the aquatic resources.
11.8.7 Caviar Production
Caviar is one of the high-value food products from fish. It is produced from the
roe of primarily sturgeon and other fishes such as herring, salmon, cod, trout and
catfish. The key challenge in caviar production is the separation of the roe from the
connective tissue which links it to the parent body. The process of removing the roe
from the connective tissue is referred to as riddling (Kim et al. 2002). This leads to
higher cost of production in terms of time and man power. The time and complexity of
this process can be reduced by using proteolytic enzymes. The enzymes break down
the connective tissue leaving behind the eggs used for caviar. Using this method
compared to conventional riddling method can achieve up to 20% improvement in
yield (Kim et al. 2002). The yield is a measure of how much of the roe is successfully
riddled without damage to the egg. The process is quite delicate; if not carefully done,
the egg is damaged; and typically, over half of the eggs are damaged in the riddling
process. The enzyme method minimizes the mechanical stress on the egg, resulting
in hire successful riddling. This has significant economic impact on the producer as
256 11 Enzymes
it reduces loss of product and wasted labor. Collagen-degrading enzymes and other
protein-degrading enzymes such as collagenase and pepsin are the enzymes used in
the riddling process for caviar production.
11.8.8 Biomarkers for Environmental Pollution

Measurement
Enzymes from fish can be used as a biomarker to measure exposure to pollutants in

the environment. An example of such an enzyme is cholinesterase which is being
investigated for use as a biomarker to measure exposure of fish to pesticides (Mena
et al. 2014). Pesticides from agricultural activities on land often get into the aquatic
environment and are absorbed by the fish. These fish are then consumed by humans
and could pose severe health threats.
11.8.9 Pharmaceutical Application
The applications of enzymes in pharmaceutical industry are centered around the

ability of these enzymes to break down tissue materials, serve as sites for processes
and induce the destruction of pathogenic organisms. Proteases, for example, due to
their activity in catalyzing the breakdown of the proteins which make up tissue such
as collagen, are used as a mild means of cleaning and removal of dead skin tissue
from wounds (Yaakobi et al. 2007) compared to manually cleaning and removal
of dead tissue from wounds, whereby the mechanical stress on the wound would
cause severe pain and discomfort to the patient. The applications of enzymes in the
pharmaceutical industry extend to other areas such as treatment of cardiovascular
diseases, inflammations and cancer (Preeti et al. 2011). Producing such enzymes
from aquatic sources provides the potential of developing more robust treatment to
address a broader range of diseases and disease-causing organisms. The diversity
of activity and stability of aquatic-sourced enzyme are particularly promising for
developing new ways to counter bacterial resistance to drugs.
One of the challenges facing commercial production of enzymes from fish waste is
the level of purity required in enzyme production. This results in a product with high
production cost and where most of the mass is not used. Compared to the part of
the fish which is consumed as food, the crude extracts from fish viscera without the
purification stage have some proteolytic activities which could potentially make them
useful in applications such as stain removal additives in detergents and dehairing of

hides for leather production (Jayapriya et al. 2014). The cost of production could be
reduced by using the crude extract without the expensive purification stage, although
this would require some compromise in terms of effectiveness and limited applica-
tions. Another alternative to the use of purified enzymes is the use of immobilized
algal cells with the enzyme present within the cells. These immobilized algae cells
could have even higher activity than the cells in the free form (Arabi et al. 2010).
Of all the enzymes obtainable from the aquatic environment, proteases are the
most widely used enzymes in industry. Their applications range from production
of detergents, food, pharmaceutics and silver recovery in X-ray to production of
leather (Jayapriya et al. 2014). Around 60% of enzymes produced in the industry
are proteases (Zhang and Kim 2010), thus making them very valuable resources.
Within this chapter, the several applications of enzymes from aquatic sources have
been discussed and much of these are indeed proteases. The use of these enzymes
cuts across several industries which depend on them; hence, they pose significance
to the economy.
Southeast Asia records around 300,000 tonnes of fish sauce production annually
since as far back as the early 1960s. The enzyme-assisted method of riddling in
caviar production has been commercialized for caviar production in some countries
like Canada, the USA and Australia (Kim et al. 2002). Companies that have commer-
cialized enzyme production from marine sources include Biotec ASA in Norway,
Carnitech in Denmark, Biotec Maximal in Norway and Isnard-Lyraz in France.
The commercialization of enzyme production from aquatic wastes and by-
products results in less of these wastes being released into the environment and
causing adverse effects. The main limitations that have been highlighted in the com-
mercialization of aquatic-sourced enzymes are the cost of production, the variability
of the enzymes from different aquatic organisms which limits large-scale production
and the availability of alternatives from terrestrial sources. While the other alter-
natives remain readily available, producers are less inclined to pursue extraction of
enzymes from the aquatic wastes. Therefore, further research is needed in developing
improved methods for commercial extraction of enzymes from aquatic sources.
In various industries, enzymes are used for a wide range of applications. They
catalyze biochemical processes such as the breakdown of starch into sugars for
production of alcohol and breakdown of proteins to produce peptides for cosmetics.
Many biochemical processes of commercial importance will either not occur at all
or occur too slowly to be commercially feasible.
The diverse nature of algae as polyphyletic organisms made up of thousands of
different species means there is potentially a wide spectrum of enzymes which can
be obtained from algae. Algae-sourced enzyme offers the advantage of being able to
grow the feedstock for enzyme production with increased productivity and without
the use of land space since algae grow at a much faster rate than terrestrial plants
and do not require land space as do terrestrial plants and animals. There is, however,
more demand for better enzymes in terms of stability, low cost and efficiency.
Microalgae in particular are attractive for enzyme production as they can be genet-
ically modified toward a higher yield of specific enzymes. Having an alternative
258 11 Enzymes
source of enzyme for the catalysis of industrial processes could remove certain limi-
tations of some commercial enzymes. For example, pH limitations of some protease
are either active at alkaline or acidic at pH.
11.10 Conclusion
Enzymes play diverse roles in industry. The aquatic environment due to the diversity
of organisms living in a wide range of conditions serves as a source of diverse group
of enzymes. Aquatic-sourced enzymes have unique characteristics of being adapted
to particularly challenging environment and are therefore likely to produce enzymes
which retain stability at extreme processing conditions which is beneficial in indus-
try. Applications of enzymes which have been discussed within the chapter include
fish processing, biodiesel and cleaning agent production. Extraction processed from
different sources has similar recovery and purification stages, and this could poten-
tially be a more robust production facility which could allow production of the
same enzyme from multiple sources. Presently, enzymes from aquatic sources are
less commonly used in industry than the enzymes from terrestrial plants, microor-
ganisms and animals. Extraction of enzymes from aquatic waste and by-products
could contribute significantly to optimal use of aquatic resources and reduction in
environmental pollution.
References
Adamec L (2010) Mineral cost of carnivory in aquatic carnivorous plants. Flora 205:618–621
Aguilera-Morales ME, Canales-Martinez MM, Avila-Gronzalez R, Flores-Ortiz CM (2018) Nutri-
ents and bioactive compounds of the Lemna gibba and Ulva lactuca as possible ingredients to
functional foods. Lat Am J Aquat Res 46(4):709–716
Amano K (1962) The influence of fermentation on the nutritive value of fish with special reference
to fermented fish products of South-East Asia. In: Heen E, Kreuzer R (eds) Fish in nutrition.
Fishing News Books, London, pp 180–197
Arabi H, Yazdi Tabatabaei M, Faramarzi MA (2010) Influence of whole microalgal cell immobi-
lization and organic solvent on the bioconversion of androst-4-en-3,17-dione to testosterone by
Nostoc muscorum. J Mol Catal B Enzym 62(3–4):213–217
Bele SD, Sharmila S, Rebecca JL (2014a) Isolation and characterization of lipase from marine
algae. Int J Pharm Sci Rev Res 27(1):191–195
Bele SD, Sharmila SS, Rebecca JL (2014b) Comparative study of different methods of extraction
of lipase from seaweeds. Res J Pharm Biol Chem Sci 5(3):1741–1748
Bell AS (2007) Major antifungal drugs. Ref Modul Chem Mol Sci Chem Eng Compr Med Chem
II 7:445–468
Bernal D, Donley JM, Shadwick RE, Syme DA (2005) Mammal-like muscles power swimming in
a cold-water shark. Nature 437(27):1349–1352
Bleakley S, Hayes M (2018) Algal proteins: extraction, application and challenges concerning
production (2017): 6(33):1–34
Brouwer P, Nierop KGJ, Huijgen WJJ, Schluepmann H (2019) Aquatic weeds as novel protein
sources: alkaline extraction of tannin-rich Azolla. Biotechnol Rep 24:e00368. https://doi.org/10.
1016/j.btre.2019.e00368
References 259
Daboor SM, Raudonis R, Cohen A, Rohde JR, Cheng Z (2019) Marine bacteria, a source for
alginolytic enzyme to disrupt Pseudomonas aeruginosa biofilms. Mar Drugs 17(5):307–319
Dutta R (2008) Fundamentals of biochemical engineering. Springer, Berlin, Heidelberg, New York,
pp 50–52. ISBN 978-3-540-779fYJ-l
Fernandes P (2016) Enzymes in fish and seafood processing. Front Biotechnol 4(59):1–14
Fleurence J (1999) Seaweed proteins: biochemical, nutritional aspects and potential uses. Trends
Food Sci Technol 10:25–28
Garcia-Carreno FL, Navarrete del Toro MA, Serviere-Zaragoza E (2003) Digestive enzymes in
juvenile green abalone, Haliotisfulgens, fed natural food. Comp Biochem Physiol B Biochem
Mol Biol 134(1):143–150
Glancy B, Balaban RS (2011) Protein composition and function of red and white skeletal muscle
mitochondria. Am J Cell Physiol 300(6):C1280–C1290
Greene RV, Griffin HL, Cotta MA (1996) Utility of alkaline protease from marine shipworm
bacterium in industrial cleansing applications. Biotechnol Lett 18:759–764
Haddar A, Agrebi R, Bougatef A, Amidet N, Sellami-Kamoun A, Nasri M (2009) Two detergent
stable alkaline serine-proteases from Bacillus mojavensis A21: purification, characterization and
potential application as a laundry detergent additive. Bioresour Technol 100:3366–3373
He W, Li L, Zhao J, Xu H, Rui J, Cui D, Li H, Zhang H, Liu X (2019) Candida sp. 99-125 lipase-
catalyzed synthesis of ergosterol linolenate and its characterization. Food Chem 280:286–293
Hong R, Su L, Wu J (2019) Cutinases catalyze polyacrylate hydrolysis and prevent their aggregation.
Polym Degrad Stab 159:23–30
Howe PE (1921) The use of sodium sulfate as the globulin precipitant in the determination of
proteins in blood. J Biol Chem 49 93–107
Jayapriya J, Sabtecha B, Tamilselvi A (2014) Extraction and characterization of proteolytic enzymes
from fish visceral waste: potential applications as destainer and dehairing agent. Int J ChemTech
Res 6(10):4504–4510
Kim H, Seo HJ, Byun DS, Heu MS, Pyeun JH (2002) Proteolytic enzymes from fish and their
utilization. Fish Sci 68:1557–1562
Kobayashi H, Hatada Y, Tsubouchi T, Nagahama T, Takami H (2012) The hadal amphipod Hiron-
dellea gigas possessing a unique cellulase for digesting wooden debris buried in the deepest
seafloor. PLoS One 7(8):e42727
Komori Y, Nikai T (2013) Gloydius halys venom metalloproteinases. In: Handbook of proteolytic
enzymes, vol 1, 3rd edn., pp 965–967
Kumar KS, Ganesan K, Rao PV S (2014) Seasonal variation in nutritional composition of Kappa-
phycus alvarezii (Doty) Doty- an edible seaweed. J Food Sci Technol. https://doi.org/10.1007/
s13197-014-1372-0
Leema RT, Sachindra NM (2018) Purification and characterization of agarase from marine bacteria
Acinetobacter sp. PS12B and its use for preparing bioactive hydrolysate from agarophyte red
seaweed Gracilaria verrucosa. Appl Biochem Biotechnol 186(1):66–84
Lima FR, Ferreira AJ, Menezes G, Miranda VFO, Dourado MN, Araujo WL (2018) Cultivated
bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae)
from floodplains in Brazil. Braz J Microbiol 49:714–722
Mena F, Azzopardi M, Pfennig S, Ruepert C, Tedengren M, Castillo LE, Gunnarsson JS (2014)
Use of cholinesterase activity as a biomarker of pesticide exposure used on Costa Rican banana
plantations in the native tropical fish Astyanax aeneus (Gunther, 1860). J Environ Biol 35(1):35–42
Mogharabi M, Faramarzi MA (2016) Are algae the future source of enzymes? Trends Pept Protein
Sci 1(1):1–6
Moreno-Garcia J, Garcia-Martinez T, Mauricio JC, Moreno J (2018) Yeast immobilization sys-
tems for alcoholic wine fermentations: actual trends and future perspectives. Front Microbiol
9(241):1–13
Narwal SK, Gupta R (2013) Biodiesel production by transesterification using immobilized lipase.
Biotechnol Lett 35(4):479–490
Nogueira AV, Rossi GR, Iacomini M, Sassaki GL, Cipriani TR (2019) Viscera of fish as raw
material for extraction of glycosaminoglycans of pharmacological interest. Int J Biol Macromol
121:239–248
260 11 Enzymes
Paulsen SS, Andersen B, Gram L, Machado H (2016) Biological potential of chitinolytic marine
bacteria. Mar Drugs 14(230):1–17
Preeti C, Dimpi G, Drukshakshi J, Jasbir S (2011) Applications of microbial proteases in
pharmaceutical industry: an overview. Rev Med Microbiol 2(4):96–101
Rashidah AR, Nazalan N, Razip S, Koay PC (2006) Lipase producing psychrophilic microorganism
isolated from Antarctica. J Bacteriol 182:125–132
Schulze WX, Sanggaard KW, Kreuzer I, Knudsen AD, Bemm F, Thorgersen IB, Brautigam A,
Thomsen RL, Schliesky S, Dyrlund TF, Escalante-Perez M, Becker D (2012) The protein com-
position of the digestive fluid from the venus flytrap sheds light on prey digestion mechanisms.
Mol Cell Proteomics 11(11):1306–1319
Seca AML, Pinto DCGA (2018) Overview on the antihypertensive and anti-obesity effects of
Seibel BA, Walsh PJ (2002) Trimethylamine oxide accumulation in marine animals: relationship
to acylglycerol storage. J Exp Biol 205:297–306
Shahidi F, Kamil YVAJ (2001) Enzymes from fish and aquatic invertebrates and their application
in the food industry. Trends Food Sci Technol 12(12):435–464
Sil’chenko AS, Kusaikin MI, Zakharenko AM, Zvyagintseva TN (2013) Isolation from the marine
mollusk Lambis sp. and catalytic properties of an alginate lyase with rare substrate specificity.
Chem Nat Compd 49(2):215–218
Silchenko AS, Kusaykin MI, Kurilenko VV, Zakharenko MA, Isakov VV, Zaporozhets TS, Gazha
AK, Zvyagintseva TN (2013) Hydrolysis of fucoidan by fucoidanase isolated from the marine
bacterium, Formosa algae. Mar Drugs 11:2413–2430
Sivasubramani K, Singh JR, Jayalakshmi S, Kumar SS, Selvi C (2013) Production and optimization
of lipase from marine derived bacteria. Int J Curr Microbiol Appl Sci 2(4):126–135
Teo CL, Jamaluddin H, Zain NAM, Idris A (2014) Biodiesel production via lipase catalysed
transesterification of microalgae lipids from Tetraselmis sp. Renew Energy 68:1–5
Tokusoglu O, Unal MK (2003) Fat replacers in meat products. Pak J Nutr 2(3):196–203
Trivedi N, Reddy CRK, Radulovich R, Jha B (2015) Solid state fermentation (SSF)-derived cellulase
for saccharification of the green seaweed Ulva for bioethanol production. Algal Res 9:48–54
Valsa AK, Thomas A, Mathew M, Mohan S, Manjula R (2003) Optimization of growth conditions
for the production of extracellular lipase of vacillus my colds. Ind J Microbiol 43 67–69
Van Krimpen M, Bikker P, van der Meer I, van der Peet-Schwering C, Vereijken J (2013) Culti-
vation, processing and nutritional aspects for pigs and poultry of European protein sources as
alternatives for imported soybean products. Wageningen UR Livestock Research, Lelystad, The
Netherlands, p 48
Velasquez MT, Ramezani A, Manal A, Raj DS (2016) Trimethylamine N-oxide: the good, the bad
and the unknown. Toxins (Basel) 8(11):326–337
Yaakobi T, Cohen-Hadar N, Yaron H, Hirszowicz E, Simantov Y, Bass S, Freeman A (2007) Wound
debridement by continuous streaming of proteolytic enzyme solutions: effects on experimental
chronic wound model in porcin. Wounds 19(7):192–200
Yancey PH, Gerringer ME, Drazen JC, Rowden AA, Jamieson AJ (2014) Marine fish may be
biochemically constrained from inhabiting the deepest ocean depths. Proc Natl Acad Sci USA
111:4461–4465
Yao Z, Wang F, Gao Z, Jin L, Wu H (2013) Characterization of a κ-carrageenase from marine
Cellulophaga lytica strain N5-2 and analysis of its degradation products. Int J Mol Sci
14(12):24592–24602
Yuan D, Lan D, Xin R, Yang B, Wang Y (2016) Screening and characterization of a thermostable
lipase from marine Streptomyces sp. strain W007. Biotechnol Appl Biochem 63(1):41–50
Zhang C, Kim S (2010) Research and application of marine microbial enzymes: status and
prospects. Mar Drugs 8:1920–1934
Zhuang J, Zhang K, Liu X, Liu W, Ji A (2018) Characterization of a novel polyM-preferred alginate
lyase from marine Vibrio splendidus OU02. Mar Drugs 16(9):295
Chapter 12
Collagen
Abstract Collagen is obtained from the bones, skins and scales of aquatic animals.
It is made up of three polypeptide chains linked together to form a tertiary structure.
It serves a structural role within the tissue where it provides flexibility within the
tissue matrix. Collagen can be processed into various forms, and it finds applications
in high-value products such as scaffolds in tissue engineering. Aquatic-derived col-
lagen serves as an alternative to porcine or bovine sourced collagen, thus eliminating
associated health risk or ethical concerns associated with the collagen sourced from
these land animals. Since collagen can be obtained from the parts of the aquatic ani-
mals that are generally not consumed as food, the production of collagen contributes
toward management of waste resources as well as optimal utilization of aquatic
resources.
Keywords Collagen · Proteins · Biopolymers · Fish · Squids
12.1 Introduction
Collagen is abundant in nature as it is present in the tissue of every animal on land

and in the sea. It is limited to animals; therefore, aquatic plants and algae do not serve
as a source of collagen. Within the aquatic environment, main commercial sources
of collagen are from fish fin, skins and scales (Olatunji and Denloye 2017). Other
aquatic sources of collagen are invertebrates such as squids, starfish and jellyfish
(Benedetto et al. 2014; Tan et al. 2013). Collagen competes with the fish oil, and
fishmeal market as the fish parts is also used in this application. One of the most
common uses of collagen in the industry is in foods where it is mostly used in its
hydrolyzed form. Collagen from fish has been proven to be interchangeable with
bovine and porcine hydrolyzed collagen (Benedetto et al. 2014), with the added
advantage of being free from the risk of diseases such as mad cow diseases which
are associated with the collagen products obtained from these sources. It also serves
as an alternative where ethical or religious concerns exist with hydrolyzed collagen
sourced from bovine or porcine.
Like other proteins, collagen is made up of amino acid repeating units which form
into polypeptide chains. They however distinguish from other proteins by their triple

262 12 Collagen
helix structure. There are various types of collagen; however, the type I collagen is
most common in vertebrates where it could make up to 78% of the collagen. In the
intact form, collagen plays mainly structural role; however, when hydrolyzed into
its polypeptide forms, it has more diverse applications which range from food to
biomedical.
The production of collagen serves some advantage to the environment as it pro-
vides a way to optimize the utilization of the global aquatic resources. Further to
this, by adding value to the aquatic resource from which it is sourced, it provides an
additional source of income to the fishermen and fish traders who can then sell the
by-products to other companies who can then convert to collagen and these can then
be converted to other products of much higher value. Some adverse environmental
impacts arise from water utilization, use of acids, salts and alkali which might be
released into the environment and cause some adverse effects as well as carbon emis-
sions. All of which can be addressed through sustainable practices such as recycling
and process optimization.
Collagen is fairly well explored aquatic biopolymer resource. Global collagen
market is estimated to have an annual growth rate of 5.2% (Research and Markets
2019). Much of this comes from its use in food and other applications are also seeing
increased interest. Companies such as Nitta Gelatin existing across the world are
involved in the production of gelatin from fish. In addition to this, researchers across
the world are also continually exploring new sources and new ways of obtaining
and making use of gelatin. Such innovations include 3D printed scaffolds for bone
regeneration and corneal replacement and these are discussed in this chapter. With
such high-end application from fish by-products which are generally considered as
waste, collagen has significant impact on present and future economies.
The rest of the chapter explores collagen as another important aquatic sourced
biopolymer, beginning with where it is sourced, the chemistry of aquatic sourced
collagen and how this differs or is similar to non-aquatic sourced collagen, the pro-
duction processes and how these processes impact the environment and the current
state of the commercial collagen production.
Collagen is relatively abundant in nature, and it is the most abundant protein in

vertebrates. It is present in humans and other primates, mammals, vertebrates and
invertebrates. It makes up the bones, skin and other connective tissue where it is
produced by fibroblast cells. Collagen makes up approximately 30% of protein in
animals, in humans it makes up 75% of the dry weight of the skin (Shoulders and
Raines 2009). Within the tissue, proteins serve structural roles alongside other tissue
components such as elastin and hydroxyapatite, depending on the particular tissue
composition. The bone tissue for example comprises a composite with collagen and
other non-collagenous proteins forming the matrix filled with minerals and forming
the disperse phase of the biological tissue matrix (Aerssens et al. 1994; Robinson
1979). Without collagen, bone will be completely brittle and without the bone miner-
als, bone would have no rigidity. Therefore, the combination of collagen and minerals
gives bone the ideal mechanical property to perform its biological function.
Collagen plays a structural role in the extracellular matrix where it provides ther-
mal and mechanical stability as well as interaction with the surrounding cells and
components. Collagen can be of several types mainly type I and type II collagen,
although there are up to 28 different types of collagen in vertebrates on land and in
water (Shoulders and Raines 2009). Type I collagen consists of two alpha-1-chains
and one alpha-2-chains in the triple helix structure (Felician et al. 2019). Type I col-
lagen is more abundant in marine invertebrates such as sea urchins, jellyfish, starfish
and squids (Benedetto et al. 2014; Tan et al. 2013; Delphi et al. 2016). Collagens
from fish parts such as scales, fins, skin and bones have been mostly identified as
type 1 collagen. Since human collagen is about 90% type 1 collagen fish sourced
collagen has the potential to have good compatibility with the human system when
used externally or internally. Whale sharks collagen is type II collagen (Jeevithan
et al. 2015). Collagen is distinct from other proteins by their right-handed triple helix
structure as well as the occurrence of the amino acid glycine at every third residue
along the polypeptide chain.
12.3 Chemistry of Collagens
The general structure of collagen is a right-handed triple helix structure. A fibrous

protein does not have a tertiary structure as this helix structure does not fold up.
Collagen has high molecular weight. For example, collagen extracted from the jel-
lyfish Rhopilema esculentum had a molecular weight of between 100 and 150 kDa
when measured using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Fourier transform infrared (FTIR) (Felician et al. 2019). This was
then broken down with enzymes to polypeptides with 10–15 kDa and then further
reduced to 25 kDa with further enzyme treatment. Different types of enzyme break
different types of bonds such that the molecular weight of the proteins can be varied
by varying the enzyme used. The molecular weight has an impact on the bioactivity
of collagen (Chi et al. 2014); therefore, the ability to modify the molecular weight
of collagen increases the diversity of their applications (Fig. 12.1).
In fish collagen which could be obtained from the skin, fins or scales, alanine and
glycine were most abundant in the stated order, while tryptophan was not present
in the collagen structure (Mahboob 2015). The other more prominent amino acids
that occur on the collagen polypeptide chains are proline and hydroxyproline. There
are cases where the polypeptides making up the collagen triple helix are identical
polymeric chains made up of the same amino acid repeating units, and this is referred
to as homotrimeric triple helix. It is however more common to have the case where
the polypeptides are not identical polymer chains; this is referred to as heterotrimeric.
264 12 Collagen
Collagen Single protein Hydrolyzed

Triple Helix
Fibre Chain collagen
Fig. 12.1 Collagen and hydrolyzed forms
Normal human collagen type 1 is heterotrimeric, and it comprises of two alpha-1-

chains and one alpha-2-chain (Chang et al. 2012). Homotrimeric collagen type 1
occurs only in fetal tissue or in cases of cancer or fibrosis.
The polypeptide chains are held together by hydrogen bonds which form between
the amine (–N–H2 ) group and the carbonyl group (–CO–) of the amide links which is
present in all the amino acids making up each polypeptide chain. Using enzymes such
as collagenase, the alpha helix structure of the collagen is separated into polypep-
tides by breaking the hydrogen bonds which hold together the polypeptide chains in
the helical conformation. These polypeptides could then be further hydrolyzed into
shorter chains by breaking some of the amide bonds using enzymes like proteolytic
enzymes such as papain and alkaline proteinase (Felician et al. 2019).
Collagen is characterized by five main peaks on the FTIR spectrometry; the amide
A, B, I, II and III at a wavelength of 3433, 2926, 1641, 1549 and 1240 cm−1 ,
respectively. The amide III bands are present when the collagen triple helix structure
is still intact (Chi et al. 2014). This can therefore be used to distinguish between
whole collagen and hydrolyzed collagen.
Solubility is an important factor in the applicability and bioactivity of collagen.
This impacts their ability to dissolve under physiological conditions and interact
with the cells to carry out their biological activity. An example of such is in trans-
dermal delivery of therapeutics using microneedles, where the drug is loaded onto
a microneedle patch made from hydrolyzed collagen. Upon insertion into the skin,
these hydrolyzed collagen microneedles dissolve in the fluid within the skin releas-
ing the compounds loaded within (Olatunji et al. 2014; Olatunji and Olsson 2015).
Lower molecular weight collagen from fish skin of Spanish mackerel for example
show faster solubility in neutral pH and at acidic pH but will however decrease in
solubility at alkaline pH (Chi et al. 2014). The increased solubility at lower molec-
ular weight is attributed to the shorter polypeptide chains having better ability to be
12.3 Chemistry of Collagens 265
surrounded by the water molecules and form hydrogen bonds. This molecular weight
and pH dependence of collagen solubility prove useful in designing products which
are targeted to be released at specific points along the alimentary canal since the pH
varies at the different parts.
FAO reported 171 million tonnes of fish produced in 2016, 54.1 million tonnes of
this being farmed finfish (valued at about 138.50 billion USD). It was stated that 88%
of this was utilized for direct human consumption (FAO 2018). Of these percentage
consumed as food, some of these also include the waste generated from the fish
food waste. The term fish here also includes other aquatic animals such as shrimps,
mollusks and lobsters but excludes aquatic plants and algae. It has also been observed
that as the world population grows the consumption of fish grows twice as much.
Since the global population is ever on a constant rise so will the demand for fish, and
hence the availability of the fish waste from which collagen can be produced.
Due to its presence in a broad range of organisms, collagen is relatively abundant.
One of the issues with availability of raw materials for commercial collagen produc-
tion is the fact that some of the sources of collagen raw materials are consumed as
food. Those which are not eaten are usually sold alongside with the food for differ-
ent reasons like preserving quality and reducing the cost of processing. For example,
fin fish are often sold and served whole, such that obtaining the waste bones along
the value chain might prove difficult. Fish waste such as bones is usually discarded
alongside organic waste, they also degrade relatively fast, post-consumer collection
of such wasted is not as feasible as collecting non-biodegradable wastes such as
plastics. One way to counter this limitation is to collect from fish processors who
descale, fillet or extract oils from the fish. Coproduction of fish collagen can also be
carried out alongside the aforementioned processes.
Fish production either from fishing from the wild or fish farming using aquaculture
forms an important part of the human diet. Aquatic sourced food is a major source
of protein in many diets across the world. A long-term sustainable production of
fish and fisheries products is a key part of sustainable development. Fish processing
results in the generation of a considerable amount of waste, about 50–70% of the
fish results in waste which includes the fins, the scales and in some cases the skin
and guts. These wastes comprise up to 30% collagen which is of high commercial
value. Aquatic-based raw material for collagen production is therefore promised to
be sustainable.
The availability of aquatic raw materials for collagen production however faces
some challenges. One of which is the challenge of overfishing and irresponsible
fishing which has resulted in decline of some fish species and threatens the long-
term availability of such resources. About 33.1% of fish available in the waters are
fished beyond biologically sustainable limits. Commercial extraction of collagen
from fish scales should therefore not seek to exert further strain on the rate of fishing
266 12 Collagen
but rather align the production rate with the waste generated from a sustainable level
of fishing. The process of extraction should also not impact adversely on the aquatic
environment within which these resources exist.
The main competition for collagen production is the fishmeal production. The
fish wastes are also used for the production of fishmeal which is high in protein
and minerals. Since in the wild, the big fishes tend to fish on other little fishes (as
well as other aquatic organisms such as planktons), these fish by-products are ideal
sources for fish meal. It is also particularly important that the waste is returning into
the food cycle to further ensure continuous production of fish. However, fishmeal
production has since been on a general decline since 1994 when global fishmeal
production was at an all time high of 30 million tonnes. Of the 171 million tonnes of
fish produced in 2016, 91 million tonnes are captured from natural stock while the
rest are farmed in aquaculture (FAO 2018). This implies a demand for fishmeal for
aquaculture is relatively high. On the other hand, collagen has a broader market which
spans pharmaceuticals, food, biomedical and cosmetics. Other than fish meal, fish
skin, particularly of larger fish, is used for producing leather for a range of products
such as shoes, bags, garments and furniture.
As of 2016, China was the world’s highest producer of fish, followed by Norway,
Vietnam and Thailand. Much of these fish are being exported to Europe or the USA
as they are the greatest consumers of fish (FAO 2018). Fresh whole fish is usually
the most preferred form of fish such that the fish moves down the distribution chan-
nel with very little processing or removal of any part of it. Much of the cost goes
into transportation and preservation. This means that much of the fish waste is not
generated until the point of consumption when the fish is cut and cooked. Therefore,
although China is the biggest producer of fish, it might not necessarily be the region
where fish waste for collagen production is highest. The countries where the fish is
being cooked will therefore be more likely generators of fish waste.
Collagen is also sourced from other aquatic animals such as the jellyfish (Cheng
et al. 2017), squids (Dai et al. 2018) and sea cucumbers (Liu et al. 2019), and these are
captured in lower quantities from natural stock and hardly farmed. They are recorded
as other sea animals make up about 938,500 tonnes annually as of 2016. However,
recent giant jellyfish blooms mean there is an increasing amount of these aquatic
nuisances to be utilized.
12.5 Extraction of Collagen
There are various methods which exist for the extraction of collagen from different
sources. A single source of collagen might have more than one method of extraction
of the collagen. Here, we review some of the existing extraction methods for some
specified sources. Some of these methods lead to the production of collagen peptides
as the process of extraction results in the breakdown of the collagen triple helix
structure. This could be desirable where the end goal is to obtain peptides since the
two steps of extraction and formation of polypeptide are achieved in one process.
12.5 Extraction of Collagen 267
Collagen content varies from species to species and also varies in different parts of
the animal. It also varies for different studies. Extraction yields of collagen from the
skin of C. catla and C. mrigala are 13 and 11.2%, respectively, while that from the
scales were 9.5 and 8.3, respectively, and 13 and 13.1%, respectively, from the fins
(Mahboob 2015). In jellyfish R. esculentum recent studies report a yield of 4.31%
(weight of dry collagen to weight of wet jellyfish) compared to other studies which
reported lower yields (Felician et al. 2019). A yield of 13.6% (dry collagen per wet
weight of skin) was obtained from acid extraction of collagen from Spanish mackerel
fish skin (Chi et al. 2014) while a 30% yield (dry weight of hydrolyzed collagen per
dry weight of fish scales) was obtained from hydrothermal extraction of hydrolyzed
collagen from fish scales of croaker fish (Olatunji and Denloye 2017). In another
study (Ahmed et al. 2019), a yield of 16.7% was reported for enzyme extraction of
collagen from skin of bigeye tuna using pepsin enzyme. Using acid extraction, the
acid-soluble collagen yield from the skin was 13.5% while yield of pepsin soluble
collagen from the scale and bones were 4.6 and 2.6%, respectively. The acid-soluble
collagen obtained from scales and bones of the tuna fish was negligible.
12.5.1 Extraction of Collagen from Combined Fish Waste
Collagen has been extracted from fish skins, scales and bones of a variety of fish
such as bigeye snapper, red snapper, carp, black drum, Nile perch and yellowfin tuna
(Ahmed et al. 2019; Olatunji and Denloye 2017; Mahboob 2015; Chi et al. 2014). Fish
skins can be sourced from fish processing factories where skinless fish fillets or other
fish products are made. Processing the skin separately from the bones and other parts
since the composition of bones and skin significantly varies. Separate extraction of
the different parts of the fish waste allows for better process optimization by tailoring
the operating conditions to the respective composition. However for practicality and
reduction of labor and process complexity, it is desirable to combine all the fish
wastes and extract collagen from the mixtures in one batch.
To extract collagen from combined fish waste both the chemical and enzymatic
method follows the same steps for pretreatment of the fish waste parts. Here, an
example method (Mahboob 2015) which was used as a general method to extract
collagen from scales, fin and fish skin is presented. Although the parts were sepa-
rated, the same process conditions were applied to all parts. The different parts were
separated prior to extraction in order to analyze them separately for collagen contents
and structure in the different parts of the fish. The pretreatment stage is targeted at
removing non-collagenous proteins so that these are not extracted alongside the col-
lagen and contaminate the extract. To achieve this, the mass is treated with an alkali,
most commonly sodium hydroxide at 0.1 M using a 10 ml solution per gram of fish
waste mass. This is carried out at a temperature of 4 °C under constant stirring for
6 h with the alkali solution changed every 2 h after which the spent alkali solution
must have been saturated with non-collagenous proteins that have been removed.
This is then washed with water until all the alkali with the non-collagenous proteins
268 12 Collagen
has been washed off and the system is neutral. The type of water used depends on the
level of purity required. Clean tap water could be used, or distilled water or ultrapure
water.
For the acid-based extraction, the fish waste was treated with acid in order to
dissolve out the acid-soluble collagen from the rest of the tissue. The first step was to
remove the fat using 10% butyl alcohol at a 1:10 ratio of solid to alcohol for a duration
of 8 h. The fats are alcohol soluble so it will be removed from the fish waste. The fish
waste is then washed with cold water to remove any residue of alcohol and fat from the
surface. The next stage is then to extract the collagen in acetic acid at a concentration
of 0.5 M and 1:15 solid to liquid ratio. The extraction is allowed a duration of 24 h. The
extraction time can be varied for different samples since the composition of collagen
and other components varies for different types of fish. Defatting prior to extraction
prevents the fat from being released alongside the collagen during the extraction. The
fat will dissolve in alcohol but collagen will not, however the fat could be released
into the acetic acid solution hence the order is also important. The collagen dissolved
in acetic acid is then precipitated out in a 0.05 M tris (hydroxymethyl) aminomethane
containing sodium chloride at a concentration of 2.6 M. To separate the precipitated
collagen from the solution, centrifugation is used at 20,000 g for 1 h. A refrigerated
centrifuge is used in order to maintain the required temperature. To further purify the
collagen sample, it can then be redissolved in 0.5 M acetic acid and then separated
by repeated dialysis. The collagen extract now needs to be dried for more effective
storage and packaging. For this mild drying method is required as excessive heat
will destroy the triple helix structure and the resulting extract will be hydrolyzed
collagen. Freeze drying can be employed here. It is commonly used in such cases
where a delicate sample needs to be dried. A temperature between 4 and 8 °C is
maintained throughout the pretreatment and extraction to prevent degradation of the
collagen.
The enzyme extraction can be carried out using the residue from the acid extraction
(Mahboob 2015). The residue contains the collagen proteins which do not dissolve
in acid but can be dissolved in pepsin. This method allows obtaining two types of
collagen from the same source thus making optimal use of the fish waste. The pH is
adjusted to that suitable for the enzyme by soaking in 0.5 M acetic acid in a solid to
liquid ratio of 1:15. The pepsin is then added and continuously stirred for a duration
of 48 h. A low temperature is also maintained at 4 °C. At the end of the extraction,
the solid residue is then separated from the liquid which contains the collagen. The
collagen is precipitated using sodium chloride, and the precipitated collagen is then
separated from the salt solution and further purification by repeated dialysis then
follows.
The type of extraction process has some effect on the collagen yield and properties.
For example, acid extraction from scales of goatfish gave collagen yield of 0.46%
while pepsin extraction gave a yield of 1.20% (dry weight basis). The acid-soluble
collagen had slightly higher glass transition temperature of 41.58 °C while that of
pepsin soluble collagen was 41.01 °C (Matmaroh et al. 2011). Sodium hydroxide
extraction results in mostly denatured collagen or hydrolyzed collagen which is a
mixture of polypeptides of different chain lengths. The extraction process therefore
Fish Waste
Alkali pretreatment
Defatting
Washing
Solid residue
Acid extraction pH adjustment
Precipitation Pepsin extraction
Centrifugation Precipitation
Purification Purification
Acid soluble collagen Pepsin soluble Collagen
Fig. 12.2 Process for acid and pepsin soluble collagen extraction from fish waste
depends on the form in which the collagen is required. Figure 12.2 summarizes the
extraction process for acid-soluble and pepsin soluble collagen extraction.
12.5.2 Hydrothermal-Based Extract of Hydrolyzed Collagen

from Fish Scales
Fish scales are particularly attractive source for collagen as it is a food source, and it
is the part of the food that is discarded as it is often not used for fishmeal or fish oil.
The extraction of hydrolyzed collagen from fish scales could be carried out using the
chemical or enzymatic method described for combined fish waste; however, another
alternative is the hydrothermal method. This method requires minimal use of acids
or alkali. It uses a combination of heat and pressure for a prolonged period of time
(Olatunji et al. 2014; Olatunji and Olsson 2015; Olatunji and Denloye 2017). In the
said method the fish scales are generally collected at the end of the day from fish
traders who sell and scale a large amount of fish daily. The scales are transported
to the laboratory where they are washed with tap water to get rid of debris. They
are then washed with sodium hydroxide with a concentration of 0.1 M in a ration
of 1:15 (weight of dry scales to volume of alkali solution) at room temperature for
1 h and stirring at intervals. This step is carried out to get rid of non-collagenous
270 12 Collagen
proteins, fish skin and other residues attached to the scales. The solid and liquid
are then separated. This can then be followed by either rinsing or addition of acid
until neutral. The scales are then washed in tap water followed by distilled water.
The scales are then dried until bone dry and they can be stored in this form until
needed. Alternatively, they can be frozen until needed depending on the facilities
available. The cleaned washed scales are then extracted in distilled water in the ratio
1–10 wet weight of scales to volume of water in a closed vessel where the pressure
is around 80 kPa. The pressure prevents evaporation of water and also allows the
water molecules to penetrate into the scales. The duration of extraction could range
between 3 and 20 h depending on the desired yield. Optimal yield is obtained at
80 °C after 8 h. Further extracts were obtained up until 20 h, however, most of the
collagens were removed by 8 h (Olatunji and Denloye 2017).
The liquid extract which contains hydrolyzed collagen polypeptides is then sep-
arated from the solid residue which is mainly made up of calcium carbonate and
chitin, using a filter cloth or sieve. Further separation with a centrifuge at 2500 rpm
for 15 min is then done to separate smaller particles. The liquid filtrate is then evapo-
rated on a hot plate or in an oven until a viscous liquid is formed. This is then solvent
cast and allowed to dry forming into sheets which can then be milled in granules or
processed into other desired forms such as pellets. The advantage of this method is
the minimal requirement for additional chemicals. This puts most of the cost of the
heat required to rupture the collagen structure to release the polypeptides.
12.5.3 Extraction of Collagen from Jellyfish
Collagen obtained from jellyfish is of particular interest due to recent findings that
polypeptides from these collagens have some antioxidant, hemostatic and antihy-
pertensive bioactivities (Zhuang et al. 2009, 2012; Cheng et al. 2017). Extraction
of collagen can be carried out using the enzyme pepsin, and this has been used for
extraction of collagen from aquatic sources such as jellyfish and starfish (Felician
et al. 2019; Tan et al. 2013) used pepsin enzyme. 203 g of jellyfish filaments which
had been washed with ultrapurified water was then homogenized in a 50% w/v acetic
acid for 10 min. The extraction is then carried out using 1% pepsin under constant
stirring at 4 °C. The extraction was carried out for a duration of 72 h. Centrifugation
at 8000 rpm for 15 min was then used to separate the extracted collagen from the
other components. The collagen was then precipitated out by adding sodium chloride
at 2 and 0.05 mol/L tris to get a pH of 7. Further centrifugation was then carried out
at 10,000 rpm for 20 min to separate the precipitate. This is then dissolved in 50%
acetic acid. Dialysis against acetic acid is at 0.1 mol/L and ultrapure water for 2 h and
2 days, respectively. Advantage of this method for extraction of collagen is that it is
done at low temperature of 4 °C such that the collagen can be obtained in its whole
form rather than degraded into collagen peptides. Such method is desirable when the
whole collagen is needed, the main cost therefore goes into use of the enzyme and
maintaining the conditions required for the enzyme to function. To obtain collagen
peptides, another enzyme is then used to break the bonds between the polypeptide
chains. Since these enzymes act on specific sites, a different enzyme is required for
this. Here, the collagenase enzyme is used at 5% w/w composition.
12.6 Environmental Implication
This section discusses some key issues associated with collagen extraction from
aquatic resources. Some of these are positive environmental impacts while some are
adverse. The overall environmental impact is therefore a sum of the positive and
negative impacts for the specific source and type of extraction process.
12.6.1 Jellyfish Blooms
In recent years, rapid rise in population of jellyfish has become an environmental

nuisance in various parts of the world including Asia, the USA, Europe and Australia
(Dong 2019). This has had devastating effects and economic losses in the fisheries
industry and other related industries. Fishermen have reported frequent fishing trips
resulting in the fishing nets filled with large amounts of giant jellyfish with little or
no fish. Most of the jellyfish caught results in wasted efforts and resources and in
some cases poses risk to the lives of the fishermen. One of the measures to address
this is to utilize the jellyfish for production of much-needed biopolymer products
like collagen. These jellyfish feed on plankton at a relatively faster rate than small
fish. Most population blooms as seen with the algae blooms, result in an eventual
decline as the food runs out and the jellyfish begin to die out and decay. This is
likely to result in another severe environmental problem in the aquatic environment.
Therefore, capturing them and utilizing them for the production of biopolymers such
as collagen is one way of abating the problem until a more permanent solution os
developed for reversing the bloom.
12.6.2 Fisheries Waste Management
Processing of fish waste for the extraction of collagen is beneficial to the environment
in the sense that it prevents the accumulation of fish waste which results in environ-
mental deterioration and health hazard. One issue with processing of fish is the fishy
smell associated with fish. This is largely due to the presence of trimethylamine
oxide TMAO (Subramaniam 2018). One of the advantages of collagen as a naturally
sourced biopolymer is that it can utilize fish waste and does not require special con-
ditions for the cultivation of fish for collagen production. Collagenous waste makes
a good fraction of the fish waste which would otherwise be discarded as waste if not
272 12 Collagen
used for fishmeal. Collagen production from fish waste therefore serves a significant
role in helping to manage the waste generated from fisheries whether captured or
farmed.
The water consumed is used in different aspects of collagen production, in the wash-
ing of the fish biomass prior to pretreatment, washing the pretreated biomass where it
has been pretreated with alkali and the water used in the main extraction. The purity
of water varies from clean tap water to ultrapure water which requires additional
energy to clean. In the washing stage using the hydrothermal method for extraction
of collagen from fish scales (Olatunji et al. 2014) as an example, water is used in an
estimated ratio of 13:1 (volume of water in ml to mass of fish waste in grams), in
the washing following pretreatment this usually requires repeated washing before the
water becomes neutral after sodium hydroxide pretreatment (in the case of extraction
from fish scales). If we assume it is washed six times after pretreatment in the same
water to fish waste ratio, this means for the cleaning and pretreatment stage about
91 ml of water is required per gram of fish scales. In the extraction process, more
water is used as the solvent for the extraction. For each gram of fish scales, 10 ml of
water is used. This makes an estimated 10 ml of water for every gram of collagen
produced from fish scales. The water from washing could be recycled either on site
or sent to water treatment plant. The water used in the extraction could be recovered
during the drying process by condensation, although this will then require further
energy and material consumption for these additional operations.
In the enzyme extraction process, less energy is consumed in heating as the extraction
is carried out at low temperature. However, energy is required for cooling. Where
the average room temperature in cold climates is around 16 °C and in hotter climates
could reach much higher temperatures of around 30 °C, energy input is required to
remove heat from the system such that at least 50 J is required per gram of water
being heated. Where hydrothermal extraction at 80 °C is used, energy is required
to heat the system from room temperature (assuming this to be about 25 °C) to
80 °C. This heating will require an input of about 230 J per gram of the water being
heated for the entire duration of the extraction. Additional energy is then utilized
in drying; this can be done either by freezing and sublimation in freeze drying or
by evaporation. Other operations requiring more energy input include operating the
centrifuge for separation at about 2500 rpm for about 15 min, the power consumed
per centrifuge cycle depends on the volume of the centrifuge and the power efficiency
of the particular unit.
12.6 Environmental Implication 273
Energy is also consumed in transporting the fish waste from the point of collection
to the processing factory. Additional transportation is then required to get the products
to the distributor or to other manufacturers for further processing into more refined
forms or for use in other products. Renewable sources of energy could be considered
for each of these stages to minimize the environmental impact.
12.6.5 CO2 Emission
In the extraction of collagen from fish scale, the step before the extraction with either
an acid, alkali or hydrothermal method is the removal of the calcified minerals from
the scale. This is mainly composed of calcium carbonate (CaCO3 ) and some calcium
hydroxyapatite (Ca10 (PO4 )6 (OH)2 ). The decalcification of scales, fins and bones is a
chemical reaction between the calcium compound and acid. For example, the reaction
between hydrochloric acid and calcium carbonate is expressed in Eq. (12.1).
CaCO3 + 2HCl → CaCl2 + H2 O + CO2 (12.1)
Given a fish scale of for example the Northern Pike fish (Esox lucius) made of
20% calcium salt (Sionkowska and Kozlowska 2014), that means for every gram of
fish scales xxx grams of CO2 is released.
12.6.6 Salts, Acids and Alkali
Where the acid-based or alkali-based extraction is used for the extraction of acid-
soluble collagen, for example, acetic acid at a concentration of 50% w/v is required
for the extraction medium in the enzyme extraction of collagen from jellyfish. More
of the same acid is used to redissolve during purification and a lower concentration
of 0.1 mol/L of acetic acid is used for the dialysis such that the acid is still required
in the enzyme extraction. In the chemical extraction, either acid or alkali can be used
for the extraction of collagen, depending on the type of collagen desired.
In the extraction of collagen from fish, skin and fins, pretreatment is usually
required to get rid of debris and non-collagenous proteins. This usually requires
treatment with 10 ml of 0.1 M sodium oxide for every gram of fish waste. To remove
fat, particularly in fish skin, alcohol is required. 10 ml of a 10% concentration of
butyl alcohol per gram of fish scales are used for this purpose. Sodium chloride is
generally used for precipitation of collagen when either low-temperature extraction
of intact collagen with acid or pepsin is being used.
Therefore, the extraction process of collagen whether enzymatic, chemical or
hydrothermal requires varying amount of additional salts, acid and alkali. Some salts
are also produced in the process of decalcification of the scales, fins and bones when
these parts are used. If these salts, acid and alkali are released to the environment they
274 12 Collagen
Table 12.1 Consumptions

Consumption Amount
for extraction of acid-soluble
collagen from fish skin Butyl alcohol 10 ml of 10% v/v per gram of
fish skin
Acetic acid 30 ml of 0.5 M per gram of
fish skin
Tris (hydroxymethyl) 10 ml of 0.05 M per gram of
aminomethane crude extract
NaCl 2.6 M 10 ml per gram of
crude extract
contribute to altering the salinity and pH of the water. There is therefore a need for
careful regulation of these processes involving large-scale use of these compounds
for collagen extraction. Table 12.1 gives a summary of typical consumption for acid
extraction of collagen from fish skin (Mahboob 2015; Matmaroh et al. 2011).
12.7 Applications
Beyond it is mainly structural role within the tissue, when orally consumed,
implanted, digested, injected or transdermally applied collagen can present a broad
range of benefits. Here we explore some of the notable once from recent literature.
These applications highlight the relevance of collagen to the economy, environment
and health.
Collagen is most often used in its partially degraded form as polypeptides. In
the triple helix form collagen play mostly structural roles, however, when unraveled
into polypeptides by breaking the secondary bonds between the peptide chains, these
have more bioactivities. The peptide chains also have higher bioavailability when
applied to the skin or ingested. Their respective bioactivity is dependent on the amino
acid composition of the polypeptide chains. The length of the polymer chains also
affects their bioactivity and bioavailability. Generally, lower molecular weights more
easily penetrate membranes and this is important in for example delivering collagen
polypeptide-based formulation into the skin. When orally ingested polypeptides are
easier to break down and transported into the circulatory system from where they
can elicit their biological activity.
12.7.1 Skin Mimicking
Testing of products which require understanding or measuring how they interact with
the skin such as topical skin care, adhesives and physical devices requires the use
of skin samples. These are either obtained from cadaver, grown in tissue culture or
obtained from other animals whose skin closely resembles that of humans in terms of
porosity, thickness, mechanical properties and moisture content. Recently, artificial
skin based on hydrolyzed collagen in the form of gelatin has been developed by
different researchers (Dabrowska et al. 2017). Aside from reducing the complexity
of growing skin in tissue culture, it also addresses ethical concerns which surround
the use of human or animal skins for testing of products. Some of these artificial
skins are designed to mimic different aspects of the skin, porosity, water content and
mechanical properties. The artificial skin reported by Dabrowska et al. (2017) was
made using a composite of cross-linked collagen and cotton fibers. This resulted in
a skin-like material which mimicked the friction properties of the skin. The friction
properties of the skin are important when designing products such as textiles and
adhesive patches which are designed to interact physically with the skin. It seems
not surprising that collagen would play a role in such material since it already serves
as one of the key components of human skin.
12.7.2 Wound Healing
Collagen aids in wound healing by protecting the wound from infection and physical
abrasion, keeping it moist thereby allowing more effective and fast healing. It can be
used in combination with other wound healing aids such as antimicrobial agent, and
it can also serve as a delivery agent for drugs which support healing and pain relief.
In addition to this, collagen peptides can induce skin rejuvenation and healing by
signaling the fibroblast cells to produce new collagen cells. They can also promote
overall cell turnover by increasing the rate of cell proliferation and migration, such
that more old worn-out skin cells are removed and newer skin cells are formed.
Complete wound healing requires reformation of new blood vessels, new skin cells
and reconstruction of the different layers of the new skin tissue. Collagen deposition
is part of the key processes in wound healing and skin repair. The other processes are
angiogenesis, granulation tissue formation and re-epithelialization. A wound healing
aid should either act as a barrier to prevent further infection of the wound, serve as a
surface for cell adhesion to promote cell proliferation and differentiation or serve as
a scaffold for rebuilding if the new tissue, or all of the aforementioned. Such material
must be biocompatible and biodegradable should not induce any adverse immune
response and must have the right mechanical properties (Muthukumar et al. 2014;
Elango et al. 2018; Jeevithan et al. 2015; Chattopadhyay and Raines 2014).
When tested on human embryonic vein cells and in vivo on adult male mice,
collagen polypeptides obtained from the jellyfish R. esculentum and hydrolyzed into
different molecular weight showed significant wound healing bioactivity. Effective
and rapid acceleration of wound healing rate was also observed in rat skin cells
treated with collagen extracted from the skin of tilapia fish (Chen et al. 2019).
In improving the effectiveness of wound healing, collagen-based wound healing
material contribute significantly by reducing the hospital stay and cost of treatment.
276 12 Collagen
It also reduces the period over which the patient has to withstand the pain and dis-
comfort from the wound hence improvement in the quality of health care (Hochstein
and Bhatia 2014).
For effective wound healing the form in which the collagen is applied to the
wound is also important. The physical environment should be one which provides
the right architecture that promotes the forming of new skin cells. Collagen wound
healing materials have been made in the form of sponges produced using collagen
obtained from scales of mrigal fish (Pal et al. 2016), dressings made from electrospun
fibers of collagen extracted from tilapia fish (Moura et al. 2014; Zhou et al. 2016).
When tested in rat models, these different forms of collagen-based wound healing
materials showed enhancement of wound healing processes such as proliferation of
keratinocytes, fibroblast migration and collagen deposition.
12.7.3 Bone Regeneration
Collagen forms the matrix within which the bone mineral, hydroxylapatite is dis-
persed. Damage to the bone often requires a membrane to hold the bone in place
during healing after which the material is removed. The membrane serves to guide
the direction of bone regrowth ensuring that the bone returns to its original shape
and form. The removal of the membrane often requires an additional surgical pro-
cedure which implies more healthcare cost and further discomfort for the patient.
Non-biodegradable bone guiding membranes are made of materials such as polyte-
trafluoroethylene (Fiorellini et al. 1998), and this has the right mechanical properties
to support the bone regeneration and is unreactive enough not to induce an unde-
sirable immune response by the body. It will also not be degraded by the matrix
metalloproteinase enzymes (MMPs) which are responsible for bone protein degra-
dation, these enzymes get rid of damaged old bone tissue and replace with the newly
regenerated tissue (Armstrong and Jude 2002). In collagen-based scaffolds used in
bone regeneration, the activity of these MMPs enzymes is inhibited by treating the
scaffold with a tetracycline which inhibits the activity of MMPs (Moses et al. 2008).
Scaffolds are used where starter cells as well as a structural guide are required to
aid tissue regeneration. Collagen-based scaffolds have been developed with the goal
to replicate the macro- and microstructure of the bone tissue. Using collagen and
hydroxyapatite composites for such scaffolds further better mimics the biological
tissue physically and chemically. Different forms of these collagen/hydroxyapatite
scaffolds have been presented for bone regeneration applications (Zhang et al. 2018;
Xia et al. 2013; Yunoki et al. 2006, 2007). However, there is limited understand-
ing of the complex mineralization process and reproducing the intricately ordered
nanocrystalline structure of the collagen/mineral bone composite is not yet achiev-
able. Further, development is still required in the area of tissue engineering to develop
tissue scaffolds which better mimic the tissue structure.
The area of bone regeneration has further advanced toward development of 3D
printed scaffolds (Govindharaj et al. 2019). The ability to 3D print tissue scaffolds
offers the advantage of making more complex and more detailed and dimensionally
precise design of these scaffolds more possible. This will result in bone regrowth
which is as close to the original tissue as possible. Materials used for these include
polycaprolactone and hydroxyapatite composite (Liu et al. 2019). Such scaffolds
have been tested in vitro and in vivo on laboratory animals (commonly regeneration
of bones in damaged rat skull regions). The use of hydroxyapatite (Ca10 (PO4 )6 (OH)2 )
is with the aim of using a material which is the same as that found in the bone tissue.
It acts as the dispersed phase in the composite while the polymer acts as the matrix.
The membranes or scaffolds could be required to remain in place for different
periods of time; therefore, the collagen material needs to be designed for specific
applications. This is done by for example varying the degree of cross-linking which
in turn affects the rate of degradation (Moses et al. 2008). Composites of collagen
with varying degree of degradation could also be used. Most of the commercially
available tissue scaffolds are based on bovine and porcine-based collagen, and fish
waste could potentially replace or augment the use of bovine or porcine collagen
in tissue regeneration. Many aquatic vertebrates including fish collagen, like human
collagen, are mostly of type 1 (Zhang et al. 2018). Collagen alone usually does not
possess sufficient mechanical properties to act as a scaffold or membrane for tissue
regeneration; it is therefore often used as a composite alongside other materials such
as carbon, minerals or other polymers. The mechanical properties can also be further
enhanced by cross-linking with crosslinkers such as glutaraldehyde or ribose (Bai
et al. 2018).
12.7.4 Biomedical Implants
The biocompatibility of collagen makes it an excellent material for implants and

tissue replacement. An example of such is in corneal implants into the eye. Corneal
repair generally requires replacement of the damaged cornea with an artificial one
which is implanted into the eyes to replace the old one. The material from which
such is made needs to be biocompatible and be retained in the eye and allow growth
and proliferation of new corneal cells. Fish scales have been tested as ideal candidate
as a biocompatible corneal replacement. Scales from the collagen-based artificial
cornea have the advantage of not requiring human corneal donor, and it eliminates
the chance of an undesired immune response. Obtaining this from a widely available
resource such as fish scales which is not limited to only a particular region also offers
the prospect of having multiple producers across the world. The arrangement of the
collagen structure is quite important in tissue regeneration as the micro- and nano-
structure must allow sufficient space for oxygen transport to the cells growing within
and also allow for cell adhesion. To address this, a similar material which already
possesses an extracellular matrix structure similar to the microporous structure of
the cornea is used. This approach was used by Lin et al. (2010). Scales from tilapia
fish were decalcified using nitric acid treatment and living behind the organic part
which comprises of collagen. With the calcified part of the scales removed, what is
278 12 Collagen
left behind is a cellular architecture which has the right cellular architecture in terms
of pore size and shape to allow for effective cell growth.
12.7.5 Microneedles
Microneedles are transdermal patches which comprise of tiny projections with

micrometer dimensions. They are designed to pierce through the upper layer of the
skin without reaching the dermis where the pain receptors exist and in so doing open
up the skin in a painless manner and creating microconduits through which therapeu-
tics can be delivered. The lower layer of the skin (dermis) is easy reach to the blood
vessels from which the drugs can be absorbed into the bloodstream and to target
cells. These microneedles can be designed for delivery via different modes. One way
is to either pierce the skin, after which it is removed and replaced by a drug loaded
patch which then enters into the lower layer of the skin through the holes created by
the microneedles. These types are referred to as solid microneedles which could be
microfabricated using biocompatible materials such as metals such as stainless steel
or polymers such as silicone and polycarbonate. These solid microneedles could also
be coated with the drugs in the form of a dry coating which will then dissolve into
the skin when inserted and comes in contact with the skin fluid. Microneedles can
also be designed to dissolve into the skin upon piercing and release the drugs loaded
within into the lower layers of the skin from where it can be absorbed. These types
of microneedles are strong and rigid when in the dry state; however, they are solu-
ble in physiological fluid. Such that when they are inserted into the skin and come
into contact with the moisture in the skin, they dissolve off. The production process
usually involves loading the drug within the polymeric formulation in the wet state
followed by forming into microneedles such that the drug is entrapped within the
microneedle structure. These are referred to as dissolving microneedles and are made
from soluble polymers such as hydrolyzed collagen (Olatunji et al. 2014), polylactic
acid (Adamczak et al. 2011) and polyvinyl alcohol (Sullivan et al. 2010). A third
mode is where the microneedle with the drug loaded is inserted into the skin, the
drug is released from the microneedles after which the microneedle is then removed.
These are referred to as hydrogel microneedles. They are made using cross-linked
polymers with the drug embedded within. Cross-linked polymeric structures have
the ability to absorb water and swell. As the water is absorbed and the cross-link
network expands, the drugs which are trapped within the polymer chains are released
into the skin while the microneedle stays intact and can be removed after the drug
is released. Another type of microneedles exists which are the porous microneedles.
These are designed to inject the drug in liquid form into the skin such that it can be
absorbed from the dermis. However, these have been of much less interest due to
the complexity of their production process and high possibility of blockage of the
micron-sized holes upon insertion.
Recently, microneedles produced using fish scale-derived hydrolyzed collagen
hydrogels have been developed (Olatunji and Denloye 2019). These are a form of
hydrogel microneedles which in the dry state are rigid enough to pierce the skin;
however, once inserted into the skin it will absorb water and release the entrapped
loaded compound into the lower layer of the skin from where it can be absorbed into
the bloodstream more easily. In vitro studies showed that these hydrogel microneedles
have sufficient strength to pierce the skin and can swell and release loaded drug
compound when in contact with water. The hydrogels were made using hydrolyzed
collagen extracted from fish scales of croaker fish using the hydrothermal extraction
method.
Delivery of collagen into the skin using microneedles also serves cosmetic pur-
pose of boosting the skin collagen content. This could be a means to address the
decreased collagen production rate associated with aging and by so doing decrease
the appearance of wrinkles. Due to its large molecular weight collagen or hydrolyzed
collagen cannot pierce the upper layer of the skin; therefore, delivering the collagen
through topical means might not achieve the same goal as the collagen will not pene-
trate the impermeable top layer of the skin (Stratum corneum). Another option is the
use of hypodermic injections to deliver collagen underneath the skin. However, this
method comes with the disadvantage of discomfort and risk of accidental injury from
the injection. Dissolving microneedles made of lower molecular weight hydrolyzed
collagen can therefore be used as painless means to deliver collagen into the skin.
This also has the advantage of self-administration by the user without requiring medi-
cal expertise unlike with injection using hypodermic needles. The microneedle could
either be made entirely of collagen (Olatunji et al. 2014) or it could be blended with
another polymer such as polyvinylpyrrolidone (Sun et al. 2014).
12.7.6 Cosmetics and Skin Care
The skin is the outermost layer of the body and by virtue of this it has a significant
role to play in appearance. People are constantly seeking to smoothen, moisturize,
color, bleach and dye or improve the appearance of the skin in one way or the other.
This makes collagen also suitable as an active compound in cosmetics and skin care
products. Collagen has become a key part of the billion dollar cosmetics industry.
They have been applied in a range of products from topical skin care to cosmeceutical
oral gels to injectables (Alves et al. 2017; Devgan et al. 2019).
Collagen plays a significant role in cosmetics either in attaining the desired phys-
ical or rheological properties in terms of thickening and gelling property, and it also
plays a role as an active ingredient, mostly in the form of collagen hydrolysate where
it is intended to penetrate into the epidermis and improve the skin mechanical prop-
erties which result in smoother and more youthful looking skin (Savary et al. 2016).
Oligomeric forms of collagen which are very short polypeptide fragments are used
in skin care products such as skin lotions where they are thought to easier penetrate
the epidermis from where they can reach the dermis and induce skin rejuvenating
effect. There are further studies into confirming the exact manner or extent to which
280 12 Collagen
this happens (Bradley et al. 2015). Collagen, like other polymers, is usually used in
low concentration in cosmetics products, usually below 1% weight fraction.
12.7.7 Food
In food, collagen is used mainly in the hydrolyzed form. One of the more common
applications is its use in jellies. In the presence of water at a temperature of around
4 °C, gelatin forms a soft aqueous jelly. However, this jelly is temperature sensitive
and melts at higher temperatures. Although not yet a commercial reality, hydrolyzed
collagen is also used in the production of food packaging films (Hanani et al. 2014).
Like all polymers when dissolved in a solvent, it increases the viscosity of the
solvent such that there is a correlation between its concentration and the thickness
of the solution in a molecular weight-dependent manner. For this reason, hydrolyzed
collagen can be used as a thickening agent in food. In this dissolved form, when
the temperature is reduced to around 4 °C, the polypeptide chains take on a coiled
helix conformation and form a three-dimensional network trapping water within the
network of polymer chains. This manifests physically as a gel. This gel is thermore-
versible, and this comes an advantage when the melt in mouth feeling is required for
the food product (Rayner et al. 2016).
The gel properties vary for different types of hydrolyzed collagen. Gel properties
can be characterized by the gel strength, bloom, gel transparency and gel elasticity.
Other gel-forming polymers such as carrageenans tend to form stronger gels that
gelatin; however, in some applications a softer gel is required while in others a
strong gel is required. Hydrolyzed collagen from aquatic sources such as fish skin
has been shown to be also suitable in the production of food jellies similar to those
made using bovine and porcine sourced hydrolyzed collagen (Gamarro et al. 2013).
Hydrolyzed collagen is used in the production of soft gels which are used in
the production of some nutritional supplements such as omega-3 and production of
herbal capsules. These gels and capsules make it easier to ingest nutrients or herbs
which are either not taken in the diet or for easier intake where the taste or flavor is
not desirable.
12.7.8 Antioxidant Activity
Collagenous proteins obtained from edible jellyfish known as R. esculentum have

been shown to possess antioxidant activity (Zhuang et al. 2009), and collagens from
fish also show some antioxidant property (Chi et al. 2014). This antioxidant property
however is dependent on the structure of the collagen.
Antioxidant property could be demonstrated by a molecule or compounds abil-
ity to scavenge free radicals, donate protons or electrons, deactivate the reactive
oxygen species or chelate transition metals which promote oxidation. Collagen
shows better antioxidant activity in the hydrolyzed form. Further, lower molecular
weight hydrolyzed collagen shows better antioxidant properties than higher molec-
ular weight once. For example, when tested in vitro using hydrogen peroxide and
2,2-diphenylpicrylhydrazyl (DPPH) radicals, hydrolyzed collagen from the skin of
Spanish mackerel fish with molecular weight of 47.82 and 5.04 kDa showed 35.82
and 65.72% DPPH scavenging activity, respectively. The scavenging activity against
hydrogen peroxide was 41.72 and 76.99%, respectively (Chi et al. 2014).
The antioxidant ability of lower molecular weight hydrolyzed collagen is
attributed to the reducing property of the peptide groups present in the hydrolyzed
forms. In the helical form, the amine and carbonyl groups of the different polypeptide
chains are engaged in intermolecular secondary bonds; however, once hydrolyzed
these groups become available to other bonds, hence reducing tendencies of
hydrolyzed collagen over the intact form of collagen. Further, hydrolysis of the
polypeptides in lower molecular weight further frees up more peptide functional
groups which can have reducing effects by donating electrons to free radicals.
12.7.9 Anti-inflammatory Activity
The skin responds to external stimuli which are harmful to the body through inflam-
mation. However in certain cases, the process of inflammation could be exagger-
ated or lead to inflammatory diseases such as psoriasis, rheumatoid arthritis and
atherosclerosis. Anti-inflammatory agents are used as a therapeutic measure to treat
such inflammatory-related diseases. Fish scale collagen polypeptides have been used
to demonstrate anti-inflammatory activity of collagen extracted from fish. The col-
lagen peptides interfered with inflammatory response by preventing the secretion of
proteins associated with inflammatory response (Ahn et al. 2017). Collagen extracted
from squid also shows anti-inflammatory effects in the treatment of osteoarthritis.
The collagen type II extracted from squid offers superior therapeutic effect against
osteoarthritis by eliminating the immunogenicity associated with treatment using
collagen sourced from terrestrial animal (Dai et al. 2018).
Collagen is a protein of much commercial value. Bones and flesh of bovine and
porcine have been the main commercial source of commercial collagen production.
With the outbreak of bovine spongiform encephalopathy and the foot and mouth
disease, for health and economic reasons have led to the rising demand for alternative
collagen sources. The aquatic environment offers such alternatives. Both marine and
freshwater are a source of diverse range of collagen-producing organisms. Some of
these are bones, skins and scales of fishes, jellyfish, starfish and sponges (Felician
et al. 2019; Olatunji et al. 2014; Khong et al. 2016).
282 12 Collagen
The global collagen market is expected to reach 4.6 billion USD by 2023. In 2018,
the market was valued at 3.5 billion USD with a 5.2% estimated growth rate annually.
This includes collagen and hydrolyzed collagen, hydrolyzed collagen making a larger
share of the market. The marine sourced collagen shows constant growth as the
industry seeks alternatives to bovine and porcine sourced collagen. Main drivers of
growth for the collagen market are the increased use in the pharmaceutical, cosmetics
and food industry (Research and Markets 2019).
Companies which are commercially producing collagen and/or collagen peptides
from aquatic sources include Nitta Gelatin which has been in the business of gelatin
since 1979. Gelita AG, Weishardt Group, Darling Ingredients, Connoils, Lapi Gela-
tine S.p.a and Gelnex are some other companies which are key producers of gelatine.
The produce gelatin and collagen peptides for various target markets include food,
cosmetics and pharmaceutical. They produce from both aquatic and non-aquatic
sources.
Collagen plays a significant role in sustaining and improving human quality of
life. Such includes wound healing, tissue repair, disease prevention and cosmetics.
As human civilization expands and we develop new technologies to keep people
alive for longer, the tendency for injuries, diseases and need to improve appearance
increases. Therefore, an ever-growing market exists for collagen in its wide range of
applications.
12.9 Conclusion
Collagen is the most abundant protein in nature. It is present in both aquatic and
terrestrial animals. Fish is the most common source of aquatic collagen, other sources
are squid and jellyfish. Aquatic sourced collagen offers the advantage of avoidance
of the risk of mad cow disease and an alternative collagen source where ethics and
religious beliefs prohibit the use of collagen from pork or animal skin. Collagen from
aquatic source also has bioactive, mechanical and rheological properties which differ
from those of the conventional porcine collagen. This allows more diverse forms of
collagen for a wider range of applications. Most of the aquatic sourced collagens are
from the waste by-products of these aquatic resources; therefore, the extraction of a
valuable biopolymer such as collagen significantly contributes to optimal utilization
of and value addition to aquatic resources.
References 283
References
Adamczak M, Ścisłowska-Czarnecka A, Genet MJ, Dupont-Gillain CC, Pamula E (2011) Surface

characterization, collagen adsorption and cell behaviour on poly(L-lactide-co-glycolide). Acta
Bioeng Biomech 13(3):63–75
Aerssens J, Dequeker J, Mbuyi-Muamba JM (1994) Bone tissue composition: biochemical anatomy
of bone. Clin Rheumatol 1:54–62
Ahmed R, Haq M, Chin BS (2019) Characterization of marine derived collagen extracted from the
by-products of bigeye tuna (Thunnus obesus). Int J Biol Macromol 135:668–676
Ahn MY, Hwang JS, Ham SA, Hur J, Jo Y, Lee SY, Choi M, Han SG, Seo HG (2017) Subcritical
water-hydrolyzed fish collagen ameliorates survival of endotoxemic mice by inhibiting HMGB1
release in a HO-1-dependent manner. Biomed Pharmacother 93:923–930
Alves AL, Marques APL, Martins E, Silva TH, Reis RL (2017) Cosmetic potential of marine fish
skin collagen. Cosmetics 4(4):39
Armstrong DG, Jude EB (2002) The role of matrix metalloproteinases in wound healing. J Am
Podiatr Med Assoc 92:12–18
Bai X, Gao M, Syed S, Zhuang J, Xu X, Zhang X (2018) Bioactive hydrogels for bone regeneration.
Bioact Mater 3:401–417
Benedetto CD, Barbaglio A, Martinello T et al (2014) Production, characterization and biocom-
patibility of marine collagen matrices from an alternative and sustainable source: the sea urchin
Paracentrotus lividus. Mar Drugs 12:4912–4933
Bradley E, Griffiths C, Sherrat M, Bell M, Watson R (2015) Over the counter anti aging topical
agents and their ability to repair and protect photo aged skin. Maturitas. https://doi.org/10.1016/
j.maturitas.2014.12.19
Chang S, Shefelbine SJ, Buehler MJ (2012) Structural and mechanical differences between collagen
homo- and heterotrimers: relevance for the molecular origin of brittle bone disease. Biophys J
102(3):640–648
Chattopadhyay S, Raines RT (2014) Review collagen-based biomaterials for wound healing.
Biopolymers 101:821–833
Chen J, Gao K, Liu S, Wang S, Elango J, Bao B, Dong J, Liu N, Wu W (2019) Fish collagen surgical
compress repairing characteristics on wound healing process in vivo. Mar Drugs 17(33):1–12
Cheng X, Shao Z, Li C et al (2017) Isolation, characterization and evaluation of collagen from
jellyfish Rhopilema esculentum Kishinouye for use in hemostatic applications. PLoS One
12:e0169731
Chi CF, Cao ZH, Wang B, Hu FY, Li ZR, Zhang B (2014) Antioxidant and functional properties of
collagen hydrolysates from Spanish mackerel skin as influenced by average molecular weight.
Molecules 19:11211–11230
Dabrowska A, Rotaru GM, Spano F, Affolter C, Fortunato G, Lehmann S, Derler S, Spencer ND,
Rossi RM (2017) A water-responsive, gelatine-based human skin model. Tribol Int 113:316–322
Dai M, Liu X, Wang N, Sun J (2018) Squid type II collagen as a novel biomaterial: isolation,
characterization, immunogenicity and relieving effect on degenerative osteoarthritis via inhibiting
STAT1 signaling in proinflammatory macrophages. Mater Sci Eng C 89:283–294
Delphi L, Sepehri H, Motevaseli E et al (2016) Collagen extracted from Persian Gulf squid exhibits
anti-cytotoxic properties on apple pectic treated cells: assessment in an in vitro bioassay model.
Iran J Public Health 45:1054–1063
Devgan L, Singh P, Durairaj K (2019) Minimally invasive facial cosmetic procedures. Otolaryngol
Clin North Am 52(3):443–459
Dong Z (2019) Blooms of the moon jellyfish Aurelia: causes, consequences and control. In: World
seas: an environmental evaluation, vol III, 2nd edn., pp 163–171
Elango J, Lee JW, Wang S, Henrotin Y, de Val J, Regenstein JM, Lim SY, Bao B, Wu W (2018)
Evaluation of differentiated bone cells proliferation by blue shark skin collagen via biochemical
for bone tissue engineering. Mar Drugs 16:350
284 12 Collagen
Felician FF, Yu RH, Li MZ, Li CJ, Chen HQ, Jiang Y, Tang T, Qi WY, Xu HM (2019) The wound
healing potential of collagen peptides derived from the jellyfish Rhopilema esculentum. Chin J
Traumatol 22:12–20
Fiorellini J, Engebretson SP, Donath K, Weber HP (1998) Guided bone regeneration utilizing
expanded polytetrafluoroethylene membranes in combination with submerged and nonsubmerged
dental implants in beagle dogs. J Periodont 69:528–535
Gamarro EG, Orawattanamateekul W, Sentina J, Gopal TKS (2013) By-products of tuna processing.
GlobeFish 112(48):1–18. FAO, Rome
Govindharaj M, Roopavathi KU, Rath SK (2019) Valorization of discarded Marine Eel fish skin
for collagen extraction as a 3D printable blue biomaterial for tissue engineering. J Clean Prod (in
press)
Hanani ZAN, Roos YH, Kerry JP (2014) Use and application of gelatin as potential biodegradable
packaging materials for food products. Int J Biol Macromol 71:94–102
Hochstein AO, Bhatia A (2014) Collagen: its role in wound healing. Podiatry Manage 103(106):109–
110
Jeevithan E, Zhang JY, Wang NP, He L, Bao B, Wu WH (2015) Physico-chemical, antioxidant and
intestinal absorption properties of whale shark type-II collagen based on its solubility with acid
and pepsin. Process Biochem 50:463–472
Khong NM, Yusoff FM, Jamilah B et al (2016) Nutritional composition and total collagen content
of three commercially important edible jellyfish. Food Chem 196:953–960
Lin CC, Ritch R, Lin MS, Ni M, Chang Y, Lu YL, Lai HJ, Lin F (2010) A new fish scale-scaffold
for corneal regeneration. Eur Cells Mater 19:50–57
Liu D, Nie W, Li D, Wang W, Zheng L, Zhang J, Zhang J, Peng C, Mo X, He C (2019) 3D printed
PCL/SrHA scaffold for enhanced bone regeneration. Chem Eng J 362:269–279
Mahboob S (2015) Isolation and characterization of collagen from fish waste material—skin, scales
and fins of Catla catla and Cirrhinus mrigala. J Food Sci Technol 52(7):4296–4305
Matmaroh K, Benjakul S, Prodpran T, Encarnacion AB, Kishimura H (2011) Characteristics of acid
soluble collagen and pepsin soluble collagen from scale of spotted golden goatfish (Parupeneus
heptacanthus). Food Chem 129(3):1179–1186
Moses O, Vitrial D, Aboodi G, Sculean A, Tal H, Kozlovsky A, Artzi Z, Weinreb M, Nemcovsky CE
(2008) Biodegradation of three different collagen membranes in the rat calvarium: a comparative
study. J Periodontol 79(5):905–911
Moura LIF, Dias AMA, Suesca E, Casadiegos S, Leal EC, Fontanilla MR, Carvalho L, de Sousa
HC, Carvalho E (2014) Neurotensin-loaded collagen dressings reduce inflammation and improve
wound healing in diabetic mice. Biochem Biophys Acta 1842:32–43
Muthukumar T, Anbarasu K, Prakash D, Sastry TP (2014) Effect of growth factors and pro-
inflammatory cytokines by the collagen biocomposite dressing material containing Macrotyloma
uniflorum plant extract—in vivo wound healing. Colloids Surf B Biointerfaces 121:178–188
Olatunji O, Denloye A (2017) Temperature-dependent extraction kinetics of hydrolyzed collagen
from scales of croaker fish using thermal extraction. Food Sci Nutr 5:1015–1020
Olatunji O, Denloye A (2019) Production of hydrogel microneedles from fish scale biopolymer. J
Polym Environ 27(6):1252–1258
Olatunji O, Igwe CC, Ahmed AS, Alhassan DOA, Asieba GO, Das DB (2014) Microneedles from
fish scale biopolymer. J Appl Polym Sci 131:40377–40388
Pal P, Srivas PK, Dadhich P, Das B, Maity PP, Moulik D, Dhara S (2016) Accelerating full thickness
wound healing using collagen sponge of mrigal fish (Cirrhinus cirrhosus) scale origin. Int J Biol
Macromol 93:1507–1518
References 285
Rayner M, Ostbring K, Purhagen J (2016) Application of natural polymers in food. In: Olatunji O
(ed) Natural polymers: industry techniques and applications. Springer, Switzerland. https://doi.
org/10.1007/978-3-319-26414-1_5
Research and Markets (2019) Collagen market by product type—global forecast to 2023, Feb 2019.
Report ID: 4756592
Robinson RA (1979) Bone tissue: composition and function. Johns Hopkins Med J 145(1):10–24
Savary G, Grisel M, Picard C (2016) Cosmetics and personal care products. In: Olatunji O (ed)
Natural polymers: industry techniques and applications. Springer, Switzerland, pp 219–261
Shoulders MD, Raines RT (2009) Collagen structure and stability. Annu Rev Biochem 78:929–958
Sionkowska A, Kozlowska J (2014) Fish scales as a biocomposite of collagen and calcium salts.
Eng Mater 587:185–190
Subramaniam S (2018) Trimethylamine oxide (TMAO): a new toxic kid on the block. J Biomol
Res Therapeut 7(1)
Sullivan SP, Koutsonanos DG, del Pilar M et al (2010) Dissolving polymer microneedle patches
for influenza vaccination. Nat Med 16(8):915–920
Sun W, Inayathullah M, Manoukian MAC, Malkovsky AV, Manickam S, Marinkovich MP,
Lane AT, Tayebi AM, Rajadas J (2014) Transdermal delivery of functional collagen via
polyvinylpyrrolidone microneedle. Ann Biomed Eng 43(12):2978–2990
Tan CC, Karim AA, Latiff A et al (2013) Extraction and characterization of pepsin-solubilized
collagen from the body wall of crown-of-thorns starfish (Acanthaster planci). Int Food Res J
20:3013–3020
Xia Z, Yu X, Jiang X, Brody HD, Rowe DW, Wei M (2013) Fabrication and characterization of
biomimetic collagen-apatite scaffolds with tunable structures for bone tissue engineering. Acta
Biomater 9(7):7308–7319
Yunoki S, Ikoma T, Monkawa A, Ohta K, Kikuchi M, Sotome S, Shinomiya K, Tanaka J (2006)
Control of pore structure and mechanical property in hydroxyapatite/collagen composite using
unidirectional ice growth. Mater Lett 60(8):999–1002
Yunoki S, Ikoma T, Monkawa A, Marukawa E, Sotome S, Shinomiya K, Tanaka J (2007) Three-
dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity. Mater Sci Eng
C 18(4):393–409
Zhang D, Wu X, Chen J, Lin K (2018) The development of collagen based composite scaffolds for
bone regeneration. Bioact Mater 3:129–138
Zhou T, Wang NP, Xue Y, Ding TT, Liu X, Mo XM, Sun J (2016) Electrospun tilapia col-
lagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and
differentiation. Colloids Surf B 143:415–422
Zhuang YL, Zhao X, Li BF (2009) Optimization of antioxidant activity by response surface method-
ology in hydrolysates of jellyfish (Rhopilema esculentum) umbrella collagen. J Zhejiang Univ
Sci B 10:572–579
Zhuang Y, Sun L, Zhang Y et al (2012) Antihypertensive effect of long-term oral administration
of jellyfish (Rhopilema esculentum) collagen peptides on renovascular hypertension. Mar Drugs
10:417–426
Chapter 13
Starch
Abstract Starch plays a significant role in the economy as it serves as a source

for two products; food and bioethanol, both of which are essential commodities. It
is also used in several other industries and discussed in this chapter. In the aquatic
environment, starch can be sourced from aquatic plants and algae. Starch-producing
aquatic organisms have a significant role to play in the production of third-generation
biofuel which does not require already limited arable land for the cultivation. The
unique chemistry of some aquatic starch forms makes them attractive for specific
industrial applications.
Keywords Starch · Polysaccharides · Carbohydrates · Polymers · Aquatic
13.1 Introduction
Starch is a carbohydrate which is more commonly associated with terrestrial food

crops such as cassava and corn which forms staple foods and biofuel feedstock in
many parts of the world. Some aquatic organisms also contain starch; for example,
aquatic sources of starch are algae, duckweeds and water hyacinths. Although starch
is a biopolymer that is not exclusive to the aquatic environment, aquatic-sourced
starch plays a significant role as an alternative source of starch in many industries.
One of the potential economic importance of aquatic-sourced starch is the poten-
tial for the use in biofuel production. While the first-generation biofuels created
the promise of a renewable source of energy (Eichelmann et al. 2016), the second-
generation biofuel promises a renewable source of energy that does not compete
with food (Negm et al. 2018), and the promise of the third generation of biofuel from
aquatic resources such as algae (Yew et al. 2019) is a supply of fermentable biomass
for ethanol production which does not threaten food security or the environment
and does not compete with terrestrial animals and humans for land space. Third-
generation biofuel from algae and aquatic plants can achieve the goal of carbon-
neutral biofuel production. This is mainly owing to the fact that the process of cul-
tivation of algae and aquatic plants removes carbon dioxide from the environment.
This is unlike in the case of fossil fuel extraction and refinery which leads to further
carbon emissions.

288 13 Starch
Food security is increasingly becoming a global concern. Starch forms an impor-

tant part of the human diet and making up over half of the global calorie intake. It is
a major source of carbohydrate and energy food. Although approximately 65 million
tonnes of starch are produced annually in the world and constantly increasing at an
annual rate of 2–3% (Sullivan-Trainor 2013; McWilliams 2017), much more starch
would be required if it is to serve as a sustainable source of biofuel as well as a
component of food and other products used by humans.
Starch is one of the major polymers being explored to solve one of the biggest
global crises of single-use plastic pollution through the development of starch-based
bioplastic packaging. It is therefore pertinent to seek alternative, preferably more
abundant source of starch which do not interfere with the human consumption of
starch and starch-based products but rather augments it where possible. Conven-
tional starch sources are maize, rice, wheat, potato, tapioca and cassava. From these,
starches for biofuel, bioplastic, food and other starch-based products must be sourced.
In addition to the existing and potential application of aquatic-sourced starch to
create products, the process of cultivating and processing of aquatic-sourced starch
should also be considered in order to understand the overall impact of aquatic-sourced
starch as a biopolymer resource. This chapter therefore discusses the sources, avail-
ability, production process, chemistry, potential applications and the impact on the
environment and global economy of starch sourced from aquatic ecosystems.
Starch, a major energy storage carbohydrate, occurs naturally in plants. It is present

in the leaves, seeds, fruits, stems, roots and tubers. It is also present in red, brown
and green macroalgae and microalgae (Prabhu et al. 2019). These photosynthetic
organisms absorb light energy from the sun and store it in chemical form as starch.
Aquatics plants like the water hyacinth, duckweed and Azolla are examples of aquatic
sources of starch. These starch-producing organisms can be found in both marine
and freshwater (Zhang et al. 2018) in varying amounts. The starch content of any
given organism depends on factors such as species and abiotic factors of the aquatic
ecosystem, and it is growing within.
Microalgae contain a considerably large amount of starch. On average, they con-
tain around 37% starch by weight. Strains such as Chlamydomonas, Chlorella,
Spirulina, Dunaliella, Scenedesmus have the most abundant starch content which
could be as much as 50% carbohydrates most of which is starch (Rehman and Anal
2018). At optimized conditions, the cultivation of the green marine microalgae strain
Tetraselmis subcordiformis with starch content of up to 62.1% dry weight has been
achieved (Yao et al. 2012). These findings suggest that not only is starch present in
microalgae but also that the rate of starch accumulation in the microalgae biomass
can be controlled and thus optimized through the limitation of nutrients.
The rate of accumulation of starch and biomass within the aquatic organism is
affected by the environmental conditions as well as the nutrients available for growth.
For example, marine red microalgae, Porphyridium marinum, grow optimally at a

light intensity of 100 µmol photons m−2 s−1 , sodium nitrate (NaNO3 ) concentration
of 1 g/L and a sodium chloride concentration of 20 g/l (Himaa et al. 2019). At this
optimal condition, these algae can accumulate up to 140.21 µg per ml which is 13%
higher than when grown in suboptimal conditions. The lower the light intensity and
salinity, the higher the starch accumulated within the microalgae.
Starch is also present in a considerable amount in aquatic plants such as water
hyacinth and duckweed. The composition of starch in aquatic plants varies from
species to species and is also affected by seasonal variation and growth conditions.
In Azolla, for example, filiculoides produces up to 36.4 g/L more sugars than another
strain of Azolla pinnata. Filiculoides biomass contains 6.05% starch while pinnata
contains 4.7%. Pinnata also has higher lignin content (Miranda et al. 2016). Azolla,
in general, has the highest lignin content of all the common aquatic plants explored
in the literature; however, this lignin content (13.2% for pinnata and 10.3% for
filiculoides) is still lower than those of terrestrial lignocellulose crops such as corn
and sugar beets.
In algae, the location of starch on the organism could vary. The starch could be
present in the chloroplast, cytoplasm or pyrenoids. They could also be present in the
form of plates or granules. For example, in the red algae Gracilariopsis, starch is
present in the form of granules within the cytoplasm; in the green algae Cladophora,
starch is present within the chloroplasts in form of granules; and in the green algae
ulva, starch is present in the form of plates around the pyrenoids and as granules in
the chloroplast thylakoid membranes.
Starch content could vary depending on the environmental conditions. Growing
in the open environment weather conditions significantly affect the starch content at
any period. Starch content also varies with the organism’s reproductive cycle. Studies
on the green algae Ulva ohnois indicated a minimal starch accumulation at the initial
growth phase; in the particular study, this occured between March and May and
maximum starch content was recorded in June. Note that the biomass accumulation
rate does not necessarily correlate with the starch accumulation rate. A faster growth
rate is not necessarily implicative of a higher starch content, and the organism could
be accumulating other compounds such as cellulose, proteins and lipids.
There are also instances where starch is degraded into other polymers within the
aquatic organism, and this has been observed in red algae. Degradation of starch
into carrageenan was observed in S. cordalis, a unicellular red algae. It appears that
under various conditions such as nutrient supply and irradiance, and the starch within
the organism is converted into carrageenan (Fournet et al. 2000). This is attributed
to the need for the organism to switch to a biochemical pathway which favors the
surrounding condition. Starch also exists in the aquatic environment in the form of
floridean starch (Himaa et al. 2019). This refers to starch sourced from a particular
strain of red algae known as the florideophyceae. It is mainly characterized by the
absence of the branched polymer of starch, amylopectin. Floridean starch is discussed
in more detail in a separate section within this chapter.
290 13 Starch
Availability of starch is limited to some starch-producing aquatic plant and algae

biomass. Within this selection lies thousands of species and these include duck-
weeds, Azolla, water hyacinth macroalgae and microalgae. Duckweed, for example,
comprises of 37 species (Appenroth et al. 2013). Here we look at the availability of
starch from the aquatic environment based on the availability of these sources and
their starch component.
Duckweeds grow almost all year round with a growth season lasting between
9 and 12 months (Ziegler et al. 2015). Duckweed of up to 39.1–105.9 tonnes per
hectares annually is achievable with a starch content of up to 31.0–45.8%. 94.7% of
this starch can be converted into bioethanol, and duckweed is therefore a relatively
abundant source of starch. Methods such as selectively breeding high-performance
duckweed strains and improving starch enrichment in the plant are among the area
of research focus toward advancing duckweed for commercial production of starch
for biofuel application (Xu et al. 2014).
Duckweeds are exceptionally high in starch compared to other aquatic and non-
edible plants in general. Duckweed species include Wolffia arrhiza (rootless duck-
weed), Spirodela polyrhiza (greater duckweed), Lemna gibba (fat duckweed), Lemna
minuta (least duckweed), Lemna minor (common duckweed), Lemna trisulca (ivy-
leaved duckweed). Duckweed annual accumulation estimated to 39.1–105.9 tonnes
of starch per hectare annually (Xu et al. 2012). They also have a relatively high
protein content. Their low lignin content is also a desirable feature as it makes the
extraction process less demanding. Accumulation of starch in duckweed is affected
by factors such as light intensity, temperature, nitrogen content and phosphorus con-
tent of the water within which they are growing. Adequate monitoring and control
of these parameters can improve the rate of starch accumulation.
Availability of biomass for starch production can be further improved by prac-
ticing a mixed culture system of aquatic farming. The productivity of biomass of
some aquatic plants can be improved by polyculture method. This involves growing
more than one strain of the plant within the same growth space. Duckweed poly-
culture comprising of Lemna aequinoctialis 5505, Landoltia punctata 5506 and S.
polyrhiza 5507 attained a starch composition of up to 28.78 g m−2 . When compared
to monocultures of each species alone, the combination of all three gave a higher
starch biomass yield. However, the use of a polyculture does not always guarantee an
improvement in starch accumulation. This highly depends on the combination of the
species. While one combination of species in a polyculture could result in improved
starch content compared to monoculture, in a different combination of species the
starch accumulation could be less. For example, a monoculture of L. aequinoctialis
is grown at a temperature of 20 °C, light intensity of 105 µmol m−2 s−1 , nitrogen
concentration of 35 mg L−1 and potassium concentration of 15 mg L−1 which are
accumulated with 14.22 g of starch per m2 of duckweed growth area. At the same
growth condition, the combination of all three species L. aequinoctialis, L. punctata
and S. polyrhiza attained a starch accumulation of 13.96 g m−2 .
13.3 Availability of Raw Material 291
Aquatic plants which produce starch can accumulate biomass at a faster rate than
terrestrial plants. Duckweeds, for example, can double in biomass between 1 and
3 days (Li et al. 2016). The effect of factors such as light intensity and temperature
on the rate of accumulation from biomass has been studied by different researchers;
however, the effects of these factors depend on other complex factors such as origin
of the plant and adaptation to the previous environment, such that a particular trend
cannot be recommended. For example, that an increase in light intensity results in
higher starch accumulation for one species sourced from a particular region might
not mean the same species from a different origin will respond in the same manner.
Starch production by some aquatic plants can be further optimized by limiting the
growth of the plant biomass to only the starch yielding parts of the plant. This results
in the faster harvest and minimizes the growth of non-starch materials. For exam-
ple, in an earlier study (Fujita et al. 1999), optimal turion growth is achieved at low
nutrient concentration and low spacing between plants that have been demonstrated
in another duckweed strain, W. arrhiza (rootless duckweed). The turion is a dormant
tissue and can remain dormant for long periods (up to a month) in the absence of
nutrients. The turion is rich in starch (65.65%) and relatively low in lignocellulosic
(12.82%), and this is desirable for isolation of the starch component and can be
grown with less nutrient compared to the whole plant. Duckweed turion can achieve
a starch productivity of up to 2.90 g/m2 daily from a turion biomass accumulation
rate of 3.78 g/m2 daily. The turion of S. polyrhiza, also commonly known as root-
less duckweed or spotless watermeal, is another highly productive starch-producing
aquatic plant. 0.34 g of ethanol is obtainable per gram of its turion. The saccharified
starch achieved a sugar to ethanol conversion of 91.67% (Xu et al. 2018).
The water hyacinth is another aquatic plant with considerable starch content.
Starch is present within different parts of the plant with the highest starch content
present in the rhizomes while the roots have the least. There is also a moderate amount
of starch present in the stolons, leaves and peduncles (Penfound and Earle 1948).
Starch content in green macroalgae ranges between 1.59 and 21.44%. The starch
content is affected by growth conditions and varies from season to season. Up to
3.43 tonnes of starch can be obtained per hectare annually from offshore cultivation
of Ulva ohnoi green algae (Prabhu et al. 2019).
Several reports have shown that starch content in aquatic plants and algae can
be significantly increased through nutrient starvation (Prabhu et al. 2019; Rehman
and Anal 2018; Korzen et al. 2016; Andrade et al. 2004). Such potential for yield
improvement through biotechnology and agricultural technology makes aquatic-
sourced starch very promising for applications such as biofuel production, a process
which requires large quantities of starch. Starch content in algae can also be boosted
by controlling the wavelength of the light under which they grow. For example, Ulva
pertusa has been shown to have improved starch content in blue light (Muthuvelan
et al. 2002). Starch content in algae could be as much as 32% on a dry weight basis
as it is found in Ulva rigida (Korzen et al. 2016). Table 13.1 gives the starch content
from some aquatic sources, and Table 13.2 lists starch production rate in terrestrial
crops.
292 13 Starch
Table 13.1 Starch content of some aquatic plants and algae

Source Starch content (% weight) References
Duckweed turion 65.86 Xu et al. (2018)
Green macroalgae 1.59–21.44 Prabhu et al. (2019)
Filamentous green algae Oedogonium 46.29 Zhang et al. (2016)
nodulosum
L. aequinoctialis (duckweed) 21.70 Li et al. (2016)
S. polyrhiza (duckweed) 23.23 Li et al. (2016)
Coculture of L. aequinoctialis and S. 23.78 Li et al. (2016)
polyrhiza
Landoltia punctata duckweed 60.03 Liu et al. (2018)
Table 13.2 Starch accumulation rate in some terrestrial plants and green algae
Plant Starch (tonnes ha−1 yr−1 ) References
Wheat 1.84 Beloshapka et al. (2016)
Rice 1.79 Beloshapka et al. (2016), Reddy and Bhotmange
(2014)
Maize 1.56 Beloshapka et al. (2016)
Potato 5.46 Rahman et al. (2016)
Cassava 10.39 Edison and Srinivas (2016)
Ulva ohnoi 2.01–3.38 Prabhu et al. (2019)
13.4 Chemistry of Aquatic Starch
Starch comprises of two polymers, amylose and amylopectin, and the monomeric
unit of these polymers is d-glucose. The glucose monomers are either linked together
in linear structure forming amylose chains or in branched structure as seen in the
amylopectin fraction of the starch. In the amylose chains, the glucose monomers are
linked together in a alpha 1–4 linkage; while in the amylopectin, there is about 5%
branching with glucose 1–6 linkages forming branches along with the amylopectin
chain. Both amylopectin and amylose are combined within a starch granular structure
with a varying level of crystallinity. The ratio of amylose to amylopectin and the level
of crystallinity of the starch granules vary for different starch sources (Robyt 2008).
Figure 13.1 shows the chemical structure of starch represented by (a) 1–6 linked
alpha glucose units and (b) a linear alpha 1–4 linked alpha glucose repeating units.
One of the important properties of starch for applications in, for example, food
and pharmaceutics is the gelatinization temperature. The gelatinization temperature
of starch from green algae U. ohnoi is reported as 69 °C. Starch is insoluble in
water and will gelatinize when exposed to water at a relatively high temperature.
This gelatinization temperature varies for different starch sources. Gelatinization
of starch is an important process, whereby starch granules swell in the presence
13.4 Chemistry of Aquatic Starch 293
Fig. 13.1 a Branched 1–6 linked alpha glucose units and b linear alpha 1–4 linked alpha glucose
repeating units
of excess water at the gelatinization temperature. This property is important in the

application of starch in, for example, film formation and uses as a thickening agent.
Gelatinization of the red algae Gracilariopsis lemaneiformis is 55.1 °C, and that of red
algae Gracilaria chilensis is 52.7 °C, while that of green algae U. ohnois is 118.4 °C.
Gelatinization temperature of starch from terrestrial plants is generally higher, for
example the gelatinization temperature of potato starch is 120.8 °C (Prabhu et al.
2019) lower values for the gelatinization temperature of potato starch of 66.2 °C have
been reported in other studies (Yu et al. 2002). This suggests that the gelatinization
temperature from algal source is possibly generally lower than that from terrestrial
plants.
In one of the rare studies to observe the starch structure in algae cells, TEM and
confocal imaging revealed starch granules between 5 and 7 µm present within the cell
cytoplasm and thylakoids of the chloroplasts in U. ohnoi green algae. TEM imaging
294 13 Starch
presented various starch conformations which included ovoid, spherical and pear-
shaped starch granules as well as irregularly shaped ones. The pyrenoids were also
found to be surrounded by starch plates (Prabhu et al. 2019). This orientation of starch
within these particular algae does not deviate from that of similar starch-containing
terrestrial plants.
Hydrolysis occurs more readily in gelatinized starch than in the native granular
starch form. This can be explained by the increased surface area of the chain as they
interact with water to form temporary network structures in the gelatinized form,
thus leaving the polymer chains more vulnerable to degradation. Hydrolysis can be
carried out using acids or enzymes. Amylase and amyloglucosidase are enzymes
used for enzymatic hydrolysis of starch. Enzymatic hydrolysis is preferred as the
enzymes are selective, and they break only the amylose and amylopectin glycosidic
bonds. However, the acid hydrolysis is non-selective and breaks all the glycosidic
bonds within the biomass. For applications, such as bioethanol production acid,
hydrolysis of all the carbohydrates is desirable; however, for more selective processes
or analysis, enzyme hydrolysis is preferred. Studies on aquatic-sourced starch such
as that from U. ohnois macroalgae (Prabhu et al. 2019) have shown that aquatic
starches are susceptible to enzymatic and acidic hydrolysis as terrestrial plant-sourced
starches. For example, high-performance ion chromatograms of starch from the green
algae U. ohnois showing major peak indicating the presence of main glucose in the
acid- and enzyme-hydrolyzed starch extracts confirm that starch from such source can
be hydrolyzed to form glucose. The susceptibility to hydrolysis is an important step
toward determining the suitability of algal starch for human or animal consumption
and for other applications.
Granule size is an important characteristic of starch. It affects properties such as
gelatinization temperature, efficiency of hydrolysis and crystallinity. Gelatinization
temperature of different sources of starch is due to the variation in the amylose and
amylopectin content and the size of the starch granules. For instance, the starch gran-
ules of the green algae U. ohnois are smaller than those of potato starch, resulting in a
higher gelatinization temperature of potato starch. Starch from microalgae Chlorella
sorokiniana has granule size of 1 µm. In addition to this, its molecular weight, A
type crystallinity pattern (crystallinity 30%) as well as amylose content is similar to
starch from cereal plants (Gifuni et al. 2017). When SEM images of starch granules
extracted from aquatic plant U. ohnoi were compared, the surfaces of the algal starch
appeared smoother compared to that of the potato-sourced starch. Typical size of
red macroalgae starch granule is ~2 to 5 µm (Yu et al. 2002) that of U. ohnoi was
observed to range an average of 6.6 µm with a relatively large standard deviation
as the granule sizes ranged from 0.7 to 27.4 µm with multiple peaks in the size
distribution characterization. Although the size of U. ohnoi starch granules is larger
than that of typical red macroalgae, this variation in the size distribution of starch
granules within species of aquatic plants is not too far from those of other typical
terrestrial-sourced starch. For instance, potato starch granules have an average size
of 45.5 µm while those of rice starch are between 2 and 7 µm (Le Corre and Brass
2010). This implies that the applicability of starch extracted from aquatic source is
not limited by the granule size. Although the cause of this variation in starch granule
13.4 Chemistry of Aquatic Starch 295
size is not fully understood, it is hypothesized to be due to the timing of the granule
initiation process during starch synthesis within the cell. Other studies have estab-
lished a relationship between the size of granules, period of formation and shape of
the granules and the amylose content of the starch (Ziegler et al. 2015).
The crystallinity of starch also varies for different sources. FTIR spectroscopy
showed that starch from green algae has a more amorphous structure, indicated by
more intense peaks at the wavelength of 1014.5 cm−1 . Starches from green algae U.
ohnoi and potato starch show the same peaks for starch in the fingerprint region of
the FTIR and thus confirming that the starch from this aquatic source has identical
chemical composition to that of terrestrial-sourced starch. Decomposition temper-
ature is another important parameter to consider in polymeric materials, and the
decomposition temperature informs processing and handling thermal parameters. At
283.7 °C, starch of green algae U. ohnois had began to degrade. This decomposition
temperature is similar to that of potato starch.
The physicochemical properties of the starch from aquatic source show many
similarities to those of common terrestrial sources such as rice, potato and cassava.
This similarity in the physical and chemical properties of starch from aquatic source
to those of terrestrial source suggests similar industrial applicabilities. Future studies
on toxicology and biocompatibility, it is required to confirm the possible range of
application to include direct consumption in food, cosmetics and medicine.
13.4.1 Biodegradation of Starch
Starch is a digestible polymer. It is broken down into its glucose units which can then
be converted into ATP to meet the energy requirement of the organisms. Looking
at the chemical structure of starch as shown in Fig. 13.1, it comprises of carbon,
hydrogen and oxygen, all of which can be safely absorbed into the environment.
Starch-producing organisms have biomechanisms for degradation of starch which
they accumulate and convert to energy when required. Humans also have biome-
chanics to degrade the starch they consume from plants and algae as food. Edibil-
ity depends on the safety of other molecules attached to the starch. Since starches
from terrestrial and aquatic sources are chemically identical, no distinction is made
between their respective degradation processes.
Starch-based products, such as bioplastics not consumed as food, need to be dis-
posed of following their usage lifespan. One method of disposal is in landfill or buried
in soil or compost heaps where they degrade and serve as a carbon source for the
soil. Biodegradation of starch buried in soil in the forest occurs within several days.
Within 15 days, fungal hyphae colonize starch films and pronounced degradation of
starch granules is detectable by 45 days based on the observations using scanning
electron microscopy (SEM) (Lopez-Llorca and Valiente 1993).
The chemical properties of starch and the degradation conditions affect the degra-
dation process. Properties such as crystallinity, molecular weight and granule size
affect the degradation rate and mechanism. Higher molecular weight starch degraded
296 13 Starch
faster; however, degraded rate does not seem to be affected by granule size. Retrogres-
sion decreases the starch degradation rate. This is due to retrograded starch having
a more ordered structure which inhibits degradation (Li et al. 2015). These findings
suggest that the rate at which starch is broken down varies for different types of
starch since as discussed in the earlier section, the molecular weight, granule size
and crystallinity vary for starch from different sources. This is important in the design
of products such as bioplastic films where the rate of degradation needs to be slow
enough to retain product stability during storage and usage life but fast enough to
allow safe degradation in the environment without accumulation.
Starch is broken down by amylase enzymes which can cleave alpha 1–4 and alpha
1–6 bonds in amylose and amylopectin. This can occur in nature either by microor-
ganisms or within the digestive systems of animals and humans which metabolize the
required enzymes. Organisms which carry out degradation of starch in natural envi-
ronment include fungi such as penicillium Appressoria and chlamydospores (Lopez-
Llorca and Valiente 1993), Trichoderma viride I (Schellart et al. 1976) and bacillus
bacteria (Wang et al. 2019). These organisms produce amylase and glucoamylase
enzymes which catalyze starch degradation and therefore serve as sources of enzymes
for commercial processes involving starch degradation such as hydrolysis of starch
to fermentable sugars for bioethanol production. Degradation of starch also occurs
through acid hydrolysis and heat treatment. This can either be desirable or undesir-
able. The acid hydrolysis of starch is, for example, desirable in the production of
bioethanol where the starch is hydrolyzed into fermentable sugars. In the processing
of starch into plastic films, undesirable thermal degradation of starch occurs dur-
ing the process of extrusion (Liu et al. 2010). This is considered undesirable and is
prevented by careful control of the process to ensure proper temperature distribution.
13.5 Floridean Starch
While starch is generally defined as consisting of amylose and amylopectin in differ-

ent ratios, a form of starch exists where the amylose content is zero. This is known
as floridean starch and found in red algae as a highly branched phosphorylated amy-
lopectin chain; floridean starch is a short-chain starch with a degree of polymerization
of 18 and a branching of 4.8 (Yu et al. 2002). The highly branched structure and low
molecular weight make it less stable than the long-chain starch with linear structure.
Ordinarily starch is found in land plants and green algae within the plastids. A
different form of starch, floridean starch is found in the cytosol of red algae and glau-
cophytes, cryptophytes, dinoflagellates and apicomplexa parasites. These organisms
use a different pathway to synthesize and store starch in a form called floridean starch
(Dauvillee et al. 2009). The storage of floridean starch within the cytosol in red algae
is similar to the manner in which fungi and animals have their storage carbohy-
drate (glycogen) stored in the cytosol. Accumulation of starch within the plastid in
photosynthesizing plants is more conventional since this is the part associated with
manufacture and storage. Although it was recently thought that floridean starch also
13.5 Floridean Starch 297
contained amylose (McCracken and Cain 1980), it was later found that floridean
starch contains only amylopectin and is limited to a specific group of red algae so
named as Florideophyceae (Dauvillee et al. 2009). The other group of red algae,
Bangiophyceae, does not contain floridean starch (McCracken and Cain 1980). The
composition of floridean starch can be up to 80% in red algae.
Floridean starch extracted from red algae in one study show granules structures of
mostly spherical orientation, although some other granule shapes are also observed
(Yu et al. 2002). The granule length was measured to be in the range 1.7–3.4 µm
and was species dependent. For example, the red algae species G. chilensis has an
average granule size of 3.4 µm while that from the red algae Gracilariopsis sp. had
an average granule size of 1.7 µm.
Although floridean starch is relatively newer concept, the Florideophyceae red
algae are widely commercially explored for food and extraction of agar and car-
rageenan. There is therefore room to integrate the extraction of floridean starch
alongside the extraction of these other polymers from the red algae. The highly
branched structure of floridean starch means they are less stable and more easily
hydrolyzed than linear form. This has significance in the area of biofuel production.
13.6 Extraction Process
Extraction of starch from the natural aquatic source generally involves separation
from the other components such as lignin, cellulose, proteins, lipids and other com-
ponents. In plants and algae with no lignin, the complexity of the extraction process
is reduced to a significant extent. Starch can also be obtained as a by-product of
other extraction processes. It is more efficient to be able to obtain multiple biopoly-
mers from the same source, particularly one that can be done in a single process
concurrently.
In the following subsections, we review the extraction processes for starch from
various aquatic sources. Some similarity exists in some of these processes, while the
variations should have some significant economic and environmental implications.
The variations in the processes are in most cases which are due to the difference in
the structural composition of the plant or algae feedstock.
13.6.1 Extraction of Starch from Microalgae
Microalgae are an important source of biopolymers, and one of the reasons for this
is the ability to control and influence the metabolism of microalgae compared to
macroalgae. Here, the extraction of starch from the microalgae C. sorokiniana is
used as an example of the extraction of starch from microalgae. The microalgae
are cultivated in a photobioreactor. It is also important to consider the process for
extraction of starch from microalgae harvested from the open aquatic ecosystem as
298 13 Starch
this is a way to utilize the microalgae generated from green tides. The limitation of
harvesting microalgae from the open aquatic ecosystem includes risk of contami-
nation and uncontrolled growth condition therefore the starch content might not be
optimized.
In the closed harvest system, the microalgae are cultivated in photobioreactors
in Bold’s Basal algae growth medium. The growth conditions are maintained at
25 °C, illumination of 300 µE m−2 s−1 , aerated at 0.02 vvm with air containing 2%
CO2 . The system was allowed to run until it reached nitrogen starvation to optimize
starch accumulation by the microalgae. The microalgae biomass is then harvested
and taken through cell disruption. This process of cell disruption disintegrates the
cell wall freeing up the components which include starch. Cell disruption can be
achieved by methods such as enzyme or alkali pretreatment of mechanically by bead
beating with 0.5 mm glass beads while suspended in ethanol. Further, cell disruption
is achieved by heating at a temperature of 75 °C. The starch is then separated from
the other component of the microalgae by centrifugation at 10,000 g for 20 min.
The starch is recovered as the solid residue and is resuspended in distilled water
and centrifugation is repeated and separation carried out through Percoll gradient to
achieve purer starch. Figure 13.2 summarizes the extraction process for obtaining
starch from microalgae.
Fig. 13.2 Process flowchart Microalgae biomass

for the extraction of starch
from microalgae
Mechanical cell disruption
Heating
Centrifugation Liquid
Solid
Washing
Centrifugation using Impurities

Percoll gradient
Solid
Purification
Starch
13.6 Extraction Process 299
13.6.2 Extraction of Starch from Duckweed
Removal of lignin is carried out by treating the biomass with 25 mM of sodium acetate
at a pH of 5.5 and autoclaving at 121 °C for 20 min. The residue from this is the starch
and cellulose component of the biomass. The starch can then be separated from the
cellulose by methods such as hydrolysis whereby the starch is removed as soluble
sugar. This can be enzymatic hydrolysis with amylase or amyloglucosidase. In this
process, 500 and 50 µL of alpha-amylase and alpha-amyloglucosidase, respectively,
are added per gram of dried biomass and heated under continuous agitation of 250 rpm
for a duration of 5 h after in which hydrolysis is completed. The hydrolyzed starch in
the form of glucose can then be separated from the cellulosic residue. This method of
the extraction of saccharified starch is used, for example, as stage in the conversion
of aquatic plant biomass for bioethanol production. This particular method has been
adapted for use in the extraction of starch for use in the production of biofuel from
duckweed (Miranda et al. 2016). The starch can also be extracted in the whole form.
13.6.3 Extraction of Starch from Macroalgae
Extraction yield from algae varies depending on the type of algae and efficiency of
the process. Here, we review a typical method used for extraction of starch from the
green macroalgae U. ohnoi. Extraction yield of 50.37% has been reported for the
green macroalgae U. ohnoi. The starch extract had 75.45% starch content per extract
mass.
In the method reported for extraction of starch from the green seaweed, U. ohnoi,
the cultivated seaweed was washed and added to distilled water in the ratio 1:20 dry
weight of biomass to water volume. The mixture of water and seaweed was then
homogenized until it forms a fine suspension. This was then followed by filtration
using nylon filters with pore sizes of 100 µm and then through 50 µm and finally
through 10 µm filters. This allowed for more effective filtration. The filtrate obtained
was then centrifuged at 4000 rpm for 6 min. The solid fraction from the centrifugation
contains mainly starch. The residual pigments and lipids are removed by washing
three times with absolute ethanol.
The extraction process can be scaled up for mass production. The process does not
deviate much from the typical process used for industrial production of starch from
terrestrial sources. Extraction yield of 50.37% has been reported for starch from the
green algae U. ohnoi. The purity obtained using this particular method was 75.45%
(Prabhu et al. 2019). This is similar to the purity of other non-aquatic starches such
as cassava, rice and potato which are 80, 75–86.8 and 78.6%, respectively (Bhat and
Riar 2016; Sjöö and Nilson 2018).
300 13 Starch
13.6.4 Extraction of Floridean Starch from Red Algae
To extract floridean starch from red algae, the algal biomass is pulverized to smaller
particle sizes. This can be achieved using equipment such as a hammer mill- or
motor-driven mortar and pestle. The pulverization is done under liquid nitrogen to
prevent oxidation. 50 mM of citrate buffer is then added to the biomass at a pH of
6.5. At this stage, the floridean starch is dispersed as smaller particles and the protein
dissolved in the liquid state since acid-soluble proteins will dissolve out of the algae
tissue at low pH. These are separated from the larger solid residues of the algae tissue
by filtration. The filtrate is then centrifuged at 13,000× g for 10 min. This separates
the dispersed starch granules which settle at the bottom from the dissolved proteins
which is decanted off. Further, purification is achieved by subsequent addition of
distilled water and centrifugation. Using this method of extraction, a yield of 1%
starch has been reported for starch extracted from the Gracilariales red algae species
(Yu et al. 2002). The process for extraction of floridean starch from red algae is
therefore similar to that used for the extraction of starch.
13.7 Environmental Impact
As a biopolymer, starch is a benign material. Within the organisms from which it is

sourced, starch serves as a source of energy and is accumulated in different parts of
the organism. The edible plants and algae can be consumed whole as dietary starch
sources with less input of energy or materials. In order to make use of starch in its
isolated form for more advanced applications such as additives in food and pharma-
ceuticals, further consumption other than the biomass is involved. In this section, the
environmental impact of some processes involved in the extraction of starch from
aquatic biomass is discussed. Table 13.3 gives a summary of the consumption in a
typical process for extraction of starch from microalgae (Gifuni et al. 2017). Some

Consumption Amount
for extraction of starch from
microalgae Microalgae biomass 2.5 g per gram starch
Water 30 ml per grama
Ethanol 30 mla
CO2 2% at 0.02 vvm
Percoll reagent 15 ml/ga
Energy Mechanical disruption
Heating 75 °C for ~1 h
Centrifugation—10,000× g for 40 min
a Estimation based on typical volume to mass ratios
13.7 Environmental Impact 301
of the environmental issues associated with the process of starch production from
aquatic resources are then discussed.
13.7.1 Waste Utilization
In the extraction of other biopolymers from algae, the starch is usually seen as
a waste. This starch could be further processed and purified for other applications
such as bioethanol production, where the starch content is relatively lower, and this is
combined with starch from other extraction process to augment the amount of starch
in the feedstock. The biochemistry of starch from aquatic plants and algae is similar to
that of terrestrial crops used in bioethanol production; therefore, combining multiple
sources of starch for bioethanol production should not pose difficulty in processing.
13.7.2 Cultivation of Aquatic Plants and Algae for Starch

Production
Cultivation of some aquatic life forms for the production of biopolymers interferes
with the balance in the food chain as some of these biomass serve as food for other
life within the aquatic system. For example, some fish feed on algae such as U.
ohnoi (Ingle et al. 2018). It is important to have a consideration for the replacement
cycle of these organisms for a truly sustainable marine biorefinery. Furthermore,
to optimize the bioaccumulation of starch, it is often required to vary the environ-
mental condition to stimulate optimal starch accumulation by the aquatic plant or
algae. This could limit the production of starch from aquatic sources to indoor cul-
tivation in controlled environment which could be at increased capital and running
costs. For example a typical indoor photobioreactor for cultivation of microalgae C.
sorokiniana for production of starch under nitrogen starvation using a photobioreac-
tor requires indoor temperature maintained at 25 °C, light intensity at 300 µE m−2
s−1 air flow of 0.02 vvm with 2% CO2 composition in an inorganic Bold basal algae
growth medium (Gifuni et al. 2017). If grown in mariculture, altering the natural
environmental conditions in the open marine or freshwater ecosystem could result
in blooms which have a detrimental effect on the entire aquatic ecosystem, and this
should be prohibited.
Aquatic plants and algae also have an advantage of, unlike terrestrial crop plants,
not requiring freshwater for cultivation. This is particularly important as freshwater
302 13 Starch
is increasingly scarce, and where available is required for consumption by humans

and farm animals. The water consumption given in Table 13.3 is the water consumed
during the extraction process. While algae do not require freshwater to grow in and
can fix nitrogen and other elements from the water for their growth, the extraction
process requires distilled water to extract purified starch.
13.7.4 CO2 Emission
One key advantage of starch source from aquatic plants and algae is that the emission
during extraction is balanced by the CO2 that the plant or algae removes from the
environment; it can be assumed to be a zero emission process. The process of cultivat-
ing microalgae is CO2 negative since CO2 is consumed in the process. For example,
in the cultivation of C. sorokiniana, the system is aerated with air containing 2% car-
bon dioxide at 0.02 vvm (Gifuni et al. 2017). Starch production from terrestrial plants
also involves the plan removing CO2 from the environment; however, aquatic plants
grow at a much faster rate than some of the terrestrial plants which commonly serve
as sources of starch (Table 13.2) some also have higher starch content (Table 13.1);
therefore, the cultivation of aquatic plants and algae for starch production results in
more CO2 removal per gram of starch produced.
For systems, where the cultivation is carried out in a bioreactor or in aquaculture
system which are within the factory where the starch is extracted, CO2 emission from
running vehicles on fossil fuel to transport the biomass to the factory for extraction
is minimized. This is particularly for starch which the bioaccumulation of starch is
optimized by using carefully controlled growth conditions and nitrogen starvation.
13.7.5 Water Remediation
Aquatic plants and algae are able to extract impurities, mainly compounds of nitrogen
and phosphorus from wastewater. Water recycling systems which implement the
cultivation of macrophytes for production of biochemicals (Muradov et al. 2014)
such as starch for bioethanol production are an efficient and low impact means of
providing freshwater for other applications such as terrestrial crop farming which
requires clean water. This provides a solution to water pollution and contributes to
ensuring food security through providing water for plant cultivation and also a source
of third-generation renewable energy.
13.8 Applications of Aquatic-Sourced Starch 303
13.8 Applications of Aquatic-Sourced Starch
Applications of starch include textiles, food, biomedical, pharmaceutical and energy

industries. Here, we take a look at some recent developments in the application of
starch, where aquatic-sourced starch could play a particular role.
13.8.1 Third-Generation Biofuel Production
In terms of biomass for bioethanol production, duckweed takes the lead of all the
aquatic plants studied so far. With a starch composition of up to 64% and the ability
to grow to twice its size with 5–6 days, this aquatic mosquito fern is an attractive
source of bioethanol production. For this reason, it has generated a lot of research
interest, and this includes how to best optimize its growth to selectively boost the
rate of production of desired metabolites and making use of its water remediation
properties in the treatment of industrial waste. Duckweed is also used in the recovery
of nutrients from water.
Algae can be used to produce either bioethanol or biodiesel as they contain lipids,
starch and other polysaccharides. Recent studies have experimented with carbon
switching in algae to direct the metabolism of the fixed carbon toward either pro-
duction of lipids or starch (Zhang et al. 2018). This is primarily achieved through
controlling the salinity of the water. This carbon switching occurs in both freshwa-
ter and marine algae. C. sorokiniana is one of the species which demonstrates this
property.
Starch from algae has gained increasing attention for its potential in serving as
the key to low-cost, low-impact and commercially profitable biofuel that is globally
available.
13.8.2 Bioplastic Production
One of the global problems facing the world today is pollution caused by the accu-
mulation of non-biodegradable plastic. Between 2015 and 2017, an estimated of
6.3 billion tonnes of plastics have been accumulated in the ocean globally and that
number continues to increase at an annual rate of 10–20 million tonnes (Urbanek
et al. 2018). In the great pacific between California and Hawaii, 79 thousand tonnes
of plastics are accumulated covering an area of 1.6 million km2 based on models
developed from the data collected from vessels and aircraft surveys (Lebreton et al.
2018). The approaches to addressing the plastic pollution problem include reduc-
ing the production and use of non-biodegradable plastics, reusing the plastics and
hence keeping them away from being accumulated in the environment as waste and
recycling the plastics into other long-lasting products and keeping them in use for
304 13 Starch
longer periods of time. In reducing the production of non-biodegradable plastics,

other replacements which serve the same purpose are increasing in demand. Starch
is one of the most widely used alternatives to non-biodegradable plastics. Starch
can be processed into plastic films which have the same mechanical properties as
non-biodegradable plastic films made up of synthetic, fossil-derived plastics such as
polyethylene. However, these starch-derived plastics degrade into compounds that
are absorbed into the environment and utilized by living organisms without causing
harm.
One of the limitations of bioplastic applications is their ability to retain their prop-
erties at the service conditions. Properties such as water absorbance and thermome-
chanical properties in bioplastics vary more with environmental conditions compared
to the non-biodegradable plastics. Plastics made from aquatic-derived starch could
potentially have higher temperature tolerance.
13.8.3 Waxy Starch
Starches with high amylopectin content and little amylose content are desirable in
the formation of waxy starch. Waxy starch is used as a food thickener, bread mak-
ing, emulsion stabilizers, coating and film forming. It is preferred for its improved
digestibility due to its branched structure. Branched polymers tend to be less sta-
ble than linear polymers, thus making waxy starch more prone to break down by
digestive enzymes. The branched nature of the waxy starches also makes then suit-
able for nanoparticles formation. These nanoparticles can then be used in a variety
of applications such as drug delivery and scaffold formation (Sarka and Dvoracek
2017). Waxy starch has preferred gelatinization properties compared with starch with
higher amylose content. It is also more resistant to retrogation and hence acts as a
more stable viscosity enhancer (Sarka and Dvoracek 2017).
Floridean starch which is found in the cytoplasm of red algae is similar in structure
to these waxy starches (Dauvillee et al. 2009) since they contain only amylopectin
with no amylose content (McCracken and Cain 1980). Although waxy starch is most
commonly sourced from maize, maize is also largely used in other applications; the
unique aquatic floridean starch could potentially replace or augment the use of maize
for production of waxy starch.
13.8.4 Food
Starch is the major staple food across the world. About 35% of the daily calo-
rie intake comes from starch (Gifuni et al. 2017). Starch is also widely used as a
thickening agent in food products. Isolated starch is also on its own a meal such as
cornmeal or tapioca. The gelatinization property in hot water makes it a good vis-
cosity enhancer. Combination of different types of starch results in a hybrid material
13.8 Applications of Aquatic-Sourced Starch 305
with new properties which leads to improved quality or a new variety of products.
Therefore, aquatic starch combined with terrestrial-sourced starch has potential for
novel formulations for food applications. With an ever-increasing global population
and decreasing arable land for food cultivation, augmenting the already existing,
mostly land-based starch sources with aquatic-derived starch would be a significant
contribution to global food security. Some studies have explored the production of
edible starch from algae. When not acting as a direct food for human consumption,
aquatic-sourced starch could also be used as a source of animal feed (Prabhu et al.
2019; Smith et al. 2010) as texture enhancers or stabilizers for animal food. As dis-
cussed in other chapters in this book, some aquatic-sourced biopolymers which do
not meet the organoleptic or purity requirements to be used for direct human con-
sumption are approved for the use for use in pet food. The feed for pets which are
not consumed as food is different for those of animal feed which will eventually be
consumed by humans.
13.8.5 Textile and Paper Industry
In the textile industry, starch is applied as a thickener in aiding the textile printing
process, and it is also used in fabric treatment to improve finish (Teli et al. 2009).
These applications are attributed to the coating property of starch and its ability to
adhere to dyes. Application of starch from aquatic resource is particularly attractive
toward developing a textile industry which integrates an aquatic-based water reme-
diation system, whereby the aquatic source of starch-based raw material could also
be used in the wastewater treatment. This allows the source of the raw material to
also be used to clean the effluent from the industrial process.
Furthermore, the use of non-edible aquatic-sourced starch would make more edi-
ble starch from other sources such as corn, cassava and potato more available for
food-based applications. This is of particular significance as the world faces the risk
of global food shortage, and it becomes more important to limit the use of food crops
for non-food applications.
Similarly, the papermaking is a non-food application of starch. Papermaking
makes use of starch as a coating binder, to aid ink retention and in finishing (Mau-
rer 2009). In such application, starch is most commonly used in the modified form.
Starch for papermaking can be sourced from extraction from high yielding aquatic
plants such as duckweed and as a by-product from the extraction of other aquatic
biopolymers such as carrageenan.
13.8.6 Pharmaceutics
In the pharmaceutical industry, starch is used as a thickening and bulking agent

in syrups, tablets and capsules. It protects the active ingredient from the first-pass
306 13 Starch
metabolism in the digestive system when administered orally such that the active
ingredient gets to the walls of the small intestine in its intact form. Usually in com-
bination with other polymers and compounds, for example, floating gel tablets made
from starch and cellulose blend in the weight ratio 3:7 used in gastric drug deliv-
ery. Starch is also used in wound dressings and tissue engineering (Liu et al. 2017).
Although starch from aquatic plants and algae is yet to be explored in pharmaceutical
applications, they have the same structure as the starch from terrestrial plants used in
these applications, the aquatic-sourced starch can potentially be used for such. This is
highly dependent on the aquatic-sourced starch meeting the same safety requirement.
13.9 Commercial Production and Applications
Present rate of biofuel production from algae cannot replace fossil fuels in terms of
cost of production. Microalgae and seaweeds are a potential source of commercial-
scale production of starch. The most economic way of commercial production of
algal starch is the use of wastewater which contains the nutrients required for algae
growth and are already the cause of uncontrolled algal pollution anyway. Current
challenges in this area are the fluctuation of wastewater which depends on the source.
As outlined in Fig. 13.3 starch production from algae can be integrated into biodiesel
and bioethanol production by using the lipids and starch from the algae. This could
be further expanded to utilize the other biopolymers, obtainable from algae. These
biopolymers are discussed in other chapters of this book.
The bioplastic market also forms a great opportunity in the production of starch
from aquatic biomass, particularly those that are not primarily consumed as food. The
annual production rate of plastics is estimated to be >320 million tonnes (Lebreton
et al. 2018). With the increasing concern and evidence of the adverse impact of the use
of the non-biodegradable plastics on the environment, this means there is an already
existing market for biodegradable plastics which can offer a better alternative to the
conventional plastics.
Presently, the commercial exploration of the production of starch from aquatic
biomass is largely limited by the fact that there still exist cheaper alternatives in
Transesterification Biodiesel
Dirty Water in Lipids
Algae
culture Photobioreactor Cell
disruption
Bioethanol
Hydrolysis Fermentation
Clean Water out Starch
Fig. 13.3 Starch and lipids extraction to obtain biofuels from algae
13.9 Commercial Production and Applications 307
terms of economic feasibility (Chia et al. 2018). However, as fossil fuels continue to
deplete further against unrelenting increasing global consumption closely associated
with population growth, alternative sources of versatile resources such as starch
become more important; therefore, all alternative sources become important.
Commercial production of starch from algae would benefit much from the tech-
nique of nutrient starvation which results in optimal yield. This would mean less
expense on nutrients unlike in terrestrial crop farming where fertilizers are con-
stantly required for plant growth. Nutrient is required to reach the desired growth
phase, and this is then followed by nutrient starvation which yields an increase in
starch production. This is accompanied by reduced biomass production and as much
of the nutrient available in this stressful period is then directed toward energy produc-
tion for basic survival. This increased production of starch by plant during nutrient
starvation accompanied by reduced biomass production could further make the starch
extraction process more effective as more starch is present per unit mass.
Starch accumulation rate in green algae growing in the wild varies with season.
An example of such variation has been detected in green algae U. ohnoi (Prabhu et al.
2019) where winter conditions seem to favor optimal starch production in Tel Aviv,
Israel. This seasonal variation in starch content implies fluctuation in the quality of
raw materials sourced from wild at different periods; therefore, commercial starch
production from aquatic source requires cultivation in controlled photobioreactors
or aquaculture or storage facilities for those sourced from the wild to ensure constant
availability throughout the year.
13.10 Conclusion
A variety of sources of starch exist in the aquatic environment. The growth rate and
starch accumulation rate of these organisms can be further optimized by adequate
control of their growth system in controlled cultivation. They can also be sourced
from natural stocks as part of efforts to utilize natural resources which otherwise
cause environmental nuisance. To date, starch remains one of the algae resources
which is yet to be industrially explored. Although aquatic plants and algae have been
explored for other biopolymers such as alginate and proteins, when considering
a zero-waste marine biorefinery, it is important to have a refinery process which
integrates the extraction of starch alongside the other extractives from the aquatic
biomass since it makes up a significant weight fraction. The low lignin content
of aquatic sources of starch makes them a promising source of fermentable starch
for bioethanol production. Therefore, despite not having as diverse applications as
other polymers reviewed within this book, its potential application in a low-cost,
high-volume and highly valued product such as biofuel gives it high commercial
significance.
308 13 Starch
References
Andrade LR, Farina M, Amado GM (2004) Effects of copper on Enteromorpha flexuosa

(Chlorophyta) in vitro. Ecotoxicol Environ Saf 58:117–125
Appenroth KJ, Borisjuk N, Lam E (2013) Telling duckweed apart: genotyping technologies for the
Lemnaceae. Chin J Appl Environ Biol 19:1–10
Beloshapka A, Buff P, Fahey G, Swanson K (2016) Compositional analysis of whole grains, pro-
cessed grains, grain co-products, and other carbohydrate sources with applicability to pet animal
nutrition. Foods 5:23–32
Bhat FM, Riar CS (2016) Effect of amylose, particle size & morphology on the functionality of
starches of traditional rice cultivars. Int J Biol Macromol 92:637–664
Chia SR, Ong HC, Chew KW, Show PL, Phang SM, Ling TC, Nagarajan D, Lee DJ, Chang
JS (2018) Sustainable approaches for algae utilisation in bioenergy production. Renew Energy
129(Part B):838–852
Dauvillee D, Deschamps P, Ral J, Plancke C, Puteaux J, Devassine J, Durand-Terrasson A, Devin
A, Ball SG (2009) Genetic dissection of floridean starch synthesis in the cytosol of the model
dinoflagellate Crypthecodinium cohnii. PNAS 106(50):21126–21130
Edison S, Srinivas T (2016) Status of cassava in India an overall view. Crops 46:7–172
Eichelmann E, Wagner-Riddle C, Warland J, Deen B, Voroney P (2016) Comparison of carbon
budget, evapotranspiration and albedo effect between the biofuel crops switchgrass and corn.
Agr Ecosyst Environ 231:271–282
Fournet I, Zinoun M, Deslandes E, Diouris M, Yves Floch J (2000) Floridean starch and carrageenan
contents as responses of the red alga Solieria chordalis to culture conditions. Eur J Phycol
34:125–130
Fujita M, Mori K, Kodera T (1999) Nutrient removal and starch production through cultivation of
Wolffia arrhiza. J Biosci Bioeng 87(2):194–198
Gifuni I, Oliveri G, Krauss IR, D’Errico G, Pollio A, Marzocchella A (2017) Microalgae as new
sources of starch: isolation and characterization of microalgal starch granules. Chem Eng Trans
57:1423–1428
Himaa HB, Dammak M, Karkouch N, Hentati F, Laroche C, Michaud P, Fendri I, Abdelkafi S (2019)
Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium
marinum. Int J Biol Macromol. https://doi.org/10.1016/j.ijbiomac.2019.01.207 (in press)
Ingle KNKN, Polikovsky M, Chemodanov A, Goldberg A (2018) Marine integrated pest
management (MIPM) approach for sustainable agriculture. Algal Res 29:223–232
Korzen L, Abelson A, Israel A (2016) Growth, protein and carbohydrate contents in Ulva rigida and
Gracilaria bursa-pastoris integrated with an offshore fish farm. J Appl Phycol 28:1835–1845
Lebreton L, Slat B, Ferrari F, Sainte-Rose B, Aitken J, Marthouse R, Hajbane S, Cunsolo S, Schwarz
A, Levivier A, Noble K, Debeljak P, Maral H, Schoeneich-Argent R, Brambini R, Reisser J (2018)
Evidence that the great pacific garbage patch is rapidly accumulating plastic. Nat Sci Rep 8:4666
Le Corre BAD, Bras J (2010) Starch nanoparticles: a review. Biomacromolecules 11:1139–1153
Li M, Witt T, Xie F, Warren FJ, Halley PJ, Gilbert RG (2015) Biodegradation of starch films: the
roles of molecular and crystalline structure. Carbohyd Polym
Li Y, Zhang F, Daroch M, Tang J (2016) Positive effects of duckweed polycultures on starch and
protein accumulation. Biosci Rep 36(00380):1–8
Liu W, Halley PJ, Gilbert RG (2010) Mechanism of degradation of starch, a highly branched
polymer, during extrusion. Macromolecules 43(6):2855–2864
Liu G, Gu Z, Hong Y, Cheng L, Li C (2017) Electrospun starch nanofibers: recent advances,
challenges and strategies for potential pharmaceutical application. J Controlled Release 252:95–
107
Liu Y, Wang X, Fang Y, Huang M, Chen X, Zhang Y, Zhao H (2018) The effects of photoperiod
and nutrition on duckweed (Landoltia punctata) growth and starch accumulation. Ind Crops Prod
115:243–249
References 309
Lopez-Llorca LV, Valiente MFC (1993) Study of biodegradation of starch plastic films in soil using
scanning electron microscopy. Micron 457–463
Maurer HW (2009) Starch in the paper industry. In: Starch, 3rd edn. Food science and technology,
pp 657–713
McCracken DA, Cain JR (1980) Amylose in floridean starch. New Phytol 88:67–71
McWilliams A (2017) Biodegradable polymers. BCC Research, Wellesley, MA, USA
Miranda AF, Biswas B, Ramkumar N, Singh R, Kumar J, James A, Lal B, Subudhi S, Bhaskar
T, Mouradov A (2016) Aquatic plant Azolla as the universal feedstock for biofuel production.
Biotechnol Biofuels 9(221):1–17
Muradov N, Taha M, Miranda AF, Kadali K, Gujar A, Rochfort S, Stevenson T, Ball AS, Mouradov
A (2014) Dual application of duckweed and Azolla plants for wastewater treatment and renewable
fuels and petrochemicals production. Biotechnol Biofuels 7(30):1–17
Muthuvelan B, Noro T, Nakamura K (2002) Effect of light quality on the cell integrity in marine
alga Ulva pertusa (Chlorophyceae). Indian J Mar Sci 31:21–25
Negm NA, Zahran MK, Elshafy MRA, Aiad AI (2018) Transformation of Jatropha oil to biofuel
using transition metal salts as heterogeneous catalysts. J Mol Liq 256:16–21
Penfound WT, Earle TT (1948) The biology of the water hyacinth. Ecol Monogr 18(4):447–472
biorefinery. Algal Res 37:215–227
Rahman M, Roy TS, Chowdhury IF (2016) Biochemical composition of different potato varieties
for processing industry in Bangladesh. Agric Sci Pract 2:81–89
Reddy DK, Bhotmange MG (2014) Viscosity of starch: a comparative study of Indian rice (Oryza
sativa L.) varieties. Int Rev Appl Eng Res 4:397–402
Rehman ZU, Anal AK (2018) Enhanced lipid and starch productivity of microalga (Chlorococcum
sp. TISTR 8583) with nitrogen limitation following effective pretreatments for biofuel production.
Biotechnol Rep 20. https://doi.org/10.1016/j.btre.2018.e00298
Robyt J (2008) Starch: structure, properties, chemistry and enzymology. In: Fraser-Reid BO, Tatsuta
K, Thiem J (eds) Glycoscience. Springer, Berlin, Heidelberg
Sarka E, Dvoracek V (2017) Waxy starch as a perspective raw material (a review). Food
Schellart JA, Visser FMW, Zandstra T, Middelhoven WJ (1976) Starch degradation by the mould
Trichoderma viride I. The mechanism of starch degradation. Antonie van Leeuwenhoek 42:229
Sjöö LM, Nilson L (2018) Starch in food: structure, function and applications, 2nd edn. Elsevier, p
58
Smith JL, Summers G, Wong R (2010) Nutrient and heavy metal content of edible seaweeds in
New Zealand. N Z J Crop Hortic Sci 38:19–28
Sullivan-Trainor M (2013) Starches/glucose, global markets. BCC Research, Wellesley, MA, USA
Teli MD, Rohera P, Sheikh J, Singhal R (2009) Application of germinated maize starch in textile
printing. Carbohyd Polym 75(4):599–603
Urbanek AK, Rymowicz W, Mironczuk AM (2018) Degradation of plastic-degrading bacteria in
cold marine habitats. Appl Microbiol Biotechnol 102:7669–7678
Wang Y, Pan S, Jiang Z, Liu S, Feng Y, Gu Z, Li C, Li Z (2019) A novel maltooligosaccharide
forming amylase from Bacillus stearothermophilus. Food Biosci 30:100415
Xu J, Zhao H, Stomp AM, Cheng JJ (2012) The production of duckweed as a source of biofuels.
Biofuels 3:589–601
Xu J, Stomp A, Cheng J (2014) The production of duckweed as a source of biofuels. Biofuels
3(5):589–601
Xu Y, Fang Y, Li Q, Yang G, Guo L, Chen G, Tan L, He K, Jin Y, Zhao H (2018) Turion, an
innovative duckweed-based starch production system for economical biofuel manufacture. Ind
Crops Prod 124:108–114
Yao C, Ai J, Cao X, Xue S, Zhang W (2012) Enhancing starch production of a marine green microalga
Tetraselmis subcordiformis through nutrient limitation. Bioresour Technol 118:438–444
310 13 Starch
Yew GY, Lee SY, Show PL, Tao Y, Law LC, Nguyen TTC, Chang J (2019) Recent advances in algae
biodiesel production: from upstream cultivation to downstream processing. Bioresour Technol
Rep 7:10027
Yu S, Blennow A, Bojko M, Madsen F, Olsen CE, Engelsen SB (2002) Physico-chemical
characterization of floridean starch of red algae. Starch/Staerke 54:66–74
Zhang W, Zhao Y, Cui B, Wang H, Liu T (2016) Evaluation of filamentous green algae as feedstocks
for biofuel production. Bioresour Technol 220:407–413
Zhang L, Pei H, Chen S, Jiang L, Hou Q, Yang Z, Yu Z (2018) Salinity-induced cellular cross-talk in
carbon partitioning reveals starch-to-lipid biosynthesis switching in low-starch freshwater algae.
Bioresour Technol 250:449–456
Ziegler P, Adelmann K, Zimmer S, Appenroth KJ (2015) Relative in vitro growth rates of duckweeds
(Lemnaceae)—the most rapidly growing higher plants. Plant Biol 17:33–41
Chapter 14
Cellulose
Abstract The most abundant polymer on earth is also present in the aquatic environ-
ment, produced by aquatic plants, algae and cellulose-producing bacteria. Cellulose
can be modified into various forms such as cellulose nanofibers and methyl cellulose,
and it finds diverse applications in various industries which include textiles, paper and
energy. The feasible commercial production of bioethanol from cellulosic biomass
is limited due to the stable structure of cellulose which makes it less susceptible to
hydrolysis. However, cellulose from aquatic biomass may serve other industries such
as textiles where the unique chemistry of aquatic-derived cellulose has some desir-
able properties. Extraction of cellulose from biomass reduces the methane released
into the environment from the degradation of dead aquatic plant and algae biomass.
Keywords Cellulose · Polysaccharide · Polymers · Hydrolysis · Cellulase
14.1 Introduction
Cellulose occurs in the aquatic ecosystem in aquatic plants, algae and cellulosic
bacteria. Aquatic plants which serve as sources of cellulose include water hyacinth,
duckweed, Azolla and many other aquatic plants whose cell wall comprises of cel-
lulose, a prominent feature in plants. Algae sources include red, brown and green
macroalgae, where cellulose also plays a structural role in the cell walls. While bac-
teria such as acetobacter are also sources of high-quality cellulose, this chapter is
focused on cellulose from aquatic sources. Therefore, much of the discussion shall
be on plant and algae sourced cellulose.
Cellulose is a biopolymer of huge industrial significance. Cellulose is the most
abundant polymer on earth, and this extends to cellulose sourced from the aquatic
ecosystem. Exploring the availability of cellulose in the aquatic environment would
further expand the ubiquity of cellulose. More importantly, the aquatic environment
serving as an additional or alternative source of cellulose for certain industrial appli-
cation could pose some significant advantage to the environment and commerce by
reducing the strain on land-based cellulose sources and potentially offering lower-
cost processing. Cellulose in aquatic organism is generally free from lignin and
hemicellulose, which are either completely absent or present in much lower amounts

312 14 Cellulose
in aquatic organisms compared to terrestrial sources such as wood. This alters the
extraction process in ways we shall explore within this chapter.
As the world population continues to increase annually, more land space is
required for housing, animal grazing and growing of plants. It becomes important to
understand the role the aquatic environment plays in replacing or augmenting the cel-
lulose from terrestrial plants. Cultivation and harvesting of aquatic-based cellulosic
biomass take a different process compared to that of terrestrial plants. The fact that
the land requirement is omitted means the production of aquatic-based cellulose does
not interfere with the occupation of land by humans for habitation, animal grazing
and crop production.
This chapter therefore explores the economic impact in terms of availability of
feedstock and applications in different industries. The environment impact is also
assessed through reviewing the extraction processes and occurrence in nature. Addi-
tionally, the chapter also explores the chemistry of cellulose and peculiar characteris-
tics of cellulose from aquatic sources. This is aimed to guide and inform exploration
of cellulose production from aquatic sources and encourage the conservation of such
resources as well as further research in the area.
Cellulose is the most abundant polymer on earth. It is present in all plants and algae
as a structural polymer and in bacteria as protection against UV radiation and harsh
chemicals. It is indigestible by humans and however can be digested by ruminant
animals and some bacteria. Within the aquatic environment, aquatic plants and algae
serve as the sources of cellulose. The rate of generation if cellulosic biomass by
aquatic plants and algae is much faster than that it terrestrial plants. Non-competition
with space used for cultivation of edible crops and for human habitation and the
bioremediation properties of some aquatic plants and algae make aquatic plants and
algae serve as particularly appealing option for cellulose-based products.
Aquatic plants have the advantage over algae as a source of cellulose being free-
floating plants. This makes their harvesting and cultivation much simpler and less
expensive. Aquatic plants which have drawn much interest over the years are Azolla,
duckweed and water hyacinth. Each one is of interest for different reasons, due to
either the resource potential or the adverse impact on human activity and the envi-
ronment. The occurrence of these plants in nature is also affected by human activi-
ties such as introducing pollutants into the water. The environmental and economic
impact of these plants can therefore be controlled by understanding their resource
potential and growth parameters.
The Azolla plant makes an interesting source of cellulose because of the plant’s
remarkable ability to grow in wastewater. Azolla metabolizes contents of wastewater
such as nitrogen and phosphorus and produces metabolites for its growth and survival.
Metabolites include cellulose, hemicellulose, starch and lipids. Azolla grows well
in rural lagoons, rivers, ponds, irrigation channels, ditches and wetlands under a
wide range of temperature conditions. It required little or no maintenance or care,

is endemic to most parts of the world and therefore occurs in abundance in nature.
Azolla can contain up to 35% cellulose by weight (Miranda et al. 2016), and it has
a symbiotic relationship with nitrogen-fixing bacteria known as Anabaena azollae.
This symbiotic relationship with A. azollae makes it possible for Azolla to grow in
water where nitrogen is not present (Kollah et al. 2016). Two most common Azolla
species are A. filiculoides and A. pinnata, and 7 species of Azolla have been identified.
Azolla has a rather unique composition of cellulose, hemicellulose starch and lipids
which is being explored as a universal biofuel feedstock (Miranda et al. 2016). This
implies that pyrolysis and hydrothermal liquefaction of Azolla biomass could result
in good yield of hydrocarbons, and the transesterification of the lipids would yield
biodiesel while the enzymatic hydrolysis of starch and cellulose would yield sugars
for bioethanol production (Miranda et al. 2016).
Duckweed is relatively well-explored aquatic plants, and they have been used
for over two decades for the remediation of industrial and municipal wastewater in
countries such as the USA, Bangladesh and Israel and in biofuel production. They
grow in slow flowing or stagnant water as floating plant. They grow best in warm
waters where they can grow all year round in some areas. Duckweeds can double in
size within 2–7 days and in as little as 20 h at optimal growth conditions. Duckweeds
have the unique property of being able to accumulate high levels of microelements
and heavy metals from wastewater (Basile et al. 2012). Although starch makes up a
higher percentage by mass in duckweed, they do contain a considerable amount of
cellulose and are therefore worth considering as a source of cellulose. Total carbo-
hydrate content of duckweed of 51.2% has been reported from composition analysis
(Zhao et al. 2014) an estimated 35% of this being cellulose. Table 14.1 lists the
chemical composition of duckweed.
Another abundant floating aquatic plant is the water hyacinth (Eichhornia cras-
sipes). Water hyacinth contains 25% cellulose, 33% hemicellulose and 10% lignin
(Thiripura and Ramesh 2012). It grows rapidly in lakes, basins and rivers in tropical
and subtropical regions throughout the world and has been identified as far back as
the 1940s (Penfound and Earle 1948). Water hyacinth has been categorized as the
worst-growing weed in the world, due to its ability to cover vast areas of water in
a relatively short period of time causing damaging effects to aquatic life and water
Table 14.1 Duckweed

Duckweed component % w/w
composition (Zhao et al.
2014) Pectin 20.3
Starch 19.9
Total carbohydrates 51.2
Hemicellulose 3.5
Phenolics 0.03%
Essential fatty acids 0.6% alpha-linolenic and
linoleic/linoelaidic acid
0.015% p-coumaric acid
314 14 Cellulose
Table 14.2 Cellulose content

Aquatic resource Cellulose (%) References
is some aquatic plants and
algae Water hyacinth 25 Thiripura and
Ramesh (2012)
Duckweed 55.2 Yadav et al. (2017)
Azolla 21.8–12.8 Miranda et al. (2016)
Green algae 20–30 Mihranyan (2010)
Red algae (Gelidium 17.2 Chen et al. (2016)
elegans)
Brown algae 11 Siddhanta et al.
(Sargassum (2011)
tenerrimum)
transportation. Water hyacinth has a higher cellulose content than Azolla. It therefore
is an important aquatic source of cellulose.
Macroalgae cell walls consist of layers of cellulose in relatively large amounts
(Munoz et al. 2014). Green algae Cladophora is a seasonal light-sensitive aquatic
filamentous macroalgae that are submergent aquatic organisms. They bloom in a
variety of temperatures on rough rock surfaces. Brown algae are widely used as
a source of alginate, and the residue from the alginate extraction can be further
treated to isolate cellulose. Table 14.2 gives yield of cellulose from different aquatic
organisms.
14.3 Chemistry of Aquatic Cellulose
The chemistry of cellulose is well established. It is a linear polymer made up of

glucose monomers in 1–4 glycosidic bonds. Cellulose is identified by a color change
when in contact with iodine and sulfuric acid. This is characteristic of cellulose as
other polymers will not give the same response, for example, alginate from marine
algae tends to be more crystalline, forming thick microfibrils when in contact with
the same (Koyama et al. 1997).
Although having the same unit structure, glucose ring, the secondary structures of
the cellulose from algae are different from that of higher plants. The cellulose in algae
is more tightly packed than in terrestrial plants. A density of 1.64 g/cm3 for cellulose
is obtained from green algae Cladophora while that of terrestrial plant is around
1.56 g/cm3 . Furthermore, cellulose from algae distinguishes itself from terrestrial
and aquatic plants by its lower level of hornification. This is thought to be associated
with its well-ordered structure. This property allows formation of well-dispersed
microfibrillated cellulose without agglomeration. Cellulose from green algae can go
through repeated cycles of hydration and dehydration with its structure intact.
Crystallinity of cellulose varies in different algae depending on what group the
algae fall into. This grouping is based on the nature of cellulose that makes up
14.3 Chemistry of Aquatic Cellulose 315
the algae cell wall. Group 1 comprises of algae which have mainly native cellulose
within their cell walls. Algae belonging to this group are the Cladophorales and some
Siphonocladales. Most algae fall into Group 2 in terms of their cell wall structure.
Group 2 comprises of algae whose cell walls are made up of mercerized cellulose
derived from native cellulose. This form of cellulose has low crystallinity. Example
of such is the Spongomorpha. The third group in this classification is Group 3 algae.
These are characterized by heterogeneous cell wall structure in which cellulose is
not the major component of the cell walls. Spirogyra and Vaucheria fall into this
group. The variation in crystallinity in cell walls is attributed to the cellulose syn-
thase complex responsible for determining the structure of the cellulose microfibrils
(Brown and Saxena 2000). Selectivity of species for cellulose extraction is therefore
necessary in order to obtain cellulose of desired crystallinity.
Hornification occurs to a much lower degree in crystalline cellulose (Fernandez
Diniz et al. 2004) due to the high level of orderliness in the microfibrils which hinder
water absorption. Such property aids formation of uniform dispersion in micro- and
nanoformulations of cellulose. This further adds to the appeal of cellulose from
green algae. Cellulose from Cladophora has a surface area of 95 m2 /g, a relatively
high surface area compared to that of industrial adsorbents which could be around
100 m2 /g. This surface area is about 100 times higher than that of pharmaceutical
grade microcrystalline cellulose which is around 1 m2 /g. This high surface area
coupled with their very low level of hornification makes green algae sourced cellulose
a potentially better candidate for production of cellulose-based aerogels that are more
resistant to humidity than those produced from native cellulose.
Cellulose has particularly excellent mechanical properties due to its linear orien-
tation, microfibrillar structure and high degree of polymerizations. Linear polymers
are able to form well-ordered structures resulting in densely packed secondary struc-
ture which prevents penetration of heat, moisture or other molecules. The superior
mechanical properties of starch fit well with the structural role it plays in the plant
cell wall. Cellulose provides rigidity and strength to the plant. The strong hydrogen
bonds present within the cellulose structure also contribute to the strong mechanical
structure of cellulose. This property also makes the hydrolysis of cellulose much
more difficult than that of starch.
Such high mechanical strength of cellulose does not seem so pertinent in algae
compared to land plants which need to grow higher above the ground and require
strong cell walls for strength. Hypothesis developed to explain the need for such
crystalline cellulose in the Cladophora algae is to retain turgor pressure where salinity
fluctuates, as is common in aquatic environment and/or withstand the drag flow
of water (Johnson and Shivkumar 2004). The cellulose from aquatic source can
therefore be said to have similar chemical structure as terrestrial cellulose but for
differences in secondary structure in some cases such as in Cladophora.
316 14 Cellulose
14.3.1 Decomposition of Cellulose
Cellulose has a very simple linear polymer structure made up of glucose repeating
units. The absence of branching gives cellulose a very stable crystalline structure
making it resistant to degradation. This makes it an excellent structural component
of the cell wall. While in plants cellulose plays mainly structural role and not utilized
for energy or cell metabolism, bacteria are able to decompose cellulose and use
as a carbon source. Humans do not produce any known enzyme for digestion of
cellulose. However, cellulose is important as a fiber in the human diet. Degradation of
cellulose by cellulose-degrading organisms involves a cocktail of enzymes working
in synergy. These enzymes are referred to as cellulases (Beguin and Aubert 1994).
Industrial production of enzymes for degradation of cellulose involves isolation of
these enzymes from cellulose degrading organisms. Ruminant animals are able to
digest cellulose at relatively faster rate due to symbiotic relationship with bacteria
present in the rumen of these animals. The rate is further assisted by the process
of regurgitation, a process whereby the food is returned to the mouth and further
particle size reduction is achieved by chewing before being returned to the rumen
for digestion (Russel et al. 2009). Cellulose can also be degraded by thermochemical
process through the use of high temperature and pressure in the absence of oxygen.
This is used in the production of biofuel from cellulose in the process of pyrolysis.
The chemical structure of cellulose from both aquatic and terrestrial sources is
identical. Therefore, the degradation process is expected to follow the same mecha-
nism. The difference in the degradation of cellulose within aquatic biomass lies in the
difference in the other components of the cell walls. In the isolated form, degradation
depends on the level of modification and the form in which it exists in the product.
The growth rate of most aquatic plants is much faster than most non-edible and
edible crops being used or considered for biofuel production. They can therefore
generate biomass at a faster rate than the terrestrial crops. One of the limitations
facing commercial utilization of cellulosic biomass for biofuel production is the
availability of raw materials. With the faster biomass accumulation rate of aquatic
plants and algae, availability of cellulosic biomass can be significantly increased.
Cultivated Azolla plant in aquaculture can grow at a rate of 2.9–5.8 g/m2 in dry
weight per day, and in the wild, it can grow at a rate of 25.6–27.4 g dry weight per m2
per day (93.4–100 t/ha-year dry weight). Azolla is one of the fastest-growing aquatic
plants with the ability to double its weight within 5–6 days. On average, cellulose
content in algae is between 20 and 30% dry weight (Mihranyan 2010), and however,
there have been reports of cellulose content in filamentous algae as high as 45%.
Duckweeds can grow at a rate of between 39.2 and 44 tonnes per hectare annually.
Duckweed contains up to 45.7% starch and for over two decades has been explored
for application in nutrient recovery from wastewater which can then be used as animal
feed. It has also been largely explored for production of biofuel through fermentation
of its non-edible starch (Miranda et al. 2016). Another cellulose contains aquatic
plants. Water hyacinth can cover a whole hectare of water space within 6 months
growing to an estimated 125 tonnes wet weight (Istirokhatun et al. 2015).
Algae blooms have been reported in different parts of the world such as in the
southern coast of California (Smith et al. 2018). This has been accompanied by
a loss of devastating amount of fish and negative impact of the aesthetics of the
location. Algae biomass for cellulose extraction is therefore beyond abundant. It is
accumulated to devastating degree. Conversion of such waste into useful polymer
products such as cellulose is beyond its economic returns but also the gains to the
environment and all life concerned.
An estimated 100–150 billion tonnes of cellulose is produced annually by cellu-
lose synthesizing plants, bacteria and algae (Hon 1994). Most plants contain cellu-
lose, lignin and starch, and however, they contain these in varying compositions. The
composition of each plant will determine if the extraction of cellulose is economical,
or it might be more economical to extract other carbohydrates from them. Azolla, for
example, can accumulate 34 tonnes of cellulose in dry weight of biomass per hectare
of land annually. Although not polymers, lipids are also potential by-products from
cellulose extraction from aquatic plants. Azolla, for example, can be a source of up
to 8 tonnes of lipids per hectares annually. This is higher than other sources of lipids
of terrestrial plants such as oil palm, soy and rapeseed. Azolla has been described as
a universal biofuel crop as its chemical composition mimics that of a combination
of terrestrial and macroalgae. However, as a source of cellulose, it has moderate
cellulose composition.
Cellulose can therefore be said to be relatively abundant in the aquatic environ-
ment since it is present in fast-growing plants and aquatic algae whose biomass accu-
mulation rate is faster than those of terrestrial plants. Actual availability of aquatic
biomass for cellulose production depends on several other factors such as demand-
driven expansion of cultivation of these sources and the cost of processing compared
to the typical sources of cellulose. Future availability of abundant aquatic plants and
algae could reduce as other applications are being discovered. For example, farmers
in tropical regions had adopted water hyacinth for use as compost fertilizer (Pol-
prasert et al. 1994). This value addition to such aquatic plant which was previously
seen as a nuisance could therefore lead to an increase in the price of the aquatic plant
as a feedstock for cellulose production.
14.5 Extraction of Aquatic Cellulose
The chemical resilience of cellulose compared to other components of the cell wall
of plants and algae serves as a basic for its extraction. The chemical-based process
for extraction involves dissolving off the other components of the cell wall in strong
acids and bases under high temperature followed by the recovery of cellulose from the
318 14 Cellulose
residual material. It is important to maintain the structural integrity of the cellulose

during the process of extraction as this eventually affects the quality and applicability
of the cellulose obtained. Properties such as crystallinity, degree of polymerization,
microfibril structure, wettability and mechanical properties are used to measure the
quality of cellulose extracted. Here, we consider extraction methods that have been
reported for the main cellulose sources in the aquatic ecosystem.
14.5.1 Extraction of Cellulose from Aquatic Plants
High moisture content of water hyacinth means the process of drying may require
more time and energy input compared to other plants. Water hyacinth has a moisture
content of around 93–96%. Following the drying, the water hyacinth fibers are heated
at 115 °C in toluene solvent for a duration of 3 h. The next step is the removal of color
by bleaching in sodium hypochlorite NaClO (3%) under heating for 2 h at 80 °C.
The hemicellulose within the cell wall is removed using the process of hydrolysis
with sodium hydroxide (1%) at a temperature of 60 °C for 2 h. Further treatment is
carried out with sodium hypochlorite at 1% at 75 °C while stirring for a duration of
3 h, to remove the residual lignin (Istirokhatun et al. 2015).
To extract cellulose from Azolla plant, in the method by Miranda et al. (2016), the
Azolla plants harvested were washed and dried in hot air oven at 70 °C overnight. This
was followed by grinding to reduce particle size. Lignin was removed by autoclaving
in sodium acetate at 121 °C for 20 min. The remaining mass contains a mixture
of polysaccharide starch and cellulose. The starch can be removed by chemical
treatment with sodium hydroxide and heat or enzymatic treatment with amylase and
amyloglucosidase which reduces the starch to glucose. Where the goal is to obtain
cellulose, the glucose can then be washed off as it is water soluble leaving behind
cellulose fibers. This process is summarized in Fig. 14.1.
14.5.2 Extraction of Cellulose from Algae
One key advantage of cellulose extraction from algae over aquatic plants such as
Azolla is the very little to no lignin content in algae. This eliminates the high energy
and chemically demanding delignification process. Although cellulose is not a main
structural polymer in brown algae, some have been reported to have no cellulose
content, for example, brown algae is made up of 32% alginate, 15% laminarin which
is the glucose polymer in brown algae, 18% mannitol and the rest ash or salts This
composition was based on average values from three harvests of S. latissima, a strain
of brown algae commonly used in the industry (Konda et al. 2015). Red algae on
the other hand contain cellulose, glucans and galactan (Wi et al. 2009; Yoon et al.
2010). Nanocellulose extraction from red algae (Gelidium elegans) involved alkaline
treatment, bleaching and acid hydrolysis. The nanocellulose extracted had diameter
14.5 Extraction of Aquatic Cellulose 319
Fig. 14.1 Extraction of

cellulose from aquatic plant Washing
Drying 70oC
Milling
Delignification Sodium acetate 121o C

20 mins
Starch Hydrolysis
Washing and
purification
Cellulose
of around 21.8 nm and a length of around 547 nm (Chen et al. 2016). Marine green
algae contain 9% cellulose, 21% hemicellulose and 1.9% lignin (Yaich et al. 2011).
Microalgae also contain cellulose (Chen et al. 2009). Extraction of cellulose from
the microalgae N. oceanica yielded cellulose nanofibrils with mechanical properties
superior to nanofibril cellulose from wood. The nanofibrils obtained had a diameter
of 9 nm and tensile strength between 3 and 4 GPa. This process of extraction from
microalgae required no delignification process as microalgae do not contain lignin.
The process of extraction is involved in deproteinization and removal of lipids fol-
lowed by purification and TEMPO-mediated oxidation under gentle mixing (Lee
et al. 2018).
Most commercially used biopolymers in algae are the phycocolloids alginate,
agar and carrageenan. The residue from the extraction of these phycocolloids con-
tains significant amount of cellulose. For example, the cellulose obtained from the
residue of red algae from which agar had been extracted yielded high-quality cel-
lulose nanocrystals (Achaby et al. 2018). This use of the cellulosic residue reduces
the waste generated from the processing of red algae for production of agar and
hence optimizes the utilization of the aquatic resource. Although discarding might
seem the least energy-intensive option following agar extraction, the conversion of
the cellulose from the waste residue into high-value products used in, for example,
biomedical industry could compensate for the additional processing cost.
Likewise, cellulose can be extracted from the residue from alginate extraction
of brown algae. Cellulose with molecular weight of 2.69 × 105 was obtained with
an average fiber length of 1.1 µm and fiber width of 4 nm. The cellulose obtained
using this method showed good biocompatibility and potential for application in food
industry demonstrated by its superior thickening property due to the ability to bind
to milk (Gao et al. 2018).
320 14 Cellulose
Compared to the extraction from aquatic plants, the extraction of nanocellulose

from terrestrial wood is more intensive. The pulp used for this process contained
lignin of 0.7% and hemicellulose 13.8%. The pulp had been pretreated to reduce
lignin content. The pulping process requires the use of either the kraft process (Lah-
nalammi et al. 2018) or the acid sulfite process (Hanhikoski et al. 2019). The kraft
process involves treatment of wood with high concentrations of sodium hydroxide
and sodium sulfide at elevated temperatures while the acid sulphite process makes
use of high concentration of sulfuric acid at high temperatures. The process to convert
pulp to nanocellulose involved suspension of wood pulp in water at a ratio of 1:100
(grams wood pulp to water). This is then followed by oxidation with 10 mmol of
sodium hypochlorite per every gram of cellulose mediated by a mixture of 2,2,6,6-
tetramethylpiperidine-1-oxyl (TEMPO) and sodium bromide. Sodium hydroxide is
then added to increase the pH to 10 and the reaction allowed to take place for 5 h to
form cellulose nanoparticles, which was then washed with water and the cellulose
nanoparticles separated by centrifugation at 14,000 rpm (Olatunji and Olsson 2015).
The extraction of cellulose from wood requires a more rigorous process to remove
the lignin and hemicellulose which are present at a higher content.
Here, some environmental issues arising from the extraction of cellulose from aquatic
plants and algae are discussed. In this section, more focus is directed at the process of
extraction as discussed in the previous section. Further environmental issues arise in
additional processes such as transportation of the raw materials to the factory and the
packaging process. These issues have been discussed in other sections of the book
and will not be discussed here to avoid repetition.
14.6.1 Energy for Drying
Most of the aquatic plants contain a considerable amount of water. Water hyacinth
contains the most amount of water ranging between 93 and 96% (Penfound and Earle
1948). Drying could be done prior to transportation to factories where the biopoly-
mers are extracted or the biomass could be transported before drying. Spoilage could
be prevented by initial drying, and the reduced moisture content cold also reduces
transportation cost. To dry a sample of duckweed, for example, required drying at
60 °C for 2 days (Chen et al. 2012) while Azolla required drying at 70 °C for sev-
eral hours overnight (Miranda et al. 2016). Drying of biomass is usually done using
electric-powered hot air ovens. This could be from hydroelectricity, nuclear or renew-
able energy-powered source. Drying could also be achieved using open air drying in
hotter climates, where high level of purity is not required at this stage.
14.6.2 Chemical Consumption
Depending on the source of cellulose, the types and quantity of chemicals con-
sumed could vary. From plants with lignified cell walls, mineral acids and alkali are
often required for delignification. The amount of chemical used also depends on the
grade of cellulose required. Cellulose with all lignin, lipids and extractives such as
proteins removed and cellulose with all the hemicellulose removed have different
requirements.
Sodium hydroxide is commonly used for removal of the non-cellulosic compo-
nents, and toluene has also been used for removal of lipids while depigmentation
and further delignification are achieved using sodium hypochlorite. Sodium acetate
at high temperature can also be used for delignification. Sodium hydroxide and
sodium acetate can be neutralized during wastewater treatment, thereby limiting the
impact on environment pH. Toluene however is an organic solvent derived from fos-
sil fuel during the production of gasoline and other products. Exposure to toluene
either through the respiratory tract, gastrointestinal tract through contamination of
soil, air or drinking water as a result of various forms of release into the environ-
ment. Toluene has been associated with neurological, reproductive and developmen-
tal adverse effects (Vulimiri et al. 2017). Being a volatile organic compound, use of
toluene in the production of cellulose from aquatic biomass will require additional
processes for treatment such as adsorption, condensation and catalytic oxidation
(Martinez de Yuso et al. 2013). Exposure limits and safety of the workers coming
in contact with the harmful chemical will also require additional precautions hence
additional cost.
Cellulose production in general requires use of a wide variety of chemicals in large
amounts for delignification. The process of extraction results in generation of solids
and some other chemicals as effluent, and some of these chemicals are unknown
due to the complex structure of lignocellulosic biomass. One advantage of cellulose
from algae and aquatic plant is the lower lignin content compared to, for example,
wood. For example, duckweed has a lignin content of 12.2% (Yadav et al. 2017).
This results in reduced requirements for the heavy chemicals and energy used in
delignification.
14.6.3 Methane Emission
While the process of cultivation of aquatic plants and algae can be considered as CO2
emission negative, the process of decomposition of the biomass in nature leads to the
emission of methane, another greenhouse gas of concern (Heilig 1994). Controlled
degradation of isolated cellulose and utilization of the decomposition products as
fuel are the ways to minimize the release of methane to the environment from the
decomposition of aquatic plants and animals. Cellulose has been shown to have a
higher biomethane potential of the three lignocellulosic compounds (Li et al. 2018).
322 14 Cellulose
More methane is produced in combined biodigestion than when isolated anaerobic

digestion of cellulose, hemicellulose and lignin.
The environmental significance of this is that allowing aquatic plants and algae to
degrade in the environment results in more methane emission into the environment.
One large-scale example of this is the production in paddy fields where the decom-
position of the straws in the paddy fields results in methane release. A proposed
solution to this is the collection of these straws and utilizing them (Fusi et al. 2014).
One way of utilizing this is extraction of the cellulose content for applications such
as those discussed in the following section. Therefore, the controlled extraction and
utilization of cellulose from these sources could contribute to reducing the release
of methane into the environment.
Furthermore, one of the solutions proposed and being adopted for addressing the
water hyacinth bloom in tropical regions is the harvesting of the water hyacinth from
the water and use as compost in farming (Polprasert et al. 1994). This is a solution that
makes use of the aquatic plant biomass in large quantities and significantly contributes
to reducing the environmental nuisance. If the composting is done properly with
adequate aeration to encourage aerobic decomposition, the process does not result
in methane emission. On the other hand, if anaerobic decomposition occurs, this
results in further release of methane. New methods have therefore been proposed to
minimize anaerobic decomposition in compost pile using semipermeable membrane
that aid aeration of the pile (Ma et al. 2018). This can be applied to the composting
of water hyacinth in order to ensure that the positive benefits of this approach to the
management of the water hyacinth is not overshadowed by the adverse impact of
methane emission.
14.6.4 Land Space Occupied
The main advantage of aquatic biomass-sourced cellulose is that there is no compe-

tition with land occupied by humans and that used for cultivation of food crops. It
also does not lead to depletion of nutrients from the soil. However, the process of
extraction of cellulose requires land space. The use of aquatic environment for culti-
vation of cellulosic biomass for commercial applications such as biofuel production
could save thousands of acres of land space which would be required to cultivate the
required amount of terrestrial-based cellulose crops.
14.6.5 Aquatic Plants and Algae Bloom
Considering the devastating effect uncontrolled growth that some aquatic plants such
as water hyacinth and algae have caused in recent times, harvesting of these aquatic
organisms goes beyond the financial gains or economic boost. Effective system to
convert these presently unmanaged resources would be of immense benefit to the
environment and recovery from the financial loss to the fisheries and associated
businesses. Algae blooms have been discussed in the previous chapters in this book.
One of the solutions is to harvest the abundant algae and aquatic plant and extract
biopolymers such as cellulose from them.
Industries which are affected by uncontrolled growth of aquatic plants and algae
include tourism, seafood, water transportation, aquatic farming, fishing and water
sports. Water hyacinth also affect activities on land by preventing access to water
as a result of blocking if irrigation channels and rivers. This blockage or reduced
access to water affects biodiversity as many animals on dry and wetlands time their
migrations, reproduction and other activities crucial to their survival in tune with the
aquatic cycle. For example, water turtles lay eggs on land and the timing is such that
the egg hatches just in time as the water level rises to carry the hatched turtles into
the water (Steen et al. 2012). If the water flow is interrupted and the turtles hatch
before the water level rises, the turtles have longer distances to reach water and may
die from dehydration. This results in a drastic drop in population as the older turtles
die out without offspring to maintain population balance.
Therefore industries gaining profits from extraction of biopolymers such as cel-
lulose from these aquatic plants and algae is a bonus, since solving the problem they
create otherwise is a global priority.
14.6.6 Wastewater Treatment
Aquatic plants and algae have water remediating effect. Duckweeds in particular have
exceptional water remediating effect. They can remove heavy metals and microele-
ments from water and retain them at high concentration within the plant up 100,000
times the concentration in the surrounding water. Water hyacinth can also remove
heavy metals such as zinc, cadmium, chromium and copper from wastewater (Sarkar
et al. 2017; Hasan et al. 2007). In the studies reported mainly, the shoot of the water
hyacinth was utilized for wastewater treatment. This leaves the rest of the plant for
extraction of biopolymers. The water hyacinth biomass used for water treatment
can be recovered, and the biopolymer also extracted from them. The heavy metals
adsorbed unto the surfaces can be removed by dissolving in acid during extraction
while the cellulose is recovered.
14.7 Applications
In this section, we take a look at some significant application of cellulose, particularly

cellulose from aquatic plant. The applications reviewed here are primarily based on
those of current non-aquatic plant-based sources of cellulose. This provides some
perspectives on the potential market for aquatic sourced cellulose in such appli-
cations. The goal is therefore to explore the potential application in the industries
324 14 Cellulose
(where none is currently existing) and the demand for such production in the global
market.
14.7.1 Textile
The textile industry is one of the earliest and one of the largest in the world.
The textile industry formed the basis for the first industrial revolution. As
of 2008, 24,025,100 tonnes of cotton, 3,097,000 tonnes of cellulosic fibers,
361,590,800 tonnes of wool, 790,100 tonnes of flax and 558,400 tonnes of syn-
thetic fibers were consumed globally (FAO 2011). There is an increasing demand for
more sustainable methods of fabric production. Aquatic plants are yet to be commer-
cially explored for production of alternative cellulose-based fabrics on commercial
scale.
Cotton has been the main cellulose-based fiber in the textiles and garment industry.
In the past seven decades, for reasons such as yield, pest on cotton plantations and
the vast land occupied for cotton plant cultivation, cheaper alternative fabrics from
cellulose such as viscose, Tencel and Modal (Shen et al. 2010) have been introduced
to the global market. Unlike cotton where the cellulose naturally grows into cellulose
fibers which are then carded and spun into long yarns that are then woven into fabrics,
man-made or semi-synthetic cellulosic fabrics are made from dissolved cellulose
extract from lignocellulosic plants, mainly wood which are then taking through the
wet fiber spinning process (Olatunji and Olsson 2016) to form cellulose fibers which
are then spun and woven into yarns and fabrics, respectively. Although having other
environmental concerns such as chemicals used in dissolving the fibers, production
of these man-made cellulose fibers requires less water in production and requires less
land space compared to cotton fibers. This could further be made more sustainable
by using cellulose from aquatic plants rather than land plant.
The process of producing cellulose-based fabrics from lignocellulosic plants
requires a variety of chemicals, and one of the most chemically and energy-
demanding stages is the lignin removal. Aquatic plants such as duckweed and water
hyacinth contain a considerable amount of cellulose, but little or no lignin duckweed,
for example, contains 12.2% (Yadav et al. 2017) while water hyacinth contains around
10% (Thiripura and Ramesh 2012).
Cladophora, a filamentous green algae, has mercerized cellulose structure
(Mihranyan 2010). This is a more amorphous, swollen form of cellulose. This makes
the cellulose adhere better to dye and have a glossier appearance. This property of
cellulose in algae presents some potential application in advanced textile technology
development.
Production of viscose fiber could consume as much as 110,000 kJ per tonne of
fiber produced from wood (Shen et al. 2010). An advancement in the man-made fiber
in the textile industry should aim at reducing the energy requirement for production
of such using aquatic sourced cellulosic fiber.
14.7.2 Bioethanol Production
Aquatic plants fall into the category of third generation energy crops. Not only
are they non-fossil, but also they are sourced from biomass and even better non-edible
biomass that is grown without competing with food or space for human and livestock
habitation or food cultivation. In addition to this, their cultivation is beneficial to the
environment as they have the capacity to remove nitrogen, phosphorous, carbon
and microelements and heavy metal from wastewater. Third generation biofuels are
characterized by their ability to yield high biomass with lower resource requirements
than conventional feedstocks. Another advantage of aquatic sources such as algae
is the lower lignin content which reduces the complexity of separating lignin from
cellulose.
Different methods have been explored for the production of ethanol which takes
advantage of the cellulose content of aquatic plants, duckweed and water hyacinth
(Bayrakci and Kocar 2014). The key attraction here is the use of plants with bioreme-
diating properties which contain polysaccharides which can be converted to glucose
for fermentation. The challenge of this is the right cocktail of enzymes and conditions
which can most effectively convert both the cellulose alongside other polysaccha-
rides of the cell wall into glucose without costly separation processes. Cellulose
from aquatic source can be saccharified into glucose using the enzyme cellulase
(Shen and Xia 2003), and however, the cost of enzyme limits the commercial feasi-
bility of such process. Thermochemical process such as pyrolysis is an alternative to
enzyme-based process for bioethanol production from cellulosic biomass. However,
this process requires high energy input which makes it commercially infeasible.
The high yield and relatively fast growth rate of algae compared to terrestrial
plants sourced as biomass made it a desirable third generation biofuel feedstock
option. However, a major drawback for biofuel production by algae is the high cost
of cultivation and harvest relative to the present price of fuel. For biomass to make
the cut as an ideal source of biofuel, it needs to be produced cheaply and contains
sufficient amount of organic compounds which can be converted to biofuel such as
ethanol or diesel.
As the world’s fossil resources begin to dwindle and the impact of using vast
land for cultivation of sources of second generation biofuels begin to manifest, third
generation biofuels are fast emerging. Aquatic-sourced cellulose is one of such.
Cellulose can be hydrolyzed to cellulase of cellobiose using enzymes such as
cellulase. In an example study, up to 65.9 g/L of glucose has been achieved after
enzymatic hydrolysis of cellulose from Azolla. This required use of a combination
of four different enzymes: cellulase and cellobiase for the hydrolysis of the cellu-
lose and amylase and amyloglucosidase for the hydrolysis of the starch. Hydrolysis
of cellulose is more difficult than that of starch as cellulose has a more resilient
unbranched polymer structure.
Fermentation of glucose from Azolla-derived cellulose using Saccharomyces cere-
visiae achieved an ethanol yield of 0.56 g/g. However, these yields vary for different
326 14 Cellulose
species of the plant. The ethanol yield from aquatic plants is comparable to those
from crops presently used for ethanol production.
For the production of bioethanol from biomass containing a mixture of cellulose
and starch, the hydrolysis can be achieved simultaneously using a cocktail of different
enzymes since the enzymes do not interfere with each other.
The hydrolysis and fermentation of cellulose biomass from Azolla plants can be
used to achieve up to 11,700 L of ethanol per hectare of aquatic space annually
(Miranda et al. 2016). Duckweed has also been successfully used at experimental
stage as feedstock for biogas production via anaerobic digestion (Yadav et al. 2017).
Although this is yet to be industrially adopted, a 1:1 mix of cow dung and duckweed
biomass yields 12,070 mL of biogas when anaerobically digested at 37 °C over
55 days duration. The yield from combination of cow dung and duckweed was higher
than when cow dung was digested alone (11,620 mL) with comparable methane
contents.
14.7.3 Cellulose Filler in Composites
Cellulose extracted from green algae, Cladophora has been used as fillers in
polyurethane foams. At 5–10% content by weight, cellulose improves the thermal
properties, elastic modulus, color retention and biodegradability and reduces the
polyurethane content by part substituting with cellulose. The functional groups of
cellulose show good affinity to that of polyurethane resulting in good compatibility
between the cellulose fiber and polyurethane matrix.
Cellulose has shown good compatibility with other hydrophobic fossil-based
polymers and has been used as a filler for different purposes. This has significant
environmental impact as it improves the biodegradability of the materials.
14.7.4 Cellulose Nanofilters
One of the challenges in developing cellulose-based nanofilters is the limitation if

native cellulose in attaining stable microfibrils in the nanometer range. Membrane
filters developed using green algae-derived cellulose have addressed this challenge
(Mitsuo and Eisuke 1996). This is due to their highly ordered structures which make
achieving well-dispersed nanoscale fibers more possible. Therefore, aquatic-sourced
cellulose has some structural advantages which make them superior option in some
applications. Nanocellulose has broad range of applications in various industries.
These include drug delivery applications where they have been used to produce
polymeric drug delivery devices like microneedles (Olatunji and Olsson 2015).
14.7.5 Drug Carrier
The high surface area and inert structure of cellulose from green algae make it a
suitable drug carrier. A good drug carrier should have sufficient surface to embed the
drug compound but without altering the integrity and activity of the drug. In addition
to the crystalline structure, the low degree of hornification makes it a good material for
producing stronger tablets than other plant-based cellulose (Stromme and Mihranyan
2002; Mihranyan et al. 2004). Nicotine loaded in liquid carrier formulation using
cellulose from green algae showed higher loading capacity and stability compared
to microcrystalline cellulose grade.
14.7.6 Papermaking
One of the oldest applications of cellulose is in papermaking. Paper is said to have

been invented by Ts’ai Lun in 105 AD china from mixture of plant and textile fibers
(Hunter 1974). Today, the papermaking process still follows the same fundamental
principle of using the fibers from plants to create thin sheets for various purposes.
Over the years, however, these clear sheets are known to be made mostly of cellulose
and the techniques have been refined to obtain more pure forms of paper. As of 2016,
FAO reports a global pulp for paper production of 107,042,000 metric tonnes which
is 92% of a production capacity of 150,273,000 metric tonnes, projected to rise to
156,850,000 metric tonnes production capacity by 2021 (FAO 2017).
The papermaking industry is also a water-intensive industry (Shen and Qian 2012),
and sourcing the cellulose from aquatic plants which have water remediating prop-
erties offers possibilities of a paper production system, whereby the aquatic plants
from with the cellulose produced are used in the cleaning of the gray water gener-
ated from the paper production process. This is a mare concept, and however, as the
paper industry develops toward more sustainable production processes, such should
be explored.
14.7.7 Production of Cellulose Derivatives
Cellulose derivatives such as carboxymethyl cellulose and cellulose acetate have

numerous applications which cut across various industries. CMC is used as a rheo-
logical property enhancer. Cellulose diacetate, for example, is used in the production
of membranes for water filtration. Cellulose extracted from water hyacinth has been
successfully used in the production of membranes which effectively filtered humic
acid from water showing that such membranes can effectively filter surface water
(Istirokhatun et al. 2015).
328 14 Cellulose
14.7.8 Conductive Cellulose Paper
Due to the large surface area of green algae sourced cellulose, it makes a good
conductive flexible paper with numerous potential applications. Cellulose in general
has good compatibility with polypyrrole (PPy). Cellulose-PPy composites possess
the mechanical properties of paper and the conductive properties of metal. This has
potential applications such as energy storage and sensors and a range of other low
cost, mass reproducible conductive devices.
Green algae cellulose-PPy composites that have been produced have recorded a
conductivity of 0.3 S/cm (Mihranyan 2010).
Cellulose extracted from water hyacinth has been used as a reducing agent and a
capping agent for the green synthesis of silver nanoparticles. Monodisperse, spherical
silver nanoparticles with size 2.68–5.69 nm are obtained using cellulose extracted
from water hyacinth as reducing agent and a capping agent (Mochochoko et al. 2013).
This further shows the broad range of applicability of cellulose in green extraction
processes for high-value products such as silver nanoparticle.
Cellulose is relatively well explored commercially. Since prehistoric times, cellulose-

based materials have been used for paper and textiles. After paperboard, man-made
cellulosic fiber is the next largest biopolymer commodity by volume (Shen and Patel
2010). In the earlier years of the textile industry, 70% of the textiles in the world
were cotton based. The textile industry depending on one plant for raw material poses
limitations such as nutrient consumption from soil and large areas of land required
for cultivation. In addition to this, cotton being a plant that is endemic to only a few
regions in the world meant dependency on importation for many and concentration
of resources in only a few areas. On the contrary, aquatic plants such as duckweed
and water hyacinth are endemic to almost all regions of the world in abundance.
The 1930s brought forth the innovation of man-made fibers sourced from cellu-
losic plants. This allowed cellulose to be extracted from, mainly wood and cotton
lint, and then processed into fabrics (Albrecht 2004). The man-made cellulosic fibers
have considerable market value, and as of 2002, global production of man-made cel-
lulose reached 2800 kt annually (Aizenshtein 2004; Lenzing AG 2006). While the
global production of cotton and petroleum-based synthetic fibers have both shown
steep rises in the past decades, man-made cellulose fiber production rate has not
shown as much increase. This can be attributed to the limited land-based resources.
Use of aquatic plants could potentially boost commercial production of man-made
cellulosic fibers.
The cellulosic fiber industry is of more significance now as land resource for grow-
ing cotton becomes scarce and the fossil resource for fossil-based synthetic fibers is
depleting. Also important is the development of more environmentally friendly pro-

duction process for production of cellulosic man-made textiles. These newer methods
eliminate the need for toxic compounds such as CS2 and minimize the total amount of
chemicals such as sodium hydroxide required for the production process (Shen et al.
2010). Aquatic-sourced cellulose from aquatic plants which are growing abundantly
and are seen as environmental nuisance means relatively low-cost feedstock for com-
mercial cellulose production. Recent studies have shown that high-quality cellulose
nanoparticles (Jaurez-Luna et al. 2019) with low aggregation can be extracted from
the water hyacinth stem.
14.9 Conclusion
Cellulose is relatively abundant in the aquatic ecosystem as it can be obtained from a

diverse range of plants and algae. Some cellulose-producing species of aquatic plants
and animals are so abundant that they have become a nuisance to water transportation
and coastal activities. Extraction of cellulose from aquatic source presents one of the
solutions to address such challenges. There needs to be a balanced system in place,
whereby the aquatic plants re-absorb the nutrients from the wastewater released by
industries such as paper and textile, among others, and then these aquatic plants in
return act as sources of polymers such as cellulose which in turn serves as a source
of fuel and feedstock for these industries. The superior quality of cellulose obtained
from aquatic plants and algae indicates future potential in their high-value application
in biomedicine and pharmacology.
References
Aizenshtein EM (2004) World production of textile raw material. Fibre Chem 36(1):3–7
Albrecht W (2004) Regenerated cellulose in chapter “Cellulose”. In: Ullmann’s encyclopedia of
industrial chemistry, 7th edn. Wiley-VCH Verlag GmbH & Co. KGaA. https://doi.org/10.1002/
14356007.a05_375.pub2
Basile A, Sorbo S, Conte B, Cobianchi RC, Trinchella F, Capasso C, Carginale V (2012) Toxic-
ity, accumulation, and removal of heavy metals by three aquatic macrophytes. Int J Phytorem
14(4):374–387
Bayrakci AG, Kocar G (2014) Second-generation bioethanol production from water hyacinth and
duckweed in Izmir: a case study. Renew Sustain Energy Rev 30:306–316
Beguin P, Aubert JP (1994) The biological degradation of cellulose. FEMS Microbiol Rev 13(1):25–
58
Brown RM, Saxena IM Jr (2000) Cellulose biosynthesis: a model for understanding the assembly
of biopolymers. Plant Physiol Biochem 38(1–2):57–67
Chen P, Min M, Chen Y, Wang L, Li Y, Chen Q, Wang C, Wan Y, Wang X, Cheng Y, Deng S,
Hennessy K, Lin X, Liu Y, Wang Y, Martinez B, Ruan R (2009) Review of the biological and
engineering aspects of algae to fuels approach. Int J Agric Biol Eng 2:1–30
Chen Q, Jin Y, Zhang G, Fang Y, Xiao Y, Zhao H (2012) Improving production of bioethanol from
duckweed (Landoltia punctata) by pectinase pretreatment. Energies 5:3019–3032
Chen WY, Lee HV, Juan JC, Phang S (2016) Production of new cellulose nanomaterial from red
algae marine biomass Gelidium elegans. Carbohyd Polym 51:1210–1219
330 14 Cellulose
FAO (2011) A summary of the world apparel fibre consumption survey 2005–2008. International
Cotton Advisory Committee
FAO (2017) Pulp and paper capacities. Rome, Italy. ISSN 0255-7665, ISBN 978-92-5-009846-3
Fernandez Diniz JM, Gil MH, Castro JAAM (2004) Hornification—its origin and interpretation in
wood pulps. Wood Sci Technol 37:489–510
Fusi A, Bacenetti J, Gonzalez-Garcia S, Vercesi A, Bocchi S, Fiala M (2014) Environmental profile
of paddy rice cultivation with different straw management. Sci Total Environ 494–495:119–128
Gao H, Duan B, Lu A, Deng H, Du Y, Shi X, Zhang L (2018) Fabrication of cellulose nanofibers
from waste brown algae and their potential application as milk thickeners. Food Hydrocolloids
79:473–481
Hanhikoski S, Niemela K, Vuorinen T (2019) Biorefining of Scots pine using neutral sodium sulphite
pulping: investigation of fibre and spent liquor compositions. Ind Crops Prod 129:135–141
Hasan SH, Talat M, Rai S (2007) Sorption of cadmium and zinc from aqueous solution by water
hyacinth (Eichhornia crassipes). Bioresour Technol 98:918–928
Heilig G (1994) The greenhouse gas methane (CH4 ): sources and sinks, the impact of population
growth, possible interventions. Popul Environ 16(2):109–137
Hon (1994) Cellulose: a random walk along the historical path. Cellulose 1(1):1–25
Istirokhatun T, Rokhati N, Rachmawaty R, Meriyani M, Priyanto S, Susanto H (2015) Cellulose
isolation from tropical water hyacinth for membrane preparation. Procedia Environ Sci 23:274–
281
Jaurez-Luna GN, Favela-Torres E, Quevedo IR, Batina N (2019) Enzymatically assisted isolation of
high-quality cellulose nanoparticles from water hyacinth stems. Carbohyd Polym 220:110–117
Johnson M, Shivkumar SJ (2004) Filamentous green algae additions to isocyanate based foams. J
Appl Polym Sci 93(5):2469–2477
Kollah B, Patra AK, Mohanty SR (2016) Aquatic microphylla Azolla: a perspective paradigm
for sustainable agriculture, environment and global climate change. Environ Sci Pollut Res
23(5):4358–4369
feasibility of Macroalgae as a potential feedstock for biorefineries. Bioenergy Res 8:1046–1056
Koyama M, Sugiyama J, Itoh T (1997) Systematic survey on crystalline features of algal celluloses.
Cellulose 4:147–160
Lahnalammi A, Sixta H, Jamsa-Jounela S (2018) Control strategy scheme for the prehydrolysis
Kraft process. Comput Aided Chem Eng 44:643–648
Lee H, Kim K, Mun SC, Chang YK, Choi SQ (2018) A method to produce cellulose nanofibrils from
microalgae and the measurement of their mechanical strength. Carbohyd Polym 180:276–285
Lenzing AG (2006) Sustainability in the Lenzing Group. Lenzing AG, Lenzing. http://www.lenzing.
com/sites/nh/english/e_index.html
Li W, Khalid H, Zhu Z, Zhang R, Liu G, Chen C, Thorin E (2018) Methane production through
anaerobic digestion: participation and digestion characteristics of cellulose, hemicellulose and
lignin. Appl Energy 226:1219–1228
Ma S, Sun X, Fand C, He X, Han L, Huang G (2018) Exploring the mechanisms of decreased
methane during pig manure and wheat straw aerobic composting covered with a semi-permeable
membrane. Waste Manag 78:393–400
Martinez de Yuso A, Izquierdo MT, Rubio B, Carrot PJM (2013) Adsorption of toluene and toluene-
water vapor mixture on almond shell based activated carbon. Adsorption 19:1137–1148
Mihranyan A (2010) Cellulose from Cladophorales green algae: from environmental problems to
high-tech composite materials. J Appl Polym Sci 119:2449–2460
Mihranyan A, Llagostera AP, Karmhag R, Strømme M, Ek R (2004) Moisture sorption by cellulose
powders of varying crystallinity. Int J Pharm 269:433–442
Miranda AF, Biswas B, Ramkumar N, Singh R, Kumar J, James A, Lal B, Subudhi S, Bhaskar
T, Mouradov A (2016) Aquatic plant Azolla as the universal feedstock for biofuel production.
Biotechnol Biofuels 9(221):1–17
Mitsuo Y, Eisuke Y (1996) (to Mitsubishi Paper Mills). Jpn Pat 08-229318
References 331
Mochochoko T, Oluwafemi OS, Jumbam DN, Songca SP (2013) Green synthesis of silver
nanoparticles using cellulose extracted from an aquatic weed; water hyacinth. Carbohyd Polym
98(1):290–294
Munoz C, Hidalgo C, Zapala M, Jeison D, Riquelme C, Rivas M (2014) Use of cellulolytic marine
bacteria for enzymatic pretreatment in microalgal biogas production. Appl Environ Microbiol
80(14):4199–4206
Olatunji O, Olsson RT (2016) Processing and characterization of natural polymers. In: Natural
polymers, industry techniques and applications. Springer, Switzerland. ISBN 978-3-319-26412-7
Penfound WT, Earle TT (1948) The biology of the water hyacinth. Ecol Monogr 18:447–472
Polprasert C, Kongsricharoern N, Kanjana Prapin W (1994) Production of feed and fertilizer from
water hyacinth plants in the tropics. Waste Manag Res 12(1):3–11
Russel JB, Muck RE, Weimer PJ (2009) Quantitative analysis of cellulose degradation and growth
of cellulolytic bacteria in the rumen. FEMS Microbiol Ecol 67(2):183–197
Sarkar M, Rahman AKML, Bhoumik NC (2017) Remediation of chromium and copper on water
hyacinth (E.crassipes) shoot powder. Water Resour Ind 17:1–6
Shen L, Patel MK (2010) Life cycle assessment of man-made cellulose fibres. Lenzinger Ber
88:1–59
Shen J, Qian X (2012) Addressing the water footprint concept: a demonstrable strategy for
papermaking industry. BioResources 7(3):2707–2710
Shen XL, Xia LM (2003) Studies on immobilized cellobiase. Chin J Biotechnol 19(2):236–239
Shen L, Worrell E, Patel KM (2010) Environmental impact assessment of man-made cellulose
fibres. Resour Conserv Recycl 55(2):260–274
Siddhanta AK, Chhatbar MU, Mehta GK, Sanandiya ND, Kumar S, Oza MD, Prasad K, Meena R
(2011) The cellulose contents of Indian seaweeds. J Appl Phycol 23:919–923
Smith J, Connell P, Evans RH, Gellene AG, Howard MDA, Jones BH, Kaveggia Palmer L, Schnetzer
A, Seegers BN, Seubert EL, Tatters AO, Caron DA (2018) A decade and a half of pseudo-nitzschia
spp. and domoic acid along the coast of southern California. Harmful Algae. https://doi.org/10.
1016/j.hal.2018.07.007
Steen DA, Gibbs JP, Buhlmann KA, Carr JL (2012) Terrestrial habitat requirements of nesting
freshwater turtles. Biol Conserv 150(1):121–128
Stromme M, Mihranyan A, Ek R (2002) What to do with all these algae? Mater Lett 57:569–572
Thiripura M, Ramesh A (2012) Isolation and characterization of cellulose nanofibers from the
aquatic weed water hyacinth—Eichhornia crassipes. J Carbohyd Polym 87:1701–1705
Vulimiri SV, Pratt MM, Kulkarni S, Beedanagari S, Mahadevan B (2017) Reproductive and devel-
opmental toxicity of solvents and gases, chap 21. In: Reproductive and developmental toxicology,
2nd edn. Academic Press, pp 379–396
Wi SG, Kim HJ, Mahadevan SA, Yang DJ, Bae HJ (2009) The potential value of the seaweed Ceylon
moss (Gelidium amansii) as an alternative bioenergy resource. Bioresour Technol 100:6658–6660
Yadav D, Barbora L, Bora D, Mitra S, Rangan L, Mahanta P (2017) An assessment of duckweed
as a potential lignocellulosic feedstock for biogas production. Int Biodeterior Biodegradation
119:253–259
Yaich H, Garnaa H, Besbesa S, Paquot M, Beckerc C, Attia H (2011) Chemical composition and
functional properties of Ulva lactuca seaweed collected in Tunisia. Food Chem 128(4):895–901
Yoon JJ, Kim YJ, Kim SH, Ryu HJ, Choi JY, Kim GS, Shin MK (2010) Production of
polysaccharides and corresponding sugars from red seaweed. Adv Mater Res 93–94:463–466
Zhao X, Moates GK, Wellner N, Collins SR, Coleman MJ, Waldron KW (2014) Chemical charac-
terisation and analysis of the cell wall polysaccharides of duckweed (Lemna minor). Carbohyd
Polym 111:410–418
Chapter 15
Polyesters
Abstract Polyesters are characterized by a structural unit made up of ester-linked

monomers. Cutin is the main polyester found in the aquatic ecosystem. It is present
in aquatic plants where it plays a role in the regulation of water permeation. It has
potential applications in packaging films, cosmetics and biopolyester production.
The understanding of the structure, function and chemistry is also useful in devel-
oping products which mimic cutin. This chapter discusses cutin obtained from the
environment. It covers the natural sources of cutin within the aquatic environment,
its chemistry, extraction process, applications, environmental implications of cutin
production and the present state of its commercial production.
Keywords Cutin · Polyester · Biopolymer · Aquatic plants · Cuticle
15.1 Introduction
The main polyesters found in aquatic environment are cutin and suberin. These
polyesters are the most native feature in plants. Although previously thought to be
absent in all aquatic plants, cutin is now known to be present in a number of aquatic
plants and makes up the bulk of the dry mass of the cuticle. Traces of suberin have
been found in the water facing part of the aquatic plant. The presence of traces of
suberin in the water facing part of aquatic plant suggests it is playing a similar role
as the terrestrial plant suberin present in the root. Suberin is present in a much lower
quantity than cutin, and therefore, this chapter will focus more on cutin as the main
polyester found in aquatic plants.
Cutin constitutes the bulk of the cuticle by mass and serves as a matrix hold-
ing the wax and other components of the cuticle. Based on the available findings
from study of the biochemistry of the cuticle, the role of cutin in aquatic plant is
thought to be UV protection and as a barrier to protect against pathogen attack
and external environmental stress. The protection against pathogen is possible due
to its physicochemical properties which discourage cell/microbe attachments and
protection against microbe is possibly due to the mechanical properties and stability.
Although methods to isolate long-chain polyester cutin are underway, methods
for extraction of cutin monomers and oligomers have been presented in literature and

334 15 Polyesters
patents. Relatively non-complex methods for extraction of cutin, which require the
use of acids and alkali treatment at high temperature, consume moderate quantities of
water. The cultivation of the feedstock for cutin production has beneficial impacts on
environment such as water remediation properties such that the process of production
of cutin would include cultivation of aquatic plants which also serve to clean the
aquatic environment.
Despite not yet being fully commercially exploited, cutin has some signifi-
cant applications in, especially the food and the pharmaceutical industry where its
mechanical properties as well as the role of the barrier/protective role of the cuticle
can be mimicked for human use. Furthermore, the side products from cutin extrac-
tion from aquatic plant cuticle are also useful. Compounds such as palmitic acid
and linolenic acids find applications in areas such as soap making, biodiesel and
antiinflammatory agent production.
In a global market that is increasingly demanding more plant-based alternatives
to the dwindling fossil-derived products, particularly those that are either injected or
indirect contact with the body or food, cutin is another one of the vast biopolymer
resources the aquatic ecosystem offers.
Cutin occurs in nature as part of plant cuticle which is a protective coating on the sur-
face plants. The cuticle protects plant from environmental stress and diseases and aids
water retention. It is adhered to the plant epidermal wall through a proteinaceous layer
with which it is interconnected. Cutin is a branched polymer, cross-linked through
ester linkages between the chains forming the polymer network. Cutin content in
most plants is between 40 and 80% dry weight of the cuticle; around 600 μg of cutin
can be obtained per m2 of a plant surface. The cuticle is measured to be between 0.5
and 40 μm thick. It is a composite of polysaccharides, biopolyester (cutin) waxes and
phenolic compounds. The cutin served as the polymeric continuous phase, holding
the other components of the cuticle. Figure 15.1 is a simplified representation of the
cutin location on the plant surface. This is simplified since the pectinaceous layer is
not always distinctly separated as the figure suggests.
Until the recent studies on cutin in aquatic plants, cutin has been widely regarded as
a feature only in terrestrial plants with a sole purpose of aiding water retention. Studies
Fig. 15.1 A representation of cuticle layer next to the epidermal wall

in recent years confirm that cutin is not only a compound restricted to terrestrial plants
but also is present in aquatic plants. This further revealed that the role of cutin in
aquatic plants is not for water retention, since aquatic plants have always abundance
of water and in fact have features to adapt them for growth in the environment. Cutin
in aquatic plants serves as a protective layer against UV radiation, pathogens and
environmental stress (Li et al. 2017). Because aquatic plants do not have the property
of terrestrial plant of being able to direct parts of their leaves to grow away from the
sun’s rays, they use cuticular matrix as a protection against UV.
Aquatic plants generally have the water facing sides and the sun-facing side.
Traces of suberin like compounds have been detected in the water facing side
(Borisjuk et al. 2018). Suberin is the polyester synthesized in the roots of vascu-
lar plants which aids in water retention. It is therefore conceivable that they are
present in the water facing sides of some aquatic plants. Further studies are yet to
fully define the roles or suberin in aquatic plants.
Cutin does not occur in all aquatic plants as not all aquatic plants have need for it.
Much is yet to be known about cutin in aquatic plants, and it has generated increasing
interest. Duckweed is one aquatic plant containing cutin which has attracted growing
interest for its potential commercial and ecological application. The cuticle contains
between 40 and 80% cutin by weight.
15.3 Chemistry of Cutin
Cutin is a polymer made up of C16 or C18 fatty acids with hydroxyl or epoxy groups
attached. The cutin polymer chains are cross-linked by ester linkages forming a
rather complex structure. It has polyester structure formed from the ester linkage
of C16 or C18 fatty acids binding with diols. The main monomer of cutin is the
10,16-dihydroxyhexadecanoic acid monomer (Graca and Lamosa 2010). Figure 15.2
shows ester linkages between this monomer and other hydroxyl compounds. Cutin
consists of different monomers covalently linked together as well as cross-linking
between different polymer chains. It is usually present in the form of a cutin matrix
with waxes and other compounds embedded within the matrix or as a layer within
the cuticle structure. Other components such as phenolic compounds could also be
present within the cutin matrix (Heredia 2003).
In a recent analysis of the biochemistry of aquatic plant cuticle, GC-MS analysis of
cutin extracted from duckweed fronds confirms the presence of a substantial amount
of fatty acids, and up to 95% of the cutin monomers were fatty acids. These include
saturated palmitic acid (C16) making up the bulk of the fatty acids. Cutin matrix
monomer also consisted of linolenic acid, an unsaturated C18 fatty acid which is
also an essential fatty acid for humans as it is not produced in the human body. Other
fatty acid components of cutin include other long-chain fully saturated fatty acids
as well as hydroxyl fatty acids, aromatic acids and phenolic ring-cinnamic acids
(Borisjuk et al. 2018). The wax fraction from the cuticle contains other valuable
compounds such as phytosterols and squalene which is a component of human skin
336 15 Polyesters
Fig. 15.2 Ester linkage between C16 monomers of cutin and a glycerol
that serves as the first point of protection against ultraviolet radiation and physical
barrier from the environment. The cutin from aquatic plants comprises of the same
compounds present as that of terrestrial plants, and however, the composition of each
compound varied for the different species. This is expected as the cutin in aquatic
plants serves different functions as those in terrestrial plants where the main function
is water retention.
Although suberin is the plant polyester found mainly in the part of the plant
below the ground, the root (Li et al. 2017), it is not expected to be present in aquatic
plants. However, traces of suberin were detected in the water facing duckweed fronds
(Borisjuk et al. 2018). Suberin from terrestrial plants is used in the production of
cork used commonly in the wine bottling (Graca 2015). Their presence in aquatic
plant could therefore be of commercial interest. However, more studies are required
to fully understand their role in aquatic plants.
An important property of polymers is to have the mechanical properties required
for their applications which may include load bearing, pulling and tearing, or serving
as a barrier against water and air. Mechanical properties of cutin from plants at
15.3 Chemistry of Cutin 337
present state of the technology are still inferior to those of the fossil-based polymers
widely used in industry. The elongation at break, young modulus and tensile strength
measured for cutin is 27%, 45 and 12.3 MPa (Heredia-Guerrero et al. 2017a, b;
López-Casado et al. 2007). Much lower than that of the petroleum-based polymers
such as polyethylene, polystyrene and polypropylene where elongation at break is
in the hundreds and the young modulus in multiple GPa. On the other hand, cutin
has a relatively high thermal degradation temperature of 200 °C, similar to that of
rubbers and polyethylene. Polymers such as polystyrene, polyvinyl chloride and
polystyrene have lower degradation temperatures. Cutin shows no melting point in
thermogravimetry analysis, thus confirming that it is a thermoset polymer. This is
expected of its cross-linked structure. It has a transition temperature of −22 °C.
Cuticle composition and cutin content vary from plant to plant. Factors which
determine cutin content include developmental stage of the plant, species, part of
the plant and the environmental conditions within which the plant is grown (Yeats
et al. 2012). Although there is a significant variation in the monomers making up the
cutin from different species and different parts of the plant, the species and anatomy
structure relation is well defined enough to allow obtaining a biomass with uniform
cutin composition. This is important in order to achieve standard controlled processes
and uniform products.
15.3.1 Biodegradation of Cutin
As a biodegradable polymer, it is essential that cutin decomposes into harmless

monomers which further decomposes into smaller molecules and finally carbon,
nitrogen and hydrogen compounds (Angst et al. 2016). Unlike other commonly
used fossil-derived polyesters such as polyethylene terephthalate that take over three
decades to degrade, cutin can be fully degraded within 3–8 months when buried in
soil (De Vries et al. 1967). The degradation of cutin by microorganisms such as
bacteria and fungi and other microorganisms involves the cleavage of ester linkages
catalyzed by cutinases released by the organisms (Kolattukudy 2001a, b; Heredia-
Guerrero et al. 2017a, b). Understanding of the mechanism of degradation of cutin
by these organisms using cutinase is key to the development of processes for degra-
dation of the fossil fuel-derived polyesters into monomers or oligomers with faster
degradation rate or further applications.
Although cutin which is present in the cuticle, is a more common feature in terrestrial
plants, cutin is also present in a diverse range of aquatic plants such as duckweed,
sea grasses and water hyacinth (Borisjuk et al. 2018). The cutin from these plants has
been shown to contain the same monomeric unit as those in land plants. Therefore,
338 15 Polyesters
the applications of cutin from land plants can also be extended to those of aquatic
plants. In aquatic plants, the cuticle switches roles from primarily a water retention
feature to serving structural protective roles. Furthermore, despite terrestrial plants
being more abundant source of plant polyester, exploring cutin in aquatic plants
provides improved understanding of the role of cutin beyond water retention. It is
also an important additional source of plant biomass for cutin production, where
aquatic plants grow in excess (e.g., water hyacinth growth in lagoons), and it serves
as a means to manage aquatic plant growth through harvesting and utilizing them.
While some aquatic plants such as the water lotus are preserved, aquatic plant
considered sacred in some parts of the world and valued for the aesthetic appeal of
its flower. Other more abundant aquatic plants such as duckweed on the other hand
are of more economic potential as a source of commercial cutin production. It grows
as a green floating plant with no flower. Duckweed is globally endemic, growing in
most parts of the world. It has a relatively fast rate of growth at 80–100 tonnes dry
mass per hectare annually. Like many other aquatic plants, it grows at a much faster
rate than terrestrial plants, up to 5 times as fast as corn (Lam et al. 2014). Although
not yet commercially cultivated or harvested for cutin production on a large scale,
this much faster rate of biomass generation indicates a potentially reliable availability
of duckweed feedstock as a non-food source of cutin which does not require land
space for cultivation.
FAO reports the global aquatic plant production rate to be 30.1 million tonnes as of
2016 (FAO 2018). Much of this present is seaweed since the commercial applications
of the macroalgae have been well established, and therefore, seaweed has a well-
established market and as such is more cultivated. As the industrial applications
of other aquatic plants such as duckweed become better established, more aquatic
farmers will be encouraged to cultivate and harvest them, thereby increasing their
market value. Cuticle from wild and cultivated plants is globally estimated at 180–
1500 kg per acre (Heredia 2003), and 8.9 × 109 acres of land are estimated to be
available for plant cultivation globally (FAO, IFAD, UNICEF, WFP and WHO 2018).
40–80% of this cuticle by weight is cutin. Duckweed is one aquatic plant which serves
as a source of cutin and the cutin content and composition in this aquatic plant has
been studied (Borisjuk et al. 2018). 80–100 tonnes dry mass of duckweed is annually
attainable per hectare of aquatic space. There is therefore potential abundant source
of raw material for cutin production to be explored considering that aquatic plants
grow at up to five times faster rate than land plants and they are produced in spaces
which do not compete with land spaces occupied by humans, in addition to the fact
that the earth is about 71% water, and there is more space for expansion of aquatic
plant.
Genetic selection can be carried out to optimize the production of specific plant
metabolites by altering the growth parameters such as temperature, ultraviolet light
radiation, pH, water salinity among others. Such can be applied to improve the cutin
production by plants, aquatic plants included . As duckweed has attracted increasing
attention for its potential applications such as bioactives and biofuel production as a
cutin producing aquatic plant, this should also raise interest in optimizing such plant
for cutin production.
15.5 Extraction of Cutin 339
15.5 Extraction of Cutin
A few methods have been presented for extraction of cutin from plants such as
tomatoes and fewer on aquatic plants. The extraction methods presented either make
use of various solvents or acid or alkali for extraction of cutin from parts of the plants
such as the leaves and peels (Borisjuk et al. 2018; Cifarelli et al. 2016; Cigognini
et al. 2015). The cuticle is separated from the plant epidermal wall by either mild
chemical treatment or enzyme treatment. Ammonium oxalate is often used in the
chemical treatment, and pectinases are used in the enzyme method to degrade the
pectinaceous layer which serves as a “glue” to adhere the cuticle to the epidermal
wall.
Extraction of cutin from duckweed as presented by Borisjuk et al. (2018) is used
to extract cutin from the cuticle of aquatic plant for the main purpose of studying
the biochemistry of cutin from species of aquatic plants. This was therefore not
presented as a commercial process for cutin extraction; rather, it was the mildest
means to obtain cuticle components for analysis. In the said process, the first step
was to remove the wax fraction of the cutin matrix. The duckweed fronds were
lyophilized and immersed in 2 ml chloroform in two consecutive processes. The
first process involves a 60 s chloroform treatment of the lyophilized fronds twice
then followed by another chloroform treatment for 30 s. This is then followed by
treatment with alkane at a concentration of 10 mg/50 ml. The wax residue is separated
at this point. This wax residue can also be a source of other useful compounds
such as squalene. The extract is then dried under a stream of nitrogen. This was
then followed by further exhaustive extraction with a mixture of chloroform and
methanol at equal volume ratios for 2 weeks with the solvent changed daily. The
residue is then transesterified with 1 N methanolic HCL for 2 h at 80 °C. Following
transesterification-saturated sodium chloride and dotriacontane were added followed
by subsequent triple extraction with hexane (Borisjuk et al. 2018).
In a method patented in 2015 (Cigognini et al. 2015), used for extraction of cutin
from tomato peels, a less laborious process using much less solvent is presented.
However, no work is yet presented which uses this method for cutin extraction from
aquatic plants. It is assumed that the same method is applicable to extract cutin from
aquatic plant leaves. The extraction is achieved with sodium hydroxide, heating and
centrifugation as follows. It can be assumed that since the cutin from both aquatic
and terrestrial plants has the same chemical components, similar methods can be
employed in their extraction since the general goal of the extraction is the isolation
of cutin from the other components of the cuticle through dissolution and partial
degradation followed by precipitation.
In the said method, tomato peels are treated in sodium hydroxide at a concentration
of 0.75 M (patent claim states that anywhere between 0.5 and 6 M can be used) and
a temperature of 100 °C or anywhere between 65 and 130 °C for a period of 2 h with
an acceptable time period range between 15 min and 6 h. The liquid alkali solution is
added at a solid to liquid alkali ratio of 1:100 tomato peels: liquid. The mass is then
filtered with sieve, and the solid residue is discarded while the brown liquid fraction
340 15 Polyesters
Fig. 15.3 Flowchart Aquatic plant biomass

showing process for
extraction of cutin from plant
biomass NaOH extract 100oC 2 hrs
Solid Filtration
Filtrate
Precipitation pH 5
Centrifugation 10,000 rpm

Supernatant
15 mins
Solid
Purification
Cutin
is kept for further processing. The next stage involves addition of hydrochloric acid
until the pH reaches between 5 and 6. This is achievable at 5% volume HCl of total
volume at a concentration of about 6 M. The acidification is required in order to
precipitate the dissolved cutaneous compounds. This is then centrifuged at an rpm
of 10,000–14,000 for 15–20 min. The liquid part is discarded while the solid residue
is repeatedly washed and centrifuged up to three times to finally obtain raw cutin.
This method of extraction is summarized in Fig. 15.3.
Using this method, the obtained cutin is made up of mostly a mixture of cutin
oligomers and monomers with a yield of 60–80%. The molecular weight of the
cutin extracted according to GC-MS analysis is 650 g/mol thus indicating a pres-
ence of mostly dimers and trimers as the molecular weight of cutin monomer,
10,16-dihydroxyhexadecanoic acid and a C16 hydroxy fatty acid (C16 H32 O3 ) is
284 g/mol. Advantage of this process is the ready availability of sodium hydroxide
and hydrochloric acid, compared to the other extraction processes which require use
of expensive solvents which also have a negative impact on health and environment.
15.6 Environmental Implication
The environmental impact of cutin stems from the harvesting of the cutin containing
plants to the process of extraction and purification. Here, we take into considera-
tion, the patented method by Cigognini et al. (2015) which requires use of acid and
alkali. It is assumed that the same process is applicable to cutin from aquatic plants.
Consumptions for the extraction of cutin are estimated in Table 15.1.

Consumptions Amount
during cutin extraction
Sodium hydroxide 0.7 M, 100 ml/g cuticle
HCl 6 M, 5 ml/g cuticle
Water >100 ml/g cuticle
Solid waste 1.5 g per gram cutin
Energy
Heating 10,000 rpm for 15 min
Centrifugation 100 °C for 2 h
15.6.1 Use of Sodium Hydroxide
The process uses sodium hydroxide at a concentration of 0.7 M and for every gram of
plant cuticle mass 100 ml of 0.7 M solution of sodium hydroxide is used. The sodium
hydroxide does not take part in the reaction; rather, it acts as a catalyst providing
the alkaline environment required for the process. Much of this is neutralized during
the acidification of the liquid filtrate after the alkali treatment in the first stage of the
extraction. However, the solid mass which is discarded remains alkali, and this will
require neutralization before it is fed into the recycle stream to the feed or added to
the next batch in a batch process. If the solid is discarded, more water will be required
to neutralize it before it is safely released to the environment. Although this is not
water directly used in the process of extraction of the cutin, it adds to the wastewater
generated and water consumption during the process of producing cutin.
Following acidification, the liquid filtrate after centrifugation can also be sent
back to the feed to extract any remaining cutin which might still be present. How-
ever, because of the alkali condition required for extraction, this higher pH allows
the precipitation of the cutin components. This filtrate which has previously been
acidified to a pH of ~5 now needs to be returned to a much higher pH of ~10, thus
requiring additional sodium hydroxide to be used. The producer must then compare
the cost of recycling to discarding. This will vary from place to place.
15.6.2 Use of Hydrochloric Acid
Hydrochloric acid is used in the acidification stage to precipitate the crude cutin
extracted using the thermal alkali treatment in the first stage. The hydrochloric acid
used makes up 5% of the liquid volume at a concentration of 6 M. Much of this
goes into neutralization reaction, and the rest goes toward reducing the pH from 10
to pH 5–6. The solid residue following centrifugation needs to then be washed until
neutral. This results in further water consumption. Part of this acidic wash water
could be used to neutralize the water used in the washing of the discarded alkali solid
residue from the first stage after alkali treatment. This mixing of high and low pH
342 15 Polyesters
streams could minimize use of further acid/alkali for neutralization prior to further
wastewater treatment and then released into the environment. The use of sodium
hydroxide and hydrochloric acid replaces the use of chloroform and organic solvents
such as hexane and methanol. Since no volatile solvents are used in the patented
method, the acids and alkali used can be treated at the wastewater treatment.
15.6.3 Wastewater Generated
Most of the water used in the process eventually becomes wastewater since the final
extract is in the solid form. The rest is evaporated during the drying process. The acids
and bases used during the extraction are all dissolved in water. A liquid to solids ratio
of 100:1 is used in the first extraction stage using high-temperature alkali treatment.
For every gram of cuticle, 100 ml of water is required. Most of the water used here
can be recycled back into the feed stream or fed in with the next batch. More water is
used in the washing of the solid residue to obtain the final product. Additional water
used in washing the waste solid residues is also directed to the wastewater stream.
The water could either be treated on site or sent to the wastewater treatment plant.
Most industrial laws require the acidic and alkali wastewater to be neutralized and
not simply diluted. The level of treatment required will depend on the country of
operations and the local laws which exist there.
The cuticle is made up of 40–80% cutin, and the rest of the cuticle comprises of
the waxes and other components of the cuticular matrix such as cinnamic acid, but
mainly waxes. The wax is made up of mostly aliphatic molecules such as ketones,
long-chain fatty acids, primary alcohols, aldehydes and ketones. These are more
soluble in acidic conditions and hence can be filtered off following acidification
while the crude cutin precipitates as a gel-like solid form (Borisjuk et al. 2018;
Cigognini et al. 2015). The plant biomass also contains other valuable biopolymers
such as starch and protein. The cuticle can be first removed, and the rest of the plant
further processed for extraction of the other biopolymers.
15.6.5 Aquatic Plant Harvesting
Cultivation of aquatic plants such as duckweed has benefits for the environment.
They are utilized in the bioremediation of wastewater due to their ability to take
up nutrients such as nitrogen and phosphate compounds from the water. Beyond
acting as a feedstock for cutin, the process of extraction of cutin also yields waxes
and essential fatty acids as by-products which have high commercial value for their
bioactivities and other applications as discussed within the chapter. Having a mixed
culture of aquatic plants could be beneficial in minimizing the risk of disease spread
and allowing for biodiversity. The monomer composition of the cutin polymer varies
for different species and plant parts; therefore, mixed cultivation of variety of species
may cause some constraints. This is due to the variation in the monomer composition
resulting in the variation in the mechanical properties of the cutin.
15.7 Applications
Much of the potential and existing application of cutin lies in its protective barrier
formation. This makes it potentially applicable in packaging for food and pharma-
ceutical products. The cutin matrix also contains other compounds which are of com-
mercial significance. This section therefore also includes the potential applications
of these compounds in the cutin matrix.
15.7.1 Sunscreen
The cuticular matrix offers protection for the plant against UV radiation, and the
phenolic compounds within the cutin matrix also provide UV attenuation. Cutin can
be extracted from the plant and used in applications which mimic the UV protection.
The waterproofing role of the cuticle could also serve in retaining skin moisture which
also minimizes sun damage to skin. This has been explored by researchers (Zhang
and Uyama 2016; Heredia-Guerrero et al. 2017a, b). The sunscreen market is a huge
one, and as consumers increasingly demand more “natural” products, plant-based
sunscreens are in demand and cutin-based sunscreens could offer an alternative.
15.7.2 Food Packaging and Additives
The food packaging industry is one where a lot of biopolymers find demand. There
is an increasing demand for not just food component but also the packaging within
which the food is enclosed, to be plant based and/or from an environmentally friendly
source. The plant role serves a similar role as is required of food packaging, to protect
the product encased within it from the environment. Nutrient retention property of
the cutin matrix has potential for application in the food industry in for example
retaining nutrients in animal or fish feed (Cheng and Stomp 2009). The application
of cutin in the food industry is based around replicating the roles cutin/cuticle plays
in nature.
344 15 Polyesters
Cutin contains some essential fatty acids which could form extracts for human feed
supplements. Phytosterols, also found in the cutin matrix, have been FDA approved
for use as anticholesterol. Phytosterols act by competing with cholesterol for active
sites for absorption into the blood stream, thereby lowering the blood cholesterol
level.
Cutin has also shown applicability in food as packaging films. Edible films with
hydrophobic properties have been produced using cutin extracted from tomatoes
(Manrich et al. 2017). At a ratio of 50/50 by eight cutin/pectin and high pH in the
alkali range, well-dispersed films of cutin-pectin blend with good water repellency
and physical properties that match those of tomato peels were achieved. The films
remained stable at different pH and showed good thermal stability. This biomimicking
cutin-based edible film has potentials in replacement of fossil-based food packaging
films. The chemical composition of cutin allows it to play protective as well as
bioactive role as edible food packaging. It is also important that such films could be
produced using the conventional low cost, reproducible film casting methods used
in industry.
Cutin can also be applied as a coating on metallic food packaging. Such protective
coating separates the metal compound from the food. This could potentially replace
bisphenol A diglycidyl ether (BADGE) resin which is presently being used for this
application. BADGE has potentially harmful effects on human health (Montanari
et al. 2014).
15.7.3 Cosmetics
The by-products of cutin extraction from the wax fraction contain squalene. These
could be further explored for production of sunscreens. The transesterification of the
fatty acids from the cutin matrix yields glycerol as a by-product. Glycerol is used in
the cosmetics industry as a skin care product. Palmitic acid is also found in the cutin
matrix in considerable amount, making up almost 95% of the fatty acids in the wax
fraction. Palmitic acid is used in cosmetics as emulsifier and in soap production. The
main source of palmitic acid at present is palm oil, a plant which only grows in a
few countries in the world, the largest producer being Indonesia and Malaysia being
second followed by countries in Western and Central Africa as well as Colombia and
Thailand (Poku 2002). An alternative source of palmitic acid using a plant source
that pretty much grows everywhere will have significant impacts on the cosmetic
market.
15.7.4 Pharmaceutical
As it is applicable in food, cutin has potential for packaging in pharmaceutical prod-

ucts. The protective edible lipophilic coating property could be applied for cap-
sules and tablets and other applications which require a biodegradable protective
hydrophobic layer. The other components of the cutin matrix also have some poten-
tial pharmaceutical applications. The cutin matrix contains palmitic acid as well as
a variety of other fatty acids, some of which have bioactive properties. One of such
is linolenic acid. Linolenic acid, a monomer of cutin, has been shown to have antiin-
flammatory effect with potential to alleviate chronic migraine based on results on a
45-year-old human female patient (Santos and Weaver 2018). The study showed that
when linolenic acid is applied to the base of the neck, it exhibits some prophylactic
effect against chronic migraine. Topically applied treatments for chronic migraine
are particularly preferred due to the convenience and the reduced side effects such
as vomiting which is experienced in some people when using orally administered
medications. This is one of the many potential applications of linolenic acid which
is a monomer from cutin.
Linolenic acid has also been shown to have some anticancer effect, and it also
has good potential for application in the treatment of osteoporosis, cardiovascular
diseases, antioxidant effect and some neuroprotective properties. However, some of
these are still being researched and are inconclusive.
15.7.5 Biomimicry of Cutin Matrix for Wastewater Treatment
The bioremediation properties of plant source of cutin matrix could be extracted or

mimicked in wastewater treatment (Ziegler et al. 2016; Zhou et al. 2018). Although
this is not a direct impact or application of the aquatic biopolymer, the process of
cultivating these plants for extraction of cutin could in the process be applied to treat
contaminated water prior to them being harvested for cutin production. Aquatic plants
can either be grown wild or cultivated using aquaculture. Some aquaculture facilities
are connected to seawater. Where the water comes in from the sea gets filtered and
treated before being released to the plants. The water is then sent back out. This is
mainly done to provide a controlled environment for cultivating aquatic plants. Such
facilities could be extended to incorporate wastewater treatment by aquatic plants
alongside cultivation of the plants for cutin extraction.
15.7.6 Biodiesel Production
Biodiesel production from triglycerides comes with one disadvantage of most triglyc-
erides are used as foods. Furthermore, the transesterification reaction results in the
formation of glycerol which needs to be separated from the main product. The fatty
acids from the cutin matrix could serve as a source of biodiesel. Studies by Borisjuk
et al. (2018) found that the wax fraction of the cutin matrix contained 95% fatty acids.
These fatty acids can be transesterified with alcohols such as methanol and ethanol
to produce biodiesel using appropriate catalysts. This will depend on obtaining a
346 15 Polyesters
suitable method which is economical and obtains free fatty acids which are suit-
able for biofuel production. Experimental attempts have been made at esterification
reactions of free fatty acids with methanol using 98% sulfuric acid as homogeneous
catalyst (Javidialesaadi and Raeissi 2013). This is particularly significant as it acts as
a source of non-edible oil for biodiesel production such that there is no competition
with oils used in food. Other studies have investigated the use of other catalysts such
as biological catalysts saccharomyces cerevisiae, ferric sulfate impregnated carbon
from waste tyres, as for production of biodiesel from fatty acids (Yongjin et al. 2014;
Hood et al. 2018). Cutin production could therefore yield fatty acids which could be
used in production of biodiesel.
15.7.7 Biopolyester Production
Another approach to apply cutin is the esterification of its monomers, dimers and
trimers into polyesters through the condensation reaction. The key to this is using
the right reaction catalysts, conditions and solvents/disperse phase. This has been
successfully carried out to the point of polyester oligomers (Gómez-Patiño et al.
2013). Using lipases as catalyst and carrying out the polymerization reaction at
low temperature using organic solvents as dispersions, short-chain polyesters were
obtained. Although these oligomers lack the desired properties to be applicable as
polyesters, such process could serve as precursors to further polymerization reactions
to obtain longer chain polyesters (Gómez-Patiño et al. 2015). Some polymerization
reactions are carried out in two stages where short-chain oligomers are obtained in
the first stage followed by a second stage to obtain a longer chain polymer. This
could be for reasons such as controlling viscosity to avoid hot spots and allow proper
mixing as viscosity increases with increasing molecular weight.
Although there exists no known commercial production of cutin in the market to date.
There is however rising research interest in the potentials for cutin from duckweed.
As a fast-growing plant with bioremediation properties, duckweed has been a plant
of interest and much of it is being grown and studied.
One of the better-explored plant polyesters is suberin found in terrestrial plants.
Suberin is commercially exploited in the production of cork, most commonly in the
production of wine stoppers. It is also used as stoppers in storage of some other
products and chemicals. The monomers of suberin are also used in several applica-
tions. There is rarely traceable amount of suberin in aquatic plants, and therefore,
it will not be considered as an aquatic biopolymer at this point. Nonetheless, the
existing applications of suberin serve as a guide to the potential applications of cutin
as research in this area advances.
15.9 Conclusion 347
15.9 Conclusion
The main polyester which can be sourced from the aquatic environment is cutin.
It is obtainable from aquatic plants. Aquatic plants have the advantage of a higher
productivity than terrestrial plants and therefore have potential as a more available
source of cutin. This biopolyester is one of the lesser explore biopolymers available
in the aquatic environment, and however, as discussed in the chapter, research studies
have explored applications of cutin in a range of applications such as packaging films
and UV sunscreens. Duckweed is one of the aquatic plants which have been explored
for cutin production with reliable availability globally.
References
Angst G, Heinrich L, Kogel-Knabner I, Muelle CW (2016) The fate of cutin and suberin of decaying
leaves, needles and roots—inferences from the initial decomposition of bound fatty acids. Org
Geochem 95:81–92
Borisjuk N, Peterson AA, Lv J, Qu G, Luo Q, Shi L, Chen G, Kishchenko O, Zhuo Y, Shi J (2018)
Structural and biochemical properties of duckweed surface cuticle. Front Chem. https://doi.org/
10.3389/fchem.2018.00317
Cheng JJ, Stomp AM (2009) Growing duckweed to recover nutrients from wastewaters and for
production of fuel ethanol and animal feed. CLEAN–Soil Air Water 37:17–26
Cifarelli A, Cigognini I, Bolzoni L, Montanari A (2016) Cutin isolated from tomato processing
by-products extraction methods and characterization. In: Proceedings of CYPRUS 2016 4th
international conference on sustainable solid waste management, pp 1–20
Cigognini IM, Montanari A, De la Torre Carreras R, Montserrat CB (2015) Extraction method of
a polyester polymer or cutin from the wasted tomato peels and polyester polymer so extracted.
WO2015/028299 A1
De Vries H, Bredemeijer G, Heinen W (1967) The decay of cutin and cuticular components by soil
microorganisms in their natural environment. Acta Bot Neerl 16:102–110
ment goals. Rome. Licence: CC BY-NC-SA 3.0 IGO
FAO, IFAD, UNICEF, WFP and WHO (2018) The state of food security and nutrition in the
World 2018. Building climate resilience for food security and nutrition. Rome, FAO. Licence:
CC BY-NC-SA 3.0 IGO
Gómez-Patiño MB, Cassani J, Jaramillo-Flores ME, Zepeda-Vallejo LG, Sandoval G, Jimenez-
Estrada M, Arrieta-Baez D (2013) Oligomerization of 10,16-dihydroxyhexadecanoic acid and
methyl 10,16-dihydroxyhexadecanoate catalyzed by lipases. Molecules 18:9317–9333
Gómez-Patiño MB, Gutiérrez-Salgado DY, García-Hernández E, Mendez-Mendez JV,
Andraca Adame JA, Campos-Terán J, Arrieta-Baez D (2015) Polymerization of 10,16-
dihydroxyhexadecanoic acid, main monomer of tomato cuticle, using the lewis acidic ionic liquid
choline chloride·2ZnCl2 . Front Mater 2:1–9
Graca J (2015) Suberin: the biopolyester at the frontier of plants. Front Chem 3(62):1–11. https://
doi.org/10.3389/fchem.2015.00062
Graca J, Lamosa P (2010) Linear and branched poly(ω-hydroxyacid) esters in plant cutins. J Agric
Food Chem 58:9666–9674
Heredia A (2003) Biophysical and biochemical characteristics of cutin, a plant barrier biopolymer.
Biochim Biophys Acta (BBA) Gen Subj 1620:1–7
348 15 Polyesters
Heredia-Guerrero AJ, Heredia A, Dominguez E, Cingolani R, Bayer IS, Athanassiou A, Benitez

JJ (2017a) Cutin from agro-waste as a raw material for the production of bioplastics. J Exp Bot
68(19):5401–5410
Heredia-Guerrero JA, Benítez JJ, Cataldi P, Paul UC, Contardi M, Cingolani R, Bayer IS, Heredia
A, Athanassiou A (2017b) All-natural sustainable packaging materials inspired by plant cuticles.
Adv Sustain Syst 1:1600024
Hood ZD, Adhikari SP, Evans SF, Wang H, Li Y, Naskar AK, Chi M, Lachgar A, Paranthaman MP
(2018) Tyre-derived carbon for catalytic preparation of biofuels from feedstocks containing free
fatty acids. Carbon Resour Convers 1:165–173
Javidialesaadi A, Raeissi S (2013) Biodiesel production from high free fatty acid-content oils:
experimental investigation of the pretreatment step. Procedia APCBEE 5:474–478
Kolattukudy PE (2001a) Suberin from plants. In: Steinbtichel A, Doi Y (eds) Biopolymers. Wiley-
VCH Verlag GmbH, Weinheim, pp 41–73
Kolattukudy PE (2001b) Polyesters in higher plants. In: Babel W, Steinbüchel A (eds) Biopolyesters.
Springer Berlin Heidelberg, Berlin, Heidelberg, pp 1–49
Lam E, Appenroth KJ, Michael T, Mori K, Fakhoorian T (2014) Duckweed in bloom: the 2nd
international conference on duckweed research and applications heralds the return of a plant
model for plant biology. Plant Mol Biol 84:737–742. https://doi.org/10.1007/s11103-013-0162-9
Li Y, Beisson F, Koo AJ, Molina I, Pollard M, Ohlrogge J (2017) Identification of acyltransferases
required for cutin biosynthesis and production of cutin with suberin-like monomers. Proc Natl
Acad Sci 104:18339–18344
López-Casado G, Matas AJ, Domínguez E, Cuartero J, Heredia A (2007) Biomechanics of isolated
tomato (Solanum lycopersicum L.) fruit cuticles: the role of the cutin matrix and polysaccharides.
J Exp Bot 58:3875–3883
Manrich A, Moreira FKV, Otoni CG, Lorevice MV, Martins MA, Mattoso LHC (2017) Hydrophobic
edible films made up of cutin and pectin. Carbohyd Polym 164:83–91
Montanari A, Bolzoni L, Cigognini IM, de la Torre Carreras R (2014) Tomato bio-based lacquer
for sustainable metal packaging. Agro Food Ind Hi Tech 25:50–54
Poku K (2002) Small scale palm oil processing in Africa. In: FAO agricultural service bulletin, vol
148. Rome. ISSN 1010-1365
Santos C, Weaver DF (2018) Topically applied linoleic/linolenic acid for chronic migraine. J Clin
Neurosci 58:200–201
Yeats TH, Buda GJ, Wang Z, Chehanovsky N, Moyle LC, Jetter R, Schaffer AA, Rose JKC (2012)
The fruit cuticles of wild tomato species exhibit architectural and chemical diversity, providing
a new model for studying the evolution of cuticle function. Plant J 69:655–666
Yongjin JZ, Bujis NA, Siewers V, Nielson J (2014) Fatty acid-derived biofuels and chemicals
production in Saccharomyces cerevisiae. Front Bioeng Biotechnol 2:32
Zhang B, Uyama H (2016) Biomimic plant cuticle from hyperbranched poly(ricinoleic acid) and
cellulose film. ACS Sustain Chem Eng 4:363–369
Zhou Y, Chen G, Peterson A, Zha X, Cheng J, Li S et al (2018) Biodiversity of duckweeds in
Eastern China and their potential for bioremediation of industrial and municipal wastewater. J
Geosci Environ Prot 6:108–116. https://doi.org/10.4236/gep.2018.63010
Ziegler P, Sree KS, Appenroth K-J (2016) Duckweeds for water remediation and toxicity testing.
Toxicol Environ Chem 98:1127–1154. https://doi.org/10.1080/02772248.2015.1094701
Chapter 16
Others Aquatic Biopolymers
Abstract In this section, we discuss other polymers which are not covered in sep-
arate chapters for different reasons. Some of these biopolymers are less explored
compared, some are more abundant in terrestrial plants and some are relatively newly
discovered. Nonetheless, they are biopolymers present in the aquatic environment
and have significance in different ways.
Keywords Bioluminescence · Biofluorescence · Phlorotannins · Lignin · Pectin ·

Mucin
16.1 Phlorotannins
These polymers are more commonly found in the Eckloma species of brown algae
such as Ecklonia stolonifera and Ecklonia cava. They are made up of phloroglucinol
repeating unit, and their molecular weight ranges between 126 Da and 650 kDa.
Phlorotannins can also be produced synthetically through the polymerization of
phloroglucinol (Seca and Pinto 2018). They are of much pharmaceutical interest
for their antihypertensive properties. For example, studies carried out on labora-
tory rats administered with oral dosage at 20 mg per kg of body weight show that
6,6 -bieckol phlorotannins act as ACE I inhibitors which prevent vasoconstriction
(Ko et al. 2017). This is important in the management of high blood pressure and
other cardiovascular diseases. Different forms of this biopolymer exist, and they
vary based on their chain length as well as the side groups and linkage between the
phloroglucinol within the chain.
16.2 Bioluminescent Proteins
76% of aquatic organisms living below the abyssopelagic zone have the ability to
produce light. The property referred to as bioluminescence, biological light, is used
by these organisms for different reasons. Some use it for protection to hide from
predators, some for luring food and some for attracting a mate. Organisms which

350 16 Others Aquatic Biopolymers
possess bioluminescence include mollusks, copepods, shrimp, zooplankton and fish

among others.
While some of the organisms produce this light through chemical reactions within
their systems, others are able to create this light through symbiotic relationships with
other organisms which they host within their bodies, mainly bacteria. They then have
means through which they switch on and off the biological light. Some of this “living
light” as it is often referred to, are continuous while some have a frequency which
could range from a fraction of a second to every 10 s. There are fish known to produce
light which remains luminous even hours after the organism itself has died.
Bioluminescence has almost exclusively been observed in marine aquatic organ-
isms (also occurs in some terrestrial organisms such as the fireflies; Kricka et al.
2019), with the exception of one freshwater snail known as the Latia neritoides (Har-
vey 1952). The possibilities that there might be some more bioluminescent aquatic
organisms that are yet to be discovered do exist.
The main proteins responsible for bioluminescence in aquatic organisms are
luciferin and luciferase, the former being the substrate which takes part in the reac-
tion, while the latter is the enzyme which catalyzes the reactions. Other essential
components necessary for the production of biological light are oxygen, ATP and
ions particularly Ca2+ and Mg+ (Kricka et al. 2019; Brasier et al. 1989). The reactions
are written out in Eq. (16.1).
O2 ,C+ Luciferase
Luciferin + ATP −−−−→ Luciferin ATP −−−−−→ Oxyluciferin + Light
2
(16.1)
There are five types of luciferin, and the most common, particularly in marine
organisms, is the coelenterazine which is found in a wide range of organisms from
copepods to fish (Altun et al. 2008).
Humans have always tried to find a way to either harvest, harness or mimic some
features of some living organisms and find ways to use them to the benefit of the
human species. This does not exclude bioluminescence. In the most basic way, some
have been known to remove parts of a bioluminescent fish and use that as fishing bait
to lure prey (Ganeri 1999; Altun et al. 2008).
16.3 Biofluorescent Proteins
Biofluorescent is the ability of an organism to absorb one form of light and use that
to emit another form of light. This differs from bioluminescence in the sense that
light is required for this process unlike bioluminescence which is the ability of an
organism to make the light even in darkness.
Biofluorescent exists in a wide range of aquatic organisms such as fish, cnidar-
ians, mantis shrimps, ctenophores, amphioxus and copepods (Sparks et al. 2014).
In these aquatic organisms, their biofluorescence has mainly been attributed to the
16.3 Biofluorescent Proteins 351
green fluorescent protein (GFP), fatty-acid-binding fluorescent proteins and GFP-

like proteins and recently bromo-tryptophan kynurenines from catshark (Park et al.
2019). Cyanobacteria such as Spirulina produce phycobiliproteins which are used
to capture light needed for photosynthesis and they also are biofluorescent (Pagels
et al. 2019). The red algae Porphyridium also produces phycobiliproteins (Li et al.
2019). The main types of phycobiliproteins are phycocyanin, phycoerythrin, allo-
phycocyanin and phycoerythrocyanin. These phycobiliproteins have also been found
to have some bioactive properties such as anti-inflammatory, anticancer and antioxi-
dant activities (Pagels et al. 2019). Such that understanding the mechanisms by which
aquatic organisms fluorescence and the polymers which are used for it has lead to
development in other areas.
16.4 Xyloglucan
In most plants, the cell wall comprises cellulose microfibrils in a matrix of hemicel-
lulose and pectin. The hemicellulose is a complex mixture of different polymers. The
most abundant of these polymers is xyloglucan. It is a polymer with a repeating unit
of beta 1-4 D glucans with every sixth glucan replaced by an alpha-d-xylose. This
xylose could further be attached to other units such as fucose, arabinose and galactose
(Hayashi and Kaida 2011). The type of units attached to the xylose varies in different
plant species and affects the property of the xyloglucan. Therefore, xyloglucan from
different species can exhibit different properties such as solubility in water. Although
predominantly in plants, xyloglucan has also been reported to be present in green
algae cell wall (Domozych et al. 2010) although only lower amount compared to the
other cell wall polymers.
Xyloglucans show some gelation properties which have been investigated for
application in developing thermoresponsive drug delivery materials (Sakakibara et al.
2019). They play mainly load-bearing roles in the cell wall, and this is based on
their interaction with cellulose. This interaction with cellulose could be explored for
production of materials with desirable mechanical properties from xyloglucan and
cellulose composites.
16.5 Pectin
Pectin is an anionic polysaccharide with a relatively complex structure. Its polymer

structure comprises beta-1,4-d-galacturonic acid where some or all of the uronic acids
present in the chain form an ester linkage with a methoxy group. This is referred to
as the methoxyl content, and it affects the gel strength and gelling temperature of the
pectin.
The most well-known application of pectin is for its use as a gelling agent in the
production of jams. They also have biomedical applications such as drug delivery
vehicles, anti-inflammatory, wound healing and anticoagulant. It is mentioned in the

chapter on polyesters that the cuticle of the plant is linked to the epidermal wall by
layer of pectin such that its abundance correlates with that of cutin in plants. Aquatic
plants are the main source of pectin in the aquatic environment. The duckweed species
Spirodela polyrhiza, for example, contains up to 57% polysaccharide most of which
is pectin and xyloglucans (Sowinski et al. 2019) in their cell wall mass. Pectin is
also present in the cell walls of certain algae belonging to the Charophyceae taxa
(Domozych et al. 2012). This class of green algae is said to be the closest related
to terrestrial plants, and this is evidenced by the presence of a cell wall with similar
polymer composition as land plants.
16.6 Lignin
Lignin is a major evolutionary feature of plants. Terrestrial plants are believed to have
developed lignified cell walls as a means of adapting to survive on land (Boyce 2004).
It is therefore not expected to be needed in terrestrial plants. However, studies have
found lignin in aquatic plants (Chen et al. 2012; Thomas et al. 2009) albeit at a lower
amount compared to terrestrial plants. Lignins are complex aromatic heteropolymers
used by the plant to bind the cellulose microfibrils which are present in the cell wall,
and it also serves as a cross-linker for other cell wall components. This cross-linked
structure provides the stiffness and fortification within the xylem tissues in the plant
secondary cell walls that are needed for plants to have the erect structure.
Azolla has one of the highest lignin content at about 10%, while lignin content
in corn, an example terrestrial plant, is about 16% (Konda et al. 2015). A. pinnata,
a particular species of the Azolla plant, has up to 13.2% lignin (also contains 4.7%
starch, 12.8% cellulose and 10.1% hemicellulose). Duckweed has a lignin content of
12.2% (Yadav et al. 2017). The discovery of the presence of lignin in primitive green
algae and red algae strays from the accepted concept that lignin is an evolutionary
feature which allowed aquatic plants to evolve and adapt to terrestrial environment
(Martone et al. 2009).
16.7 Peptides from Frog Skin
Recent years have seen the increasing interest in the peptides within the skin of
some frog for their bioactive properties. These peptides include magainin-AM2,
tigerinin-1R and esculentin-2CHa. One of the most attractive bioactivities of these
frog skin-derived peptides is the antidiabetic activities. Peptides from frog skin can
stimulate insulin release, improve the sensitivity of the insulin, reduce the glucagon
concentration in the pancreas and the blood and also promote the proliferation of
the beta cells. It also has some lipid-lowering effects and promotes the expression
of genes coding for insulin secretion. These have been confirmed in in vivo studies
16.7 Peptides from Frog Skin 353
on mice (Conlon et al. 2018). Furthermore, recent studies have also shown that
the bioactivity of peptides from frog skin also extends to antimicrobial activity.
Peptides from frog skin have the ability to kill a range of human influenza virus strains
(Crowe 2017). However, these were based on studies carried out at the cellular level.
Peptides from the skin of frog also have potential to serve as antioxidant agents as
recent studies point to antioxidant activity in particular peptide called the antioxidant
peptide, antioxidant-I. These antioxidant proteins which were obtained from the
skin of the Physalaemus nattereri frog have also been shown to possess antioxidant
activity (Barbosa et al. 2018). This peptide also shows the ability to improve the
protective response to hypoxia (low oxygen levels). Other bioactivities which have
been recently detected in frog skin peptides include activity against cystic fibrosis
and cystic fibrosis-related MRSA (methicillin-resistant S. aureus) (Yuan et al. 2019).
Frog skins have been designed to protect the organism from external factors such
as temperature, moisture, UV light, pathogenic microbes and mechanical stress.
Much of these properties can be attributed to proteins present in the skin secretions
which serve this purpose. Although some live on land, frogs spend a substantial part
of their life in or around water, particularly at the tadpole stage, and therefore, they
are considered here as aquatic inhabitants.
Other polysaccharides present in aquatic plants include glucoronan present as
minor polysaccharides in sea lettuce (Kim et al. 2011; Lahaye and Robic 2007),
Rhamnus, arabinogalactans and mannans which are produced by green algae strains
Monostroma and Codium (Tabarsa et al. 2012; Kim et al. 2011; Li et al. 2015).
Cyanobacterium, the only known photosynthesizing eukaryotes, produces glycogen,
a storage carbohydrate (Chao and Bowen 1971; Yoo et al. 2007). Laminarin and
mannitol produced by brown algae have been explored for fermentation ethanol
production, and the microbes for such have been identified by Horn et al. (2000a, b).
16.8 Conclusion
Most of the polymers discussed in this section are either present in less abundance
to those that have been discussed in dedicated chapters in this book or they have
generated less commercial or research interest. Nonetheless, these contribute to the
diversity of biopolymers in the aquatic ecosystem. Even the polymers present in
small traces are of importance as they aid in expanding knowledge on the origins of
certain organisms.
References
Altun T, Celi F, Danabas D (2008) Bioluminescence in aquatic organisms. J Anim Vet Adv 7(7):841–
846
Barbosa EA, Oliveira A, Placido A, Soccodato R, Leite JRSA (2018) Structure and function of a
novel antioxidant peptide from the skin of tropical frogs. Free Radic Biol Med 115:68–79
Boyce CZ (2004) Evolution of xylem lignification. Proc Natl Acad Sci 101:17555–17558
Brasier AR, Tate JE, Habener JF (1989) Optimized use of the firefly luciferase assay as a reporter
gene in mammalian cell lines. Biotechniques 7(11):1116–1122
Chao L, Bowen CC (1971) Purification and properties of glycogen isolated from a blue-green alga,
Nostoc muscorum. J Bacteriol 105(1):331–338
Chen Q, Jin Y, Zhang G, Fang Y, Xiao Y, Zhao H (2012) Improving production of bioethanol from
duckweed (Landoltia punctata) by pectinase pretreatment. Energies 5:3019–3032
Conlon JM, Mechkarska M, Abdel-Wahab YH, Flatt PR (2018) Peptides from frog skin with
potential for development into agents for type 2 diabetes therapy. Peptides 100:275–281
Crowe JE (2017) Treating flu with skin of frogs. Immunity 46:517–518
Domozych DS, Sorensen I, Pettolino FA, Bacic A, Willats GTW (2010) The cell wall polymers of
the Charophycean green alga Chara corallina: immunobinding and biochemical screening. Int J
Plant Sci 171(4):345–361
Domozych DS, Ciancia M, Fangel JU, Mikkelsen MD, Ulvskov P, Willats GTW (2012) The cell
walls of green algae: a journey through evolution and diversity. Front Plant Sci 3:1–7
Ganeri A (1999) Creatures that glow. McClanahan Book Co., p 30
Harvey EN (1952) Bioluminescence. Academic Press, New York, p 649
Hayashi T, Kaida R (2011) Functions of xyloglucan in plant cells. Mol Plant 4(1):17–24
Horn SJ, Aasen IM, Østgaard K (2000a) Ethanol production from seaweed extract. J Ind Microbiol
Horn SJ, Aasen IM, Østgaard K (2000b) Production of ethanol from mannitol by Zymobacter
palmae. J Ind Microbiol Biotechnol 24(1):51–57
Kim JK, Cho MI, Karnjanaprakorn S, Shin IS, You SG (2011) In vitro and in vivo immunomod-
ulatory activity of sulfated polysaccharides from Enteromorpha prolifera. Int J Biol Macromol.
49:1051–1058
Ko S, Kang MC, Kang N, Kim H, Lee SH, Ahn G, Jung W, Jeon Y (2017) Effect of angiotensin
I-converting enzyme (ACE) inhibition and nitric oxide (NO) production of 6,6 -bieckol, a marine
algal polyphenol and its anti-hypertensive effect in spontaneously hypertensive rats. Process
Biochem 58:326–332
feasibility of macroalgae as a potential feedstock for biorefineries. Bioenergy Res 8:1046–1056
Kricka LJ, Smith ZM, Adcock JL, Barnett NW (2019) Bioluminescence. In: Encyclopedia of
analytical science, 3rd edn., pp 291–299
Lahaye M, Robic A (2007) Structure and functional properties of Ulvan, a polysaccharide from
green seaweeds, Biomacromolecules 8:1765–1774
Li N, Mao W, Yan M, Liu X, Xia Z, Wang S (2015) Structural characterization and anticoagulant
activity of sulfated polysaccharide from green alga Codium divaricatum. Carbohydrates Poly
121:175–182
Li T, Xu J, Wu H, Jiang P, Chen Z, Xiang W (2019) Growth and biochemical composition of
Porphyridium purpureum SCS-02 under different nitrogen concentrations. Mar Drugs 17(124):1–
16
Martone PT, Estevez JM, Lu F, Ruel K, Denny MW, Somerville C, Ralph J (2009) Discovery of
lignin in seaweed reveals convergent evolution of cell-wall architecture. Curr Biol 19(2):169–175
Pagels F, Guedes CA, Amaro MH, Kijoa A, Vasconcelos V (2019) Phycobiliproteins from
cyanobacteria: chemistry and biotechnological applications. Biotechnol Adv 37(3):422–443
References 355
Park HB, Lam CY, Gaffney JP, Weaver JC, Krivoshik SR, Hamchand R, Pieribone V, Gruber DF,
Crawford JM (2019) Bright green biofluorescence in sharks derives from bromo-kynurenine
metabolism. iScience (in press). https://doi.org/10.1016/j.isci.2019.07.019
Sakakibara NC, Sierakowski MR, Ramirez RR, Chassenieux C, Riegel-Vidotti I, de Freitas
RA (2019) Salt-induced thermal gelation of xyloglucan in aqueous media. Carbohyd Polym
223:115083
Seca AML, Pinto DCGA (2018) Overview on the antihypertensive and anti-obesity effects of
Sowinski EE, Gilbert S, Lam E, Carpita NC (2019) Linkage structure of cell-wall polysaccharides
from three duckweed species. Carbohydrate Poly 223(115119): 284–298
Sparks JS, Schelly RC, Smith WL, Davis MP, Tchernov D, Pieribone VA, Gruber DF (2014) A
covert world of fish biofluorescence: a phylogenetically widespread and phenotypically variable
phenomenon. PLoS One 9(1):1–9 e83259
Tabarsa M, Han JH, Kim CY, You SG (2012) Molecular characteristics and immunomodulatory
activities of water-soluble sulfated polysaccharides from Ulva pertusa. J Med Food 15:135–144
Thomas EK, Gao L, Huang Y (2009) A quantitative and qualitative comparison of aquatic and terres-
trial plant lignin phenols: critical information for paleoecological reconstructions. In: American
Geophysical Union fall meeting 2009
Yadav D, Barbora L, Bora D, Mitra S, Rangan L, Mahanta P (2017) An assessment of duckweed
as a potential lignocellulosic feedstock for biogas production. Int Biodeterior Biodegradation
119:253–259
Yoo S, Keppel C, Spalding M, Jane JL (2007) Effects of growth condition on the structure of glycogen
produced in cyanobacterium Synechocystis sp. PCC6803. Int J Biol Macromol 40(5):498–504
Yuan Y, Zai Y, Xi X, Ma C, Wang L, Zhou M, Shaw C, Chen T (2019) A novel membrane-disruptive
antimicrobial peptide from frog skin secretion against cystic fibrosis isolates and evaluation of
anti-MRSA effect using galleria mellonella model. Biochim Biophys Acta (BBA) Gen Subj
1863(5):849–856
Chapter 17
Future Perspectives
The aquatic ecosystem serves as host to a diverse range of organisms which produce a
diverse range of polymers. Due to the ubiquity of polymers, in the process of exploring
the different polymers in the aquatic ecosystem from raw materials, application to
final products, we have inevitable touched on different industries. These include
the polymers used in the medical field such as HIV microbicides to those used in
the energy industry in the production of the third-generation biofuels. We have also
covered the diverse chemistry of the different polymers and explored a range of
extraction processes. In the process, some current issues of the aquatic environment
are discussed, and we discuss possible solutions to addressing such. In addition to
serving as a source of biopolymers, understanding the role of the polymers within the
organisms from which they are sourced and the role in the aquatic environment has
also enhanced our understanding of certain biochemical process. For example, the
role of polymers allows organisms to survive in extreme environments and protection
against diseases.
Although the production rate of some of these biopolymers goes into thousands
of tonnes annually, this is still very low in comparison to the current production rate
of synthetic non-biodegradable plastics which are produced in hundreds of millions
of tonnes annually. Through disseminating knowledge and aiding the development
of understanding of the aquatic biopolymers, further interest will be generated in
commercial production of these biopolymers from aquatic resources, particularly
those which can be produced from waste resources or underutilized excess resources.
Extraction of biopolymers from aquatic resources should focus more on sustain-
able utilization of waste generated from fisheries’ industry with the goal of value
addition to fish resource and waste minimization rather than farming of aquatic
resources for the sole purpose of production of biopolymers. In other words, food
production from aquatic waste should be synergized with biopolymer production.
With valuable resources such as biopolymers being produced from aquatic resources,
the risk of over exploitation should be avoided as this then defeats the whole idea of
having a renewable source of polymers.
358 17 Future Perspectives
From the review of the environmental impact of the process for producing these
biopolymers, we see that many of these are not as benign as their end product.
Therefore, future industries need to focus efforts in ensuring the materials and energy
source used in the extraction, purification and packaging of these polymers which are
as sustainable as possible. Otherwise, the use of more fossil fuels and non-renewable
source of energy and materials in the production of renewable polymers is rather
counterproductive.
The allure of aquatic biopolymers is the third-generation polymers which do not
compete with humans and terrestrial life for land space, utilizes waste materials and
contributes to lowering the CO2 levels in the atmosphere. In addition to these, aquatic
biopolymers also serve as a source of polymers with more diverse chemistry thereby
opening doors of possibilities for a range of materials and bioactive compounds and at
the same time providing solutions to existing global problems such as food shortage,
plastic pollution and deterioration of the global aquatic ecosystem.
As our fossil source of polymers are continually depleting, the organisms of the
ocean, seas, rivers and lakes promise a renewable source of polymers which either
already do, or could potentially, play a significant role in human survival. The ideal
aquatic polymer resource will remove carbon dioxide from the atmosphere, take up
nutrients from the aquatic environment it grows in thereby cleaning wastewater, will
be completely renewable, require minimal amount of energy and chemicals to extract
and be as effective in use as the terrestrial- or fossil-derived polymers.
Table 17.1 gives a summary of the polymers which has been covered within this
book; their sources, applications and repeating units form their polymeric struc-
ture. The applications listed include both the existing applications and the potential
applications which are still in early stage research.
17 Future Perspectives 359
Table 17.1 Summary of aquatic polymers covered within this book

Polymer Source Applications Repeating unit
(existing/potential)
Cellulose Aquatic plants Textiles, biofuel, paper, Glucose
and algae nanomaterials
Chitin Crustaceans, fish Water treatment, Acetyl glucosamine
scales, fungi antimicrobial, packaging
films, biomedical sutures,
antioxidant, chelation,
paper, antiacne,
antiwrinkle
Alginate Brown algae Food additives, textile Mannuronic acid and
printing, tissue guluronic acid
engineering
Fucoidan Brown algae Anticancer, tissue Fucose, xylose, ribose
engineering,
antipathogenic,
anticoagulant,
antiinflammatory
Ulvan Green algae Immune modulation, Sulfated rhamnose and
antioxidant, anticancer, xylose
anticoagulant disaccharide/glucuronic
acid
Starch Aquatic plants Food thickener, biofuel, Glucose
and algae bioplastics
Laminarin Brown algae Anticancer, biofuel, Glucose
immunomodulatory
Polyester (cutin) Aquatic plants Bioplastics, sunscreen C16 and C18
agent ester-linked fatty acids
Agar Red algae Tissue culture, gelling Galactose and
agent l-galactopyranose
Carrageenan Red algae Gelling agent, Galactose and
toothpastes d-galactopyranose
Collagen Fish, jellyfish, Topical agents, tissue Amino acids
squids engineering, drug
delivery
Enzymes Fish viscera, Biofuel catalysts, food Amino acids
fungi, bacteria, processing
algae, aquatic
plants

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Springer Series on Polymer and Composite Materials

More information about this series at http://www.springer.com/series/13173

ISSN 2364-1878 ISSN 2364-1886 (electronic)

Lagos, Nigeria Ololade Olatunji

1 Introduction to Aquatic Biopolymers . . . . . . . . . . . . . . . . . . . . . . . 1

3.3 Chemistry of Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

5.4 Availability of Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . 101

6.7.3 Meat and Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

7.7.6 Antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

9.5 Extraction of Laminarin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

10.7 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

11.8.5 Fish Processing: Skin and Scale Removal . . . . . . . . . 254

13 Starch ......................... . .. . . . . . .. . . . . . . . . . . . . 287

14.6.5 Aquatic Plants and Algae Bloom . . . . . . . . . . . . . . . . 322

16.3 Bioﬂuorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

Polymers refer to macromolecules made up of repeating units of smaller molecules

Keywords Biopolymers · Marine · Freshwater · Littoral · Pelagic · Estuaries

© Springer International Publishing 2020 5

2.2 Types of Aquatic Ecosystems

Table 2.1 Classification of

Table 2.2 Classification of

Table 2.3 Different Lake zones

Table 2.4 Different zones of the ocean

2.2.2 Springs and Aquifers

2.2.3 Rivers and Streams

Rivers serve as a means of transportation, food, recreation and biodiversity. Large

A large standing body of water surrounded by land is referred to as a lake. Over a

2.2.8 The Coral Reefs

The coral reef is commonly referred to as an underwater city as it is made up of

2.3 Aquatic Organisms

2.3.1 Aquatic Plants

Fig. 2.5 An illustration of floating, submerged and emergent aquatic plant

Algae are polyphyletic; they are a group of photosynthesizing organisms which

(a) Water Surface

2.3.3 Aquatic Animals

2.3.4 Carnivorous Aquatic Plants

2.3.5 Aquatic Insects

2.3.6 Aquatic Birds

2.3.7 Aquatic Microorganisms

Since water serves as a source of food, means of transportation, source of energy,

2.4 Abiotic Components of the Aquatic Ecosystem

Kostakioti M, Hadjifrangiskou M, Hultgren SJ (2013) Bacteria biofilms: development, dispersal

Abstract Chitin occurs in a variety of organisms in aquatic ecosystems. It is a poly-

Keywords Chitin · Polymer · Chitosan · Crustaceans · Shrimps

© Springer International Publishing 2020 31

3.2 Occurrence in Nature

Chitin is a naturally occurring amino polysaccharide. It is a structural feature in the

Beta -1,3/1,6 Water

Alpha - 1,3 Alkali

Beta - 1,3 Alkali

Fig. 3.1 Occurrence of chitin within the mushroom structure

3.3 Chemistry of Chitin

The repeat unit of chitin is an acetylglucosamine structure linked by a beta 1-4

Fig. 3.2 Chitin structure showing the acetylated glucosamine unit

For broader applicability, it is important for a polymer to be moldable in order to be

Chitin exhibits polymorphisms in three different forms, α, β and γ. The α and β

3.3.4 Variation with Source

3.3.6 Molecular Weight

Another important reaction of chitin is the depolymerization into monomers. Chitin

3.4 Availability of Raw Materials