THE INSTITUTE OF CHEMICAL TECHNOLOGY, PRAGUE FACULTY OF FOOD AND BIOCHEMICAL TECHNOLOGY

DEPARTMENT OF FERMENTATION CHEMISTRY AND BIOENGINEERING

BACHELOR THESIS

Biosynthesis of alginate and its applications

Author:

Tereza Pikalová

Supervisor of the thesis: Prof. Ing. Karel Melzoch, CSc.

Consultant: Prof. Ing. Karel Melzoch, CSc.

Study programme: Food Technology and Biotechnology

Study field: Biochemistry and Biotechnology

Hereby I confirm, that in this bachelor thesis I have used only sources, that I cite and state in the list of literature. In Prague 6.6.2008 Signed

VYSOKÁ ŠKOLA CHEMICKO-TECHNOLOGICKÁ V PRAZE FAKULTA POTRAVINÁŘSKÉ A BIOCHEMICKÉ TECHNOLOGIE

ÚSTAV KVASNÉ CHEMIE A BIOINŽENÝRSTVÍ

BAKALÁŘSKÁ PRÁCE

Biosyntéza alginátu a jeho využití

Vypracovala:

Tereza Pikalová

Vedoucí bakalářské práce: Prof. Ing. Karel Melzoch, CSc.

Konzultant: Prof. Ing. Karel Melzoch, CSc.

Studijní program: Potravinářská a biochemická technologie

Studijní obor: Biochemie a biotechnologie Prohlašuji, že jsem v předložené bakalářské práci použila jen pramenů, které cituji a uvádím v seznamu použité literatury. V Praze dne 6.6.2008 podpis

Summary This thesis covers the topic of alginate biosynthesis and applications. Alginate is a naturally

occuring biopolymer from the group of hydrocolloids, having good gelling properties used in

such diverse commercial areas as the food industry, textile industry, paper industry, and high-

tech biomedical applications. Alginate is currently produced from brown algae

(Phaeophyceae) by a chemical process that results in alginic acid derivatives such as sodium

alginate, calcium alginate or propylene glycol alginate. The price of the product is currently

rising, especially due to high energy prices and the pollution of the sea environment where

the algae are harvested, which results in costly purification steps. Brown algae are the only

source of alginate that is utilized on a commercial scale. It has been noticed, however, that

certain bacteria of the genre Pseudomonas and Azotobacter also produce alginate as an

exopolysaccharide layer known as a biofilm, or cyst, respectively. These bacteria offer a new

source of quality alginate that can be produced by fermentation in controlled conditions

which can guarantee stable and predictable properties. Through genetic manipulation of the

bacteria, the product can be tailored to suit following applications. Although for general use

in food and other industries algal alginate is of sufficient quality, the biomedical applications

require ultra-pure alginate with well-defined properties, to ensure biocompatibility. For such

applications, the bacterial alginate could be a competitor to algal alginate especially

considering the recent market reviews on alginates.

Souhrn

Tato práce se věnuje tématu biosyntézy a využití alginátu. Alginát je v přírodě se vyskytující

biopolymer ze skupiny hydrokoloidů. Má dobré želírovací schopnosti, kterých se využívá v

mnoha různorodých odvětvích, kupříkladu v potravinářském průmyslu, textilním průmyslu,

papírenském průmyslu a v nejmodernějších medicínských aplikacích. V současnosti se

alginát vyrábí z hnědých řas (Phaeophyceae) chemickým procesem, jehož výsledkem jsou

deriváty kyseliny alginové jako například alginát sodný, alginát vápenatý nebo propylen

glykol alginát. Jediným zdrojem alginátu, který je komerčně dostupný na trhu, jsou hnědé

řasy. Cena alginátu stále roste, a to hlavně kvůli dlouhodobému znečištění mořského

prostředí, ze kterého jsou řasy sklízeny, což vede k nutnosti používat při produkci alginátu

dodatečné purifikační metody, a v poslední době také kvůli vysokým cenám energie. Bylo ale

zjištěno, že některé bakterie z rodu Pseudomonas a Azotobacter jsou také schopné

produkovat alginát, a to jako exopolysacharid, známý a popsaný jako biofilm resp. cysta.

Tyto bakterie se nabízejí jako nový zdroj kvalitního alginátu, který může být vyráběn

fermentací v kontrolovatelných podmínkách, které zajistí požadované vlastnosti výsledného

produktu. Pomocí genetických manipulací prováděných na bakteriích může být tento produkt

připraven na míru požadované aplikace. Ačkoli průmyslový alginát dosahuje dobré kvality,

pro medicínské využití je vyžadován alginát ultra-čistý s dobře definovanými stabilními

vlastnostmi, aby byla zajištěna biokompatibilita. Pro takové náročné aplikace by v budoucnu

mohl bakteriální alginát konkurovat alginátu z řas, zejména pokud uvážíme poslední

hodnocení trhu s alginátem a výhody bakteriálního alginátu ohledně jeho vlastností a

nezávislosti produkce.

Acknowledgment

I would like to express thanks to the staff of the laboratory of Molecular genetics and

biochemistry at The Norwegian University of Science and Technology in Trondheim,

especially to Helga Ertesvåg, who was kindly guiding me through my first steps in molecular

genetics and helped me with shaping the basis of the thesis.

I would also like to thank my thesis supervisor, Professor Karel Melzoch, for allowing me to

continue the work I brought from Norway and helping me to design the thesis.

At last, I would like to thank my english speaking friends, Aron and Nicolas, who greatly

helped me correcting and editing the thesis and supported me during the finishing steps.

List of contents

1 INTRODUCTION ........................................................................................................... 1 2 NATURAL SOURCES OF ALGINATE ........................................................................ 2 2.1 ALGINATE FROM ALGAE .......................................................................................... 2 2.1.1 Alginate processing from algae ................................................................................. 3 2.1.2 Processing of raw seaweed ........................................................................................ 5 2.1.3 The acid method ........................................................................................................ 5 2.1.4 The calcium chloride method .................................................................................... 5 2.2 ALGINATE FROM BACTERIA .................................................................................... 7 2.2.1 Azotobacter spp. ........................................................................................................ 7 2.2.2 Pseudomonas spp. ..................................................................................................... 8 3 STRUCTURE AND PROPERTIES OF ALGINATE..................................................... 9 3.1.1 Primary structure ....................................................................................................... 9 3.1.2 Secondary structure ................................................................................................... 9 3.1.3 Tertiary structure ..................................................................................................... 10 3.1.4 Structure of bacterial alginate ................................................................................. 12 3.1.5 Viscosity .................................................................................................................. 12 3.1.6 Gel forming ............................................................................................................. 13 3.1.7 Solubility ................................................................................................................. 13 3.1.8 pH stability .............................................................................................................. 13 3.1.9 Biocompatibility ...................................................................................................... 13 4 BIOSYNTHESIS OF ALGINATE ................................................................................ 15 4.1 Metabolic processing ..................................................................................................... 15 4.1.1 From carbon source to mannuronic acid ................................................................. 15 4.1.2 Polymerization ........................................................................................................ 16 4.1.3 Acetylation .............................................................................................................. 17 4.1.4 Epimerization .......................................................................................................... 18 4.1.5 Export ...................................................................................................................... 18 4.1.6 Lysis ........................................................................................................................ 18 4.2 Biosynthesis - control of alginate production in bacteria .............................................. 18 4.2.1 General rules for reactor set-up ............................................................................... 19 4.2.2 Optimalization steps ................................................................................................ 20 4.2.2.1 Simulating conditions in a biofilm .......................................................................... 20 4.2.2.2 Influence of high osmotic pressure.......................................................................... 20 4.2.2.3 Control of oxygen levels during the cultivation ...................................................... 21 4.2.2.4 Influence of shear rate on alginate production ....................................................... 23 5 APPLICATIONS OF ALGINATE ................................................................................ 24 5.1 FOOD INDUSTRY ....................................................................................................... 24 5.1.1 Advantages of using alginate in food products ....................................................... 24 5.1.2 Practical examples of alginate usage in food .......................................................... 24 5.2 MOLD-MAKING .......................................................................................................... 26 5.3 IMMOBILIZATION AND ENCAPSULATION .......................................................... 29 5.3.1 Cell encapsulation and cell therapy ......................................................................... 29 5.3.2 Soil bioremediation ................................................................................................. 31 5.3.3 Water treatment ....................................................................................................... 31 5.3.4 Biotechnological production ................................................................................... 32 5.4 WELDING ..................................................................................................................... 32 5.5 PAPER INDUSTRY ...................................................................................................... 32 5.6 COSMETICS ................................................................................................................. 33 6 ALGINATE MARKET ................................................................................................. 34

6.1 Current situation ............................................................................................................ 34 6.1.1 Market value of alginate .......................................................................................... 34 6.1.2 Price trends .............................................................................................................. 34 6.2 Future prospects of the alginate market ......................................................................... 35 6.2.1 Worth of the global alginate market ........................................................................ 35 6.2.2 Market and science .................................................................................................. 35 6.2.3 Bacterial alginates from a commercial perspective................................................. 35 7 Conclusion ..................................................................................................................... 36 8 Literature ........................................................................................................................ 38

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1 INTRODUCTION

In this work I will concern myself with the class of materials derived from alginate. Alginates

have been used since 1881 (lit.1), but only later, in the 1950's, the methods of biotechnology

were applied to optimize their properties, and the scope of applications has been widening

ever since. However, the properties of naturally produced alginate vary due to unstable

growth conditions of the algae, such as weather and nutrient availability. Therefore, we are

looking for a means of production of alginate with stable properties and properties optimized

for our application at hand.

The demand for alginate is continuously rising, especially due to high-tech applications.

Commercially, alginate is being produced solely from seaweed. However, in recent years, an

alternative has emerged: alginate produced by bacteria. Bacterial alginate resembles algal

alginate in structure and properties and has some other advantages. Production of bacterial

alginate has not yet proven economically competitive, though this may change as prices of

alginate continue to rise.

I will present a comparative study on algal alginate versus bacterial alginate, describe the

main issues of alginate production and applications, and attempt to predict the future of

alginates.

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2 NATURAL SOURCES OF ALGINATE

2.1 ALGINATE FROM ALGAE

Alginates were discovered and patented in 1881 by E.C.Stanford1 , and have since become an

important industrial product, which is obtained commercially by harvesting brown seaweeds

(Phaeophyceae) of the genera Ascophyllum, Durvillaea, Ecklonia, Laminaria, Lessonia,

Macrocystis and Sargassum. The seaweeds used for alginate production are also called

alginophytes. The natural habitats of seaweeds in Europe are the cold waters around France,

Norway, Scotland and Iceland. Worldwide the most important sources are in Australia,

California, Chile and India. In Japan Laminaria japonica is cultivated for food purposes and

the surplus is used in alginate production. In China cultivating on rope rafts has been

introduced in vast areas for the cheap mass production of low grade alginate2.

Figure 1: Map of natural habitats of alginate producing algae3.

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The biological function of alginate in brown algae appears to be a structure-forming

component, as the intercellular polymer gel matrix gives the plant mechanical strength and

flexibility similar to pectin in higher plants.

Figure 2: Lessonia trabeculata, held by John Sanderson, Kelco Co.; "scoubidou" - device for harvesting seaweed mounted on a boat4.

2.1.1 Alginate processing from algae

First, the algae must be harvested from the sea, which was historically done by collecting

seaweed from the shores by hand, or by cutting the seaweed at sea using a scythe. These

methods have a very low ecological impact, and have therefore been reintroduced in some

places. But most seaweed is today harvested by boats with cutting and mowing devices. In

Norway and in Ireland, the Ascophyllum nodosum and Laminaria hyperborea are being

harvested by cutting the whole plant but the last 25 cm, which is left for regrowth. In France a

whirling blade device called "scoubidou", which is mounted on a boat (Figure 2), is used to

cut and drag the Laminaria digitata seaweed from the sea4. Algae are usually locally

harvested and processed, whereas Durvillaea is harvested in Australia and imported to

Scotland. This is economically possible only due to its high alginate content (about 40%)4.

4

From the sea the seaweed is transported to processing factories, where sodium alginate and

other alginate products are produced, where acids and hydroxides are added to the algae.

These react with the raw alginate salts (calcium, sodium and magnesium) within the seaweed

cells. The whole process, as described below, can be seen in Figure 3.

Figure 3: Alginate production process - from raw seaweed to sodium alginate4.

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2.1.2 Processing of raw seaweed

The first step is mechanical preparation of the seaweed by cutting and milling. Next, a

solution of hot alkali, usually sodium carbonate, is added to the cut seaweed to extract

alginate from the cell walls of the algae as sodium alginate. This primary extract contains

water-insoluble particles, mainly cellulose, and must be purified. The particles can be

separated from the suspension by filtering, but because the suspended particles are very small

and would clog the filters, diatomaceous earth is added to facilitate the filtration5. Some

producers pump air through the suspension, in order to gather the impurities at the top of the

vessel. There the impurities are collected, and the clean solution is pumped into a cloth filter.

In the next step acid or calcium chloride is added.

If alginate of medical purity needs to be produced, it is necessary to go through some

additional purification steps. These include mechanical washing of the stems of the seaweed

and eliminating endotoxins and proteins by chemical and physical means, according to

various protocols 6.

2.1.3 The acid method

In this method, acid is added to the solution to make soft gel clumps of alginic acid. This gel

contains only 1-2% of alginate and the rest is water. To concentrate the alginate contents

centrifugation or flotation is used. Filtration cannot be used because the gel is too jelly-like

and would clot the filter. The concentrated gel usually contains about 7-8% of alginate. The

alginic acid is mixed with ethanol or isopropanol to a ratio of water:alcohol = 1:1.

Subsequently, sodium carbonate (or other suitable alkali) is added to neutralize the pH and a

paste of sodium alginate is formed. The paste is insoluble in the water-alcohol mixture, and

can be easily extracted, dried and milled into particles of required granularity7,8.

2.1.4 The calcium chloride method

To the primary sodium alginate solution calcium chloride is added, and a soft fibrous gel is

formed by a gentle mixing. The fibrous material is then sieved and washed with water to

remove excess calcium chloride. Then acid is added to the remaining fibrous calcium

alginate, which changes into alginic acid but remains in a fibrous state. These alginic acid

6

fibres are then mixed with an alkali and pressed to a paste, which is dried and milled into

required granularity. The calcium chloride method requires an additional step compared to

the acid method, but the fibrous gel is easier to handle than the colloid in the acid method.

The acid method also requires large amounts of alcohol, which is expensive. Other

compounds than sodium alginate can be used for neutralization (see Figure 4) depending on

the final product7,8.

Figure 4: Commercial salts and ester produced from alginic acid3. The choice of the production process can influence the viscosity and quality of the alginate

product. The decision must be based on the specific characteristics of the algae source. For

example, Macrocystis (Australian kelp) normally gives a medium-viscosity alginate, but can,

with a special extraction procedure, yield a high-viscosity alginate5. Laminaria digitata

(French kelp) gives a soft- to medium-strength gel, while Laminaria hyperborea (Nordic Sea

kelp) gives rigid gels4. The characteristics of the algae also vary over time. Therefore,

producers of alginate are always trying to keep algae from different sources in stock, which

can be mixed to compensate for this variation.

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2.2 ALGINATE FROM BACTERIA

So far, only two bacterial genera, Pseudomonas and Azotobacter, have been found to produce

alginate9. Both genera produce alginate as an exopolymeric polysaccharide during their

vegetative growth phase, but its biological function differs.

2.2.1 Azotobacter spp.

The aerobic nitrogen fixing soil bacterium Azotobacter vinelandii undergoes, in unfavourable

environmental conditions, a differentiation process leading to a desiccation- and radiation-

resistant cyst. In contrast to endospores, the cyst is not heat-resistant and not completely

dormant. The mature cysts are surrounded by two capsule-like layers (exine and intine,

Figure 5) each containing a high proportion of alginate in order to maintain structural

integrity. These layers also help to protect nitrogenase from oxygen during nitrogen fixation

in aerobic conditions10. Azotobacter chorococcum11 has been reported to produce alginate as

well.

Figure 5: Azotobacter vinelandii cyst, Ex-exine alginate containing layer, In-intine alginate containing layer, V-vesicular, PHB-polyhydroxybutyrate granules, CB-central body12.

8

2.2.2 Pseudomonas spp.

In Pseudomonas aeruginosa, one of the best characterized bacterial human pathogens, the

alginate seems to be an important virulence factor during the infectious process of human

epithelia. Infections of the respiratory tract with P. aeruginosa strains, which produce

copious quantities of alginate, are the major contributing factor causing high morbidity and

mortality in cystic fibrosis patients. P. aeruginosa infections are also common in patients

with severe burns or other extensive skin damage. The production of large quantities of

alginate results in the development of a characteristic mucoid phenotype and is often

associated with the formation of large bacterial conglomerates known as biofilms13. Other

Pseudomonades used in alginic production are the species P. putida, P. fluorescens, P.

syringae, and P. mendocina mucoid strains and mutants.

Figure 6: A photograph sequence of Pseudomonas strain S61 growing on glass slides in the form of biofilm. Attached cells can be seen after 3 hours of growth. After 5 hours the development of exopolysaccharide (alginate) is clearly seen, as marked by the arrowheads14.

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3 STRUCTURE AND PROPERTIES OF ALGINATE

1.1 STRUCTURE OF ALGINATE

3.1.1 Primary structure

Alginates are long, unbranched polymeric chains consisting of β-D-mannuronic acid (M) and

its C5 epimer α-L-guluronic acid (G). They are linked together by 1,4 glycosidic bonds15. The

difference between the two almost similar uronic acids is that they form different chair

conformations, such that the bulky carboxyl group is in equatorial position. The equatorial

position is energetically more favorable, consequently, the glycosidic bonds in a polymer

consisting only of β-D-mannuronate will be in equatorial position and in α-L-guluronate in

axial position. These axial bonds allow less flexibility16. Bacterial alginates are additionally

O-acetylated on the 2 and/or 3 hydroxyl group of the D-mannuronic acid residues. The count

of mannuronic and guluronic acid residues is usually presented as MG ratio. High MG ratio

means high mannuronic acid contents and vice versa.

Figure 7: Models of the structure of guluronic and mannuronic acid residues15.

3.1.2 Secondary structure

As to the chain structure, the monomers can be arranged in various sequences. These may be

homopolymeric, consisting only of M residues or G residues, or heteropolymeric consisting

of alternating sequences of M and G. Thus, the regions where M residues are dominant will

form a threefold helix, while the regions where G residues are dominant will form a twofold

helix16. It has been proposed that the structure of M homopolymers is further stabilised by the

formation of hydrogen bonds between the hydroxyl group at C3 in one ring with the ring of

oxygen of an adjacent residue. The G homopolymer structure is stabilised by a different set

10

of hydrogen bonds; the carboxyl residue bonds with the hydroxyl group at C3 on the prior

residue and at C3 of the subsequent residue (see Figure 8). Thus the G homopolymer is more

stable15.

Figure 8: Secondary structure of homopolymeric guluronic (G) and mannuronic (M) acid chains and heteropolymeric (GM) chain. The dashed line depicts the stabilizing bonds15.

Alternating GMGM heteropolymers have both axial-equatorial and equatorial-axial

glycosidic bonds, so the polymer takes a rather disorderly conformation. They are further

stabilised by hydrogen bonds between carboxyl group on mannuronate and C2 and C3

groups on guluronate. These intramolecular bonds give the heteropolymeric chain overall

greater flexibility than to the G homopolymeric chain.

3.1.3 Tertiary structure

In the presence of certain divalent or multivalent cations such as Ca2+, Mg2+, Sr2+ or Ba2+,

alginate readily forms a gel. This occurs because the H3O+ molecules bond to free carboxyl

groups of the M and G residues and are exchanged for the ions. The ions sit in pockets of

oxygen ligands from hydroxyls at C3 and C2 carboxyl groups and 1,4 O-linkages15. Such an

11

arrangement is very strong and stable. The poly-G zones then form an “egg-box”-like

conformation, polyguluronate being the “box” and Ca2+ ions being the “eggs”.

Figure 9: Ball-and-stick model of polymerized guluronic acid residues, linked with calcium ions, forming an "egg-box"-like conformation15.

Figure 10: Schematics of formation of a calcium alginate gel, ^^^^^^ represents poly –G blocks and ●●●●●● calcium ions16.

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3.1.4 Structure of bacterial alginate

The structure of bacterial alginate resembles the structure of algal alginate with one

exception, which is O-acetylation of mannuronic acid residues. Some of the D-mannuronate

residues are even O-acetylated in both the O-2 and O-3 positions46. The O-acetylation is no

problem if the alginate is to be commercially used, because the O-acetyl groups can be easily

removed in alkaline conditions44. The difference between Azotobacter sp. and Pseudomonas

sp. alginate is that the latter is free of consequent guluronic acid residues (GGG)17.

Table I: Comparison of alginates produced by different organisms; FG-guluronic acid relative contents, FM – mannuronic acid relative contents, FMG – MG ratio.

Fractional composition

Organism FG FM FMG Reference

Laminaria hyperborea (stipe) 0.70 0.30 0.10 lit.18

Azotobacter vinelandii TL 0.45 0.55 0.02 lit.17

Pseudomonas fluorescens (10255) 0.26 0.74 0.40 lit.17

1.2 PROPERTIES OF ALGINATE

The characteristics of alginic acid are a high viscosity in solution, ability to form gels in the

presence of certain divalent cations, adhesive and biocompatible properties and the ability to

entrap other materials in the gel. These characteristics are the keys to develop new industrial

materials.

3.1.5 Viscosity

Alginic acid behaves at low molecular weight like Newtonian liquid (velocity gradient is

directly proportional to the shear stress). With increasing degrees of polymerization and

calcium ion concentrations in the solution, it starts to behave like thixotropic liquid (the faster

it moves, the less viscous it becomes)19. This property can be used in preparation of non-drip

paints20.

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3.1.6 Gel forming

The main functional property of alginate is its ability to form water insoluble and

thermostable gels. Gelling depends on the affinity of ion binding (Mg2+ < Ca2+ < Sr2+ <

Ba2+)15. When producing an alginate gel, ions must be dispersed evenly, so that the gel is

homogeneous21. Generally a gel with a high G content will be stiff but brittle, and

thermostable but prone to syneresis (spontaneous separation of the solid and liquid part of the

gel) during freeze-thaw processes, while a gel with a high M content alginate will be softer

and more elastic, with good freeze-thaw behaviour15. However, at either low or very high

Ca2+ concentrations alginates with a high M content produce stronger gels. As long as the

average chain length is not particularly short, the gelling properties correlate with the average

G block length (optimum about 12) and not necessarily with the MG ratio which may be

primarily due to alternating MGMG chains, where the gel stiffness will be medium22.

3.1.7 Solubility

Alginic acid is soluble in water. However, after addition of cations it turns into a more or less

water insoluble gel. It dissolves very slowly in basic solutions of sodium carbonate, sodium

hydroxide, and trisodium phosphate. Solubility is thus dependent on pH, ionic strength and

the nature of the ions present. When considering the molecular weight (length of the chains),

lower molecular weight calcium alginate chains, with less than 500 residues, show higher

water binding properties15.

3.1.8 pH stability

Alginate is generally stable in the pH range 5-10, while maximum viscosity occurs between

pH 6-8. At a pH of about 3.5 the viscosity of alginic acid solution increases, and a so-called

acid gel is formed3. Such gel is less stiff, and is therefore used in production of soft food gels.

At very low pH, alginic acid precipitates due to increased intermolecular association.

3.1.9 Biocompatibility

Alginate, being a naturally occuring polymer, is non-toxic to the human body. As food

additive it has been approved by both FDA and EFSA, and as a biomaterial the trials show

great biocompatibility and biodegradability. One study23 suggested that the presence of

14

contaminants had a greater impact on the immunity response than the alginate composition

(MG ratio). The extent of contamination, however, appeared to be related to the alginate

composition; it was observed that the high M alginate presented a higher content in

polyphenol, endotoxins and proteins compared to the other alginates tested. Incompatibility

may cause slight skin allergies when alginate is used in creams or bandages, and

inflammatory reaction when internally transplanted in the form of microcapsules24. However,

such reactions are only temporary. Another threat is the accumulation of pollutants,

especially heavy metal cations, in areas where seaweed is produced25. Such polluted seaweed

can be purified, or alternatively, bacterial alginate can be used.

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4 BIOSYNTHESIS OF ALGINATE

4.1 METABOLIC PROCESSING

Two bacterial genra producing alginate are Pseudomonas and Azotobacter. In both

organisms, alginate is metabolized from mannuronic acid residues, then polymerized and

epimerized, while being transported through the outer membrane out of the cell, where it

forms a biofilm or a cyst. In algae, the pathway resembles in many aspects the bacterial

biosynthesis pathway 26.

Figure 11: Biosynthetic pathway of alginate in bacteria9

4.1.1 From carbon source to mannuronic acid

As a carbon source, pseudomonads are able to utilize hundreds of compounds, including

some polycyclic hydrocarbons toxic to humans27. The carbon nutrients run through Entner-

Doudorff (KDPG) pathway, citric acid cycle (TCA) and gluconeogenesis. After these steps,

the product fructose-6-phosphate undergoes isomerisation with the help of phosphomannose

isomerase28, into mannose-6-phosphate, which is converted to mannose-1-phospate by

phosphomannomutase. Mannose-1-phosphate is activated by GDP-mannose phosphorylase

which results in formation of GDP-mannose. GDP-mannose dehydrogenase drives the

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reaction forward by efficiently removing GDP-mannose from the pathway, oxidizing it to

GDP-mannuronic acid9. The process is schematically depicted in Figure 11.

4.1.2 Polymerization

Polymerization of GDP-mannuronic acid residues into polymannuronate occurs on the

cytoplasmic membrane and in the periplasm. Several proteins are participating in this

process. The function of the complex is to polymerize alpha-linked GDP-mannuronic

residues into a beta-linked product. The reaction is not yet fully understood, but it resembles

other polymerization reactions such as the forming of chitin or cellulose29, 30. The process is

illustrated in Figure 12. The nascent polymannuronate chain is modified as it transverses the

periplasm, the main three modification processes being acetylation, epimerization and lysis.

Figure 12: Proposed model of the putative multi-enzyme complex involved in alginate polymerization, modification and export. Mannuronic acid residues are formed in the cytoplasm, epimerized, polymerized and transported through the periplasm and the outer membrane to the cell exterior9.

17

4.1.3 Acetylation

The acetylation of mannuronic residues is characteristic for bacteria, and one of the

differences between algal and bacterial alginate. The acetylation of hydroxylic groups at C2

or C3 position of mannuronic residues (see Figure 13) prevents the nascent polymannuronate

chain from being degraded by the alginate lyase31. Furthermore, it blocks the mannuronic

residues from being epimerized into guluronic acid residues. The acetylation also enhances

the ability of the alginate to bind water molecules, which helps the bacteria to survive dry

periods (Azotobacter cyst). The figure below depicts a comparison of infra-red spectra of

three different alginates. It can be seen that the bacterial alginate is acetylated, while the algal

alginate is not.

Figure 13: Infrared spectrum of (a) exopolysaccharide produced by A. vinelandii NCIB 9068, (b) exopolysaccharide produced by P. aeruginosa, and (c) commercial algal sodium alginate. Spectra a) and b) contain additional peaks at 1250 cm-1 and 1720 cm-1, which correspond to absorption by O-acetyl ester32.

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4.1.4 Epimerization

Epimerization is the source of G residues in bacterial alginate. C5-mannuronate epimerase is

an epimerization enzyme in both Pseudomonas spp. and Azotobacter spp. The microorganism

utilizes a set of extracellular epimerases to adjust the G content according to its needs33.

Methods using bacterial epimerases in vitro show great promise in tailor-made biosynthesis

of alginate34. It has been discovered, that the seaweed Laminaria digitata is also utilizing a

set of extracellular epimerases26, resembling the epimerases of Azotobacter vinelandii.

4.1.5 Export

Finally, the mature alginate chain is exported from the periplasm out of the cell. The

responsible protein is a porin in the outer membrane, selective for the alginate chain35.

4.1.6 Lysis

Different alginate lyases cleave the glycosidic bonds between mannuronic and/or guluronic

residues. For the bacteria, this is a means of control of the length and molecular weight of the

polymer9 and cleaning of the periplasm from misguided alginate36. Modification of these

enzymes could lead to improvements of bacterial alginate production37. In cystic fibrosis

patients, the alginate lyase could be responsible for spreading the disease throughout the body

of the patient. The cleaving of the alginate helps to detach the biofilm containing bacteria38,

thus enabling the bacteria to travel in the bloodstream and infect new tissues.

4.2 BIOSYNTHESIS - CONTROL OF ALGINATE PRODUCTION IN BACTERIA

The biosynthesis is controlled by a genotypic switch consisting of sigma factor (sigma(22),

algU or algT) and Muc proteins. Mutations in any of the muc genes is associated with a

change from a non-mucoid to a mucoid phenotype39. The switch is operating in response to

both intra- and extracellular factors like osmotic pressure, oxygen level, nutrient availability,

water activity, temperature, shear stress, cell maturity and other. All these factors can be

utilized to optimize the production of alginate and will be discussed further.

19

4.2.1 General rules for reactor set-up

Alginate has been produced in both batch and continuous cultures in small scale reactors of

working volume up to 3 litres. Sucrose or glucose is used as a carbon source. Other general

nutritional conditions are described in the referred studies. Phosphate seems to be an

important factor: a lack of phosphate impairs the respiratory activities of the microorganisms,

with the consequence of maximizing alginate production40. In the case of A. vinelandii,

nitrogen limitation seems to increase alginate production in combination with controlled

oxygen levels31. Limitation by iron and/or molybdate gave the highest specific rates for

alginate production in one study42. Some results of the experiments with different growth

conditions are given in Table II.

Table II Alginic acid final concentration, yields (Yalg) and volumetric productivities (Qalg) of different Azotobacter strains in cultures with a controlled (Cont.) or uncontrolled (Uncont.) dissolved-oxygen concentration (pO2). NFM Nitrogen-free medium, NRM nitrogen-rich medium. References to the table data can be found in lit. 41.

Strain pO2 Medium Condition Alginate Yalg Qalg

(g l-1) (g g-1) (g l-1h-1) Mutant C-14 of NCIB 9068

Uncont. NFM Phosphate-rich 6.2 0.31 0.06

Mutant SM52B of NCIB 9068

Uncont. NFM Phosphate-limited 5.0 0.25 0.05

NCIB 9068 Uncont. NRM Phosphate- rich 5.8 0.145 0.053

DSM 576 Uncont. NRM Phosphate-limited 4.9 N/A 0.2

DSM 576 Cont (2%) NRM Phosphate-rich 3.0 0.15 0.09

DSMZ 93-541b Cont (2%) NFM Phosphate-limited 4.9 0.12 0.2

As alginic acid is formed, the pH of the medium is dropping, so automatic NaOH dosers are

used to buffer the pH at 7.0. The growth temperature does not affect the alginate yield and

properties as much as oxidative stress or shear rate, and recommendations vary according to

the microbial strain and other factors. The optimal growth temperature for P. mendocina is

29°C (lit.43). With P. aeruginosa, experiments have been run at 37°C, to simulate pathogenic

growth in human body44. As in other biotechnological processes, antifoaming agents, aeration

and even distribution of nutrients is necessary. The viscosity increase during the batch

process can be regulated by addition of alginate lyases that will lower the length of polymer

chains, thus lowering the viscosity of the medium. On the other hand, if we are interested in

20

producing high-viscosity alginates (high molecular mass) it may be advantageous to add

proteolytic enzymes or betonite to decrease the polymer degradation45. To ensure even

distribution of nutrients and oxygen, different aeration and mixing techniques are utilized46.

4.2.2 Optimalization steps

As can be seen from the preceding paragraphs, many different approaches can be pursued to

grow alginate producing bacteria. However, to ensure the highest yield, optimalization steps

should be considered. They resemble the natural conditions in which the organism has been

observed to accumulate alginate.

4.2.2.1 Simulating conditions in a biofilm

Pseudomonas spp. (mucoid strains) naturally cover themselves in a biofilm, within which the

conditions are much more favourable than in a floating plankton state. Alginate is produced

by Pseudomonas spp. as the main part of this biofilm. A polysaccharide matrix of alginate is

synthesized intensively already 15 minutes after adhesion of the bacteria to a surface. In a

biofilm, cells react differently on stimuli than cells cultivated in a liquid media47. Analysis of

proteins in cells showed that more porin proteins, transport proteins and alginate synthesis

proteins are synthesized when the bacteria is part of biofilm48. This leads to an increase in

alginate production. Thus, using a fermentor simulating natural environment, alginate

synthesis is enhanced. One study48 reached excellent results using this method, doubling the

yield of alginate compared to a planktonic grown population.

4.2.2.2 Influence of high osmotic pressure

When osmotic pressure is introduced upon a bacterial culture, the bacteria need to protect

themselves from cell rupture and lysis. This process is called osmoregulation.

Osmoregulation processes are manifold, such as exporting ions from the cell, accumulating

compatible solutes or building a protective alginate barrier49. Thus, exposing cell cultures to

high salt concentrations can lead to enhanced alginate production.

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4.2.2.3 Control of oxygen levels during the cultivation

This feature is significant especially for alginate producing Azotobacter strains. Azotobacter

vinelandii, being a rare aerobic nitrogen fixing soil bacterium, produce alginate in order to

protect an oxygen-sensitive nitrogenase. The production is related to the pO2 (percent of O2 in

the air), with a peak at a pO2 of 5% (see Figure 14).

Figure 14: Specific alginate production rate (qalg) of A. vinelandii as a function of O2 tension (pO2) in phosphate-limited continuous culture at different dilution rates (D)50.

22

As can be seen from Figure 14, the alginate layer will not thicken as the pO2, rises further.

Instead, the L-guluronic acid contents of the alginate will increase (Figure 15). Alginate

formed at a pO2 of 2.5% has a much lower L-guluronic acid content (45%) than alginate

formed at a pO2 of 20% (88% of L-guluronic acid)50. In the case of P. aeruginosa, oxidative

stress also influences the alginate production. Normally, P. aeruginosa lives in

microaerophilic conditions. If exposed to aerobic conditions, P. aeruginosa turns into a

mucoid phenotype and begins to produce alginate44. This is a means for P. aeruginosa to

protect itself from the phagocyte-derived reactive oxygen intermediates upon infection of

cystic fibrosis patients39.

Figure 15: Changes in the molecular weight (A) and the L-guluronic acid content (B) of alginate produced at different pO2 values50.

23

4.2.2.4 Influence of shear rate on alginate production

In a bioreactor shear rate lowers as the biomass is accumulated. Thus, the agitation speed

must be increased to maintain the shear rate constant. It has been shown, that the shear rate

affects both alginate yield and composition. The highest yield is at about 600rpm (Figure 16),

while the highest L-guluronic acid content is at 800 rpm (lit.50). Interestingly, this

corresponds with the phenomena observed in algae growing in strong sea currents. Such

algae have higher L-guluronic acid contents.

Figure 16: Effect of agitation speed on the biomass production and the concentration, yield, and composition (expressed as weight percentages of L-guluronic acid content) of alginate in a phosphate-limited chemostat culture50.

24

5 APPLICATIONS OF ALGINATE

5.1 FOOD INDUSTRY

The bulk of alginate is used in food industry. Both alginic acid and its salts are approved food

additives. Table III shows a summary of alginate based food additives.

Table III: Different alginate based food additives, their E-codes, names and functions51.

E-code Additive name Technological function

E 440 Alginic acid Thickener, stabilizer

E 401 Sodium alginate Thickener, stabilizer, gelling substance

E 402 Potassium alginate Thickener, stabilizer

E 403 Ammonium alginate Thickener, stabilizer

E 404 Calcium alginate Gelling substance, anti-foaming agent

E 405 Propane-1,2-diol alginate or

propylene glycol alginate (PGA) Thickener, emulsifier

5.1.1 Advantages of using alginate in food products

Developers of food products often select alginate as a key ingredient because it provides

several important functional attributes to food, and yet is extremely flexible in terms of

processing. Alginates are notable for providing viscosity, forming true gels without heating

and being thermally stable. They are also effective at both highly acidic and neutral pH

levels. Besides providing viscosity and forming gels, alginates are useful as suspending

agents, foam and emulsion stabilizers, for providing excellent adhering properties, and as

coagulating agents. A special type of alginate, propylene glycol alginate (PGA), is a true

emulsifying agent as well, and has exceptional acid stability3.

5.1.2 Practical examples of alginate usage in food

Alginic acid derivatives are used mostly as texture enhancers, especially thickeners,

emulsifiers and stabilizers for soups, creams, ice creams (to prevent crystallization), jams,

pastes, purees, dairy products as cheese spreads and flavored milk. In beverages production,

25

especially beer, but also fruit beverages, propylene glycol alginate (PGA) is added to enhance

foam stability3. In high oil content food such as salad dressing or mayonnaise, alginate can be

replace starch as stabilizer. This is especially suitable for dietary products, because the oil

contents can be lowered, while the product still keeps “oily” texture52.

Another interesting use of alginate is as edible protective coatings prolonging the shelf-life of

food products. Frozen fish such as mackerel and herring are protected from oxidative

deterioration by calcium alginate gel, the gel filling the air pockets between the fish. In

addition to prevent lipid oxidation, the elimination of air pockets in the boxes reduces

freezing time by 20–25% (lit.53).

Alginate is also widely used in production of restructured food, especially fruit and canned

food. When alginate is mixed with fruit puree and pH decreased, a firm gel is created, which

results in a nutritious food with increased values of fracturability, hardness, gumminess and

chewiness54.With addition of other substances, confectionery and food decoration products

can be made. In the 90's a culinary branch called “molecular gastronomy” evolved55, which is

nowadays becoming an acknowledged branch of science. It studies physical and chemical

processes of cooking and alginate has become one of their favored study objects.

Figure 17: Examples of usage of alginate in food. 1) a molecular gastronomy product94, 2) beverage containing propylene glycol alginate as foam stabilizer95, 3) a classic example – olives filled with restructured pimento pepper pieces95.

26

Another industrially important use of alginate is in animal fodder, for example canned pet

food or fish fodder56. In fish fodder pellets, alginate is used to form a coating to prevent

disintegration, dissolving or sedimentation of the unused food, which would be wasteful and

promote unwanted growth of aquatic vegetation.

5.2 MOLD-MAKING

Calcium alginate proved useful in making models of teeth in dental practice, limbs and other

body parts in prosthetics. In Figure 18 we can see molds of teeth, which have been taken

from the mouth of a patient and are ready to be filled with casting material to continue the

process of making a dental prosthesis.

Figure 18: Dental mold from alginate for preparation of teeth prosthesis57.

1.3 REACTIVE DYE PRINTING

Textile printing is an important method of decorating textile fabrics. The colouration is

achieved either with dyes or pigments in a printing paste. Printing pastes are viscous liquids

and thickeners provide the viscosity. The reactive dyes, formerly marketed under the

trademark Procion, are the major dye printing system for cellulose fibres. The dye undergoes

a chemical reaction with the cellulose fabric. Thus, the thickener has to be nonreactive with

both the fabric and the dye. Suitable candidates are starches, CMCs, and guar derivatives

and some synthetic derivatives of polyacrylic acid58. The problem with these thickeners is

the hydroxyl group, which often reacts with the dye thus lowering the colour yields. Sodium

alginate also contains hydroxyl groups, but the reaction between alginate and dye is limited

27

by mutual anion repulsion of the alginate's carboxyl groups and the dye's sulphonic acid

groups59. Also, alginate-based thickeners are easy to wash out from the fabric.

Figure 19: Example of textile printing results59. .

1.4 ANTACIDS

Antacids provide relief from the symptoms of heartburn by neutralising the stomach acid.

Alginate swells when in contact with stomach acid, creating a ‘raft’ which floats to the top of

the stomach, providing a barrier between the stomach and the oesophagus, thus preventing

acid refluxing into the oesophagus. An example is Gaviscon tablets60, which are usually

prescribed together with a conventional (non-alginic) antacid.

1.5 WOUND DRESSINGS AND BANDAGES

Wound dressings based on alginic material are well known for their superior proprieties

compared to standard bandages. If a fine jet of sodium alginate solution is forced into a

solution of calcium chloride, calcium alginate is formed as fibres. These fibres are then

woven into a bandage. Calcium alginate, being a natural haemostat, is applicable on bleeding

wounds. It helps the blood coagulate twice as fast as a traditional cloth bandage. Because the

calcium ions are slowly exchanged for sodium ions from the blood and skin, the dressing is

28

forming a soluble sodium alginate gel, thus providing a moist environment. Furthermore, the

dressing can be removed easily and reduces the pain experience of the patient when changing

the dressing. Calcium alginate dressings provide a significant improvement in healing split

skin graft donor sites61. As an alternative to standard alginate dressing, new alginate dressings

have also been on trial. Results indicate that alginate gel has good biocompatibility for

regenerating axon outgrowth. In combination with chitosan, alginate can produce a nerve

conduit or scaffold that helps the nerve cells to grow62. Such technique is a promising

alternative to conventional treatments for peripheral nerve repair. The alginate dressing can

be used clean or mixed with antimicrobial substances as silver, zinc, antibiotics, tea tree oil,

honey or other substances with healing effects.

Figure 20: Schematic representation of alginate dressing healing properties61.

29

5.3 IMMOBILIZATION AND ENCAPSULATION

The principle of encapsulation is that a cell, microorganism, protein or nucleic acid is

enclosed in an alginate bead, which acts as a semipermeable membrane, so that the

encapsulated body can communicate with the outer environment while still being protected

from degradation and other harmful effects.

5.3.1 Cell encapsulation and cell therapy

Cell therapy is currently being used in diabetes treatment, in a research project known under

the name The Chicago project63. Treatments for other illnesses like hemophilia64, some forms

of cancer65 and renal failure66 are being investigated. The cells can also be genetically

manipulated to recognize tumors and destroy them, or to produce antibodies or enzymes that

the organism is missing67.

Figure 21: Cell microencapsulation. a) Nutrients, oxygen and stimuli diffuse across the membrane, whereas antibodies and immune cells are excluded. b) Pre-vascularized solid support system to facilitate optimal nutrition of the enclosed cells68.

Encapsulated cells can be of various origins. They can come from the patient himself, in

which case they are subsequently modified and retransplanted, or from a healthy donor or

even a immunosuppressed cell culture. When the cells originate from a human, the method is

called allografting. In the case of a cell culture from an animal (for example rat pancreatic

islets) it is called xenografting68.

30

One major benefit of cell therapy is that the encapsulated protein-expressing cells may

deliver a steady (and potentially more "physiological") concentration of the protein69,

compared to direct injection. Other practical benefits can be an increase in the concentration

of the drug in a localized area, or just making it easier for the patient to administer the drug.

It's easier to get cells implanted once per year than to remember to take a pill or an injection

once per day.

Figure 22: An example of cell encapsulation. Ovarian follicle encapsulated in a calcium alginate bead. Scale bar represents 50 µm. Ovarian follicles produce follicle-stimulating hormone (FSH), regulating the development, growth, pubertal maturation, and reproductive processes of the human body. Cell therapy can help to provide FSH to patients with Kallmann syndrome, hypothalamic suppression or other FSH deficiency diseases70.

The beads are formed thanks to the gelling properties of alginate. A suspension of cells and

alginate is by various methods71,72 funneled through a thin capillary, usually covered with

Teflon to reduce adhesion. The microbeads formed in this way can be made of uniform size

(as small as 50 µm in diameter) and mass, which makes them suitable for medical purposes

due to increased biocompatibility.

The alginate can be modified prior to encapsulation by mannuronan-C5-epimerase33, an

31

enzyme altering the MG ratio. It is also possible to modify alginate beads by adding polymer

layers, usually of poly-L-lysine, thus forming an additional PLL-AG layer73. Also introducing

sodium ions into the media has shown promising influence on the smoothness of the surface

of the bead74.

Figure 23: Left: alginate beads funneled through a narrow channel; Right: alginate beads flowing in a reservoir75 .

Such additional steps may slow down production of the beads, but gives the possibility to

adjust the characteristics of the beads to suit the specific application. Each lab has its own

preferences and so far there are few standardized protocols of making alginate beads for

medical applications.

5.3.2 Soil bioremediation

Large areas all over the world are spoiled with toxic substances, such as organic pollutants or

heavy metals. Such an environment must be cleansed before it can provide good milieu for

agricultural production. The latest techniques include bioremediation by microorganisms.

Suitable microorganisms are often naturally occurring in the polluted environment and with

some (genetic) modification, new strains can be produced, which can degrade the pollutants

effectively or bind heavy metals76,77. For these microbial remediation methods to be efficient,

the bacteria must be resistant to high concentrations of the pollutant, be easy to handle and

have a long shelf-life for storage. These parameters can be improved by encapsulating the

bacteria in alginate beads78.

5.3.3 Water treatment

One basic technique in water treatment is flocculation, where contaminants clump together

and can be easily removed be sedimentation or flotation. This process is facilitated by

flocculating agents, such as activated silica or polyelectrolytes. But these agents can be

potentially toxic and cannot be used for cleaning for example drinking water79. Alginate,

being a natural polymer and binding positively charged compounds, is a good choice in water

treatment industry.

32

Alginate in the form of alginic acid has been reported to be an excellent flocculation agent for

metal ions. Different metal cations and commercial dyes adsorb to the -OH and -COOH

groups of alginic acid, and can subsequently be removed together with the alginate. One

study has shown that alginate in combination with chitosan significantly improves removal of

ions such as Cu2+, Pb2+, Ag+ and Cd2+, by facilitating separation of the coagulant from the

water80. Alginate, in the form of calcium alginate beads, treated with Fe(III) oxides, was

reported as a convenient remover of oxyanionic pollutants as arsenate, selenite and

chromate81.

5.3.4 Biotechnological production

In both modern and traditional biotechnological production, bacteria, yeasts, molds or

purified enzymes are used to produce or modify chemical substances. To prolong the

production cycle, the organism can be encapsulated in a calcium alginate bead, which

protects the organism. This method is commercially used for encapsulating probiotic bacteria

for use in dairy products85, and research is being conducted to extend its use to amylase

production by Bifidobatrium bifidum82, citric acid production by Aspergillus niger

83, and

glucose oxidase and catalase coimmobilization84.

5.4 WELDING

In welding, alginates are used as a plasticizer, stabilizer and binding agent in the covering

material of both organic and mineral welding rods3. High gel strength alginates are used for

top end rod applications to adhere the flux to the metal core in the extrusion process. The use

of alginate is significantly more ecological than traditional agents.

5.5 PAPER INDUSTRY

Paper and other cellulose products are often sized, i.e. treated with a specific substance in

order to improve the characteristics of the paper which are important for printing, such as

surface porosity and hydrophobicity. Hydrophobic surface size agents improve printability

primarily by decreasing paper sheet absorbency and enhancing surface resistance to liquid

(ink) penetration. Such sizing agents are often recruited from anionic polymers such as

33

carboxylmethylcellulose, carboxymethyl guar, alginic acid or pectin. Other compounds are

necessary to size the paper sheet according to the needs of the printer86. Altogether, sizing

leads to improved imaging and contrast with various printing technologies.

5.6 COSMETICS

Cosmetics is a vital industry and developers are constantly looking for improvements and

new ingredients. Alginic acid and its derivates can be used in many ways, such as

encapsulating material for the active ingredients87; as humectant88, thickener and stabilizer for

creams, soaps and body lotions; and as the functional compound, being anti-inflammatory87,

for face or body masks.

34

6 ALGINATE MARKET

6.1 CURRENT SITUATION

Besides alginate production, seaweed is used in other areas, especially as food, animal

fodder, fertilizer and biomass for methane production, thus dragging the alginate price

upwards by higher demand for seaweed in general. Because cultivation of brown seaweed is

not economically convenient, the main price increase of brown seaweed is created by the

increased price of energy.

6.1.1 Market value of alginate

The price of raw alginate materials as alginic acid, sodium alginate or calcium alginate has

been estimated from the available data between 5 – 20 USD/kg. The price varies according to

the quality. Medical quality, pure, sterile alginate can cost over 40,000 USD/kg (lit.89).

6.1.2 Price trends

Since the year 2000, cheap low-grade alginates from China have emerged on the market,

resulting in slight price drop. However, since 2005, the price for raw alginate has been

growing90, because the main hydrocolloid producer ISP Alginates (UK) Ltd. announced rise

in the price of alginates. The price increase was 5% in 2005 (lit.91), 15% in 2007 (lit.92) and

another 15% in the first quarter of 2008 (lit.93). Other big hydrocolloid producers such as

Danisco Cultor (Denmark), FMC BioPolymer (USA), Fuji Chemical Industry Co. Ltd.

(Japan), China Seaweed Industrial Association (China), Algisa, Compania Industrial de

Alginatos S.A. (Chile) are also expected to elevate prices of their products. However, for

producers of end-user products such as food, cosmetics or pharmaceuticals, alginate makes

up only a minor part of their production costs, so the price of the end product is not expected

to follow the increase.

35

6.2 FUTURE PROSPECTS OF THE ALGINATE MARKET

6.2.1 Worth of the global alginate market

The alginate market was worth 195 million USD in the year 2000 and in 2005 it was worth

213 million USD. It has been estimated90 that the value can grow to 240 million USD before

the year 2010.

6.2.2 Market and science

The technological base of modern alginate products is situated in Norway and the USA,

helped by the vicinity of high quality seaweed from the North Atlantic Ocean. In 2002, the

Norwegian alginate producer Pronova merged with FMC BioPolymer96. FMC BioPolymer

has many patents related to high-tech applications of alginates, thanks to the close

cooperation with university laboratories in Norway.

6.2.3 Bacterial alginates from a commercial perspective

Bacterial alginates have not yet found their way to the market, but there are indices

suggesting that bacterial alginate may in the future replace algal alginate especially in the

medical and nanotechnology fields of use.

36

7 CONCLUSION

Most alginate is consumed by well-established industrial applications, where the quality

requirements are well met through algal alginate after sufficient purification. For some

high-tech applications, notably medical, bacteria are being evaluated as a source of alginate

of better quality. Cell encapsulation is an interesting example from this field, a process in

which protein-producing cells are enclosed in alginate beads prior to being surgically

implanted. Recent progress gives hope for better cures for diabetes and cancer.

Brown algae are difficult to cultivate, and in practice the production of algal alginate is

dependent on natural resources, which to a high extent determines the location of the

processing facilities. There is currently no risk of endangering these sources by

overharvesting, but the costly and lengthy production process could make algal alginate more

expensive over the coming years.

Although methods of analyzing and purifying alginate are becoming more exact and

sophisticated, the scientific community has not yet set any international standards regulating

the quality of alginate (notably the MG ratio and limits for exopolysaccharides, proteins and

trace elements) that would be suitable for high-tech applications in medical fields. Such

standardization would lead to greater understanding and cooperation between different

laboratories and companies involved in alginate research.

Bacterial alginates could in the future become competitive to algal alginates, especially

because of place and time-independent production, possibility of controlling the properties of

biosynthesized alginate, and lower ecological impact. However, the production of bacterial

alginates is not yet competitive to algal alginates and there have not been any published

attempts to mass-produce bacterial alginate.

Some challenges in bacterial alginates production are:

− To develop higher understanding of the genetics behind alginate production, in order

to manufacture tailor-made alginates for specific needs.

− To scale-up the fermentation process and optimize the yield of alginate, thus making

37

production of bacterial alginate economically competitive to algal alginate.

− To standardize medical and nanotechnology protocols and facilitate the

communication between different research groups, thus speeding up the process of

commercialization of scientific projects.

− To promote bacterial alginate usage, possibly in connection with other materials.

If these issues were resolved, bacterial alginates could have a bright future in the service of

man, even though the path remains long.

38

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