Production and applications of microbial exopolysaccharides 7.1 Introduction 7. 2 Origin and composition 7.3 Exopolysaccharide structure 7. 4 Medium composition for exopolysaccharide production 12 7.5 Fermentation 7.6 Product recovery 211 7.7 Physical properties 213 7. 8 Biosynthesis 217 7.9 Applications of microbial exopoly accnaniaes 223 Summary and objectives 230
Production and applications of microbial ex0 polysaccharides 7.1 Introduction 1 94 7.2 Origin and composition 7.3 Exopolysaccharide structure 7.4 Medium composition for exopolysaccharide production 7.5 Fermentation 7.6 Product recovery 7.7 Physical properties 7.8 Biosynthesis 7.9 Applications of microbial exopolysaccharides Summary and objectives 194 198 201 205 21 1 213 217 223 230
194 Chapter 7 Production and applications of microbial exopolysaccharides 7.1 Introduction Commercial applications for polysaccharides include their use as food additives medicines and industrial products. Although plant polysaccharides(such as starch, agar and alginate) have been exploited commercially for many years, microbial exopolysaccharides have only become widely used over the past few decades. The diversity of polysaccharide structure is far greater in micro-organisms compared to plants and around 20 microbial polysaccharides with market potential have been described. However, micro-organisms are still considered to be a rich and as yet underexploited source of exopolysaccharides chapter Successful commercial application of microbial exopolysaccharides depends on overview exploiting their unique physical properties. These properties concern the rheology of exopolysaccharides in solution and their ability to form gels at relative heir concentrations. The physical properties of exopolysaccharides arise largely from gh molecular weight; their molecular confirmations determined by their primary structure;associations between molecules in solution. In this Chapter we will commence by considering the origin, or natural sources of exopolysaccharides, and their molecular composition. We will then consider how the molecular composition determines exopolysaccharide structure and, in turn, how structure determines the unique physical properties of exopolysaccharides. The commercial applications of the unique physical properties of exopolysaccharides will then be discussed. We shall see that microbial exopolysaccharides are widely used as viscosifier and as gelling agents They are, for example, used in the food industry as stabilisers, adhesive, thickening agents and foam stabilisers. Other industrial applications include their use as thickening agents in the printing industry and as gels for improved petroleum recovery in the oil industry. The prospect of those approaching commercial use in food, medical and industrial areas will also be considered. Later in the Chapter, the biosynthesis of exopolysaccharides and, in particular, the genetics and regulation of synthesis will be discussed. Much of our knowledge in this area comes from studies on the bacterium Xanthomonas campestris the industrial producer of the commercially very important exopolysaccharide xanthan Our understanding of the genetics of exopolysaccharide synthesis is advancing rapidly and offers opportunities, not only to improve yields of exopolysaccharides, but also to modify their composition and thus their structure and properties, giving rise to new applications. Finally, we will consider industrial fermentation of microbial exopolysaccharides, including medium formulation and product recovery, particular in relation to xanthan 7.2 Origin and composition Many different types of carbohydrate-containing molecules are located on the surface of microbial cells. Some of these are components of the microbial cell wall limited to certain types of micro-organisms; such as bacterial peptide lipopolysaccharides, echoic acids and yeast mannans. Other polysaccharides glycan
194 Chapter 7 Production and applications of microbial exopolysaccharides 7.1 Introduction Commercial applications for polysaccharides include their use as food additives, medicines and industrial products. Although plant polysaccharides (such as starch, agar and alginate) have been exploited commercially for many years, microbial exopolysaccharides have only become widely used over the past few decades. The diversity of polysaccharide structure is far greater in mimrganisms compared to plants and around 20 microbial polysaccharides with market potential have been described. However, micro-organisms are still considered to be a rich and as yet underexploited source of exopolysaccharides. Successful commercial application of microbial exopolysaccharides depends on exploiting their unique physical properties. These properties concern the rheology of exopolysaccharides in solution and their ability to form gels at relatively low concentrations. The physical properties of exopolysaccharides arise largely from: their high molecular weight; their molecular confirmations determined by their primary structure; associations between molecules in solution. In this Chapter we will commence by considering the origin, or natural sources of exopolysaccharides, and their molecular composition. We will then consider how the molecular composition determines exopolysaccharide structure and, in turn, how structure determines the unique physical properties of exopolysaccharides. The commercial applications of the unique physical properties of exopolysaccharides will then be discussed. We shall see that microbial exopolysaccharides are widely used as viscosifiers and as gelling agents. They are, for example, used in the food industry as stabilisers, adhesive, thickening agents and foam stabilisers. Other industrial applications include their use as thickening agents in the printing industry and as gels for improved petroleum recovery in the oil industry. The prospect of those approaching commercial use in food, medical and industrial areas will also be considered. Later in the Chapter, the biosynthesis of exopolysaccharides and, in particular, the genetics and regulation of synthesis will be discussed. Much of our knowledge in this area comes from studies on the bacterium Xanthomanas campestris the industrial producer of the commercially very important exopolysaccharide xanthan. Our understanding of the genetics of exopolysaccharide synthesis is advancing rapidly and offers opportunities, not only to improve yields of exopolysaccharides, but also to modify their composition and thus their structure and properties, giving rise to new applications. Finally, we will consider industrial fermenta tion of microbial exopolysaccharides, including medium formulation and product recovery, particular in relation to xanthan. market pomtential chapter overview 7.2 Origin and composition Many different types of carbohydrate-containing molecules are located on the surface of microbial cells. Some of these are components of the microbial cell wall and are limited to certain types of micro-organisms; such as bacterial peptidoglycan, lipopolysaccharides, techoic acids and yeast mannans. Other polysaccharides are not
Production and applications of microbial exopolysaccharides wall components but are either associated with surface macromolecules or are totally dissociated from the microbial cell. These are extracellular polysaccharides, also known as exopolysaccharides, and they show considerable diversity in their composition and structure Exopolysaccharides occur widely, especially among bacteria, and include free-living saprophytes and animal and plant pathogens. They are produced by most microalgae but relatively few yeasts and filamentous fungi produce exopolysaccharides. Although diversity plants produce a wide range of polysaccharides, their diversity is considerably less than those produced by micro-organisms. The number of different sugars found in olysaccharides is an indicator of diversity of structure and is eight fold higher(around 200)in those of microbial origin compared to those of plant origin Although exopolysaccharides do not normally have a structural role, they do form structures that can be detected by either t or electron microsco Exopolysaccharides may form part of a capsule closely attached to the microbial mUco Surface, or appear as loose slime secreted by the cell but not directly attached to it Exopolysaccharide producing cells usually form mucoid colonies on solid media and uid cultures of these cells may become very viscous. However growth conditions can fluence the composition, physical properties and organisation of exopolysaccharide 7.2.1 Composition Exopolysaccharides are mainly composed of carbohydrates(see Figure 7.1) common The sugars commonly found in microbial polysaccharides are extremely diverse and sugars include most of those found widely in animal and plant polysaccharides D-glucose D-galactose;> pyranose forms, ie D-mannose: · L-rhamnose; · L-fucose; However, whereas eukaryotic polysaccharides may contain pentoses such as D-xylose and D-ribose, they are only very rarely found in microbial polysaccharides Draw the ring structures of L-fucose(6-deoxy-L-galactose) and L-rhamnose (6-deoxy-l-mannose). Use the information in Figure 7.1 to help you do this
Production and applications of microbial exopolysaccharides 195 wall components but are either associated with surface macromolecules or are totally dissociated from the microbial cell. These are extracellular polysaccharides, also known as exopolysaccharides, and they show considerable diversity in their composition and Structure. Exopolysaccharides occur widely, especially among bacteria, and include free-living saprophytes and animal and plant pathogens. They are produced by most microalgae but relatively few yeasts and filamentous fungi produce exopolysaccharides. Although plants produce a wide range of polysaccharides, their diversity is considerably less than those produced by micro-organisms. The number of different sugars found in polysaccharides is an indicator of diversity of structure and is eight fold higher (around 200) in those of microbial origin compared to those of plant origin. Although exopolysaccharides do not normally have a structural role, they do form structures that can be detected by either light or electron microscopy. Exopolysaccharides may form part of a capsule closely attached to the microbial cell surface, or appear as loose slime secreted by the cell but not directly attached to it. Exopolysaccharide producing cells usually form mucoid colonies on solid media and liquid cultures of these cells may become very viscous. However, growth conditions can influence the composition, physical properties and organisation of exopolysaccharide. dimity mucoid ~&nies 7.2.1 Composition Exopolysaccharides are mainly composed of carbohydrates (see Figure 7.1). The sugars commonly found in microbial polysaccharides are extremely diverse and include most of those found widely in animal and plant polysaccharides: common Sugars pyranose forms, ie e D-glucose; e D-mannose; furanose forms, ie e L-rhamnose; e L-fucose; However, whereas eukaryotic polysaccharides may contain pentoses such as D-xylose and D-ribose, they are only very rarely found in microbial polysaccharides. Draw the ring structures of L-fucose (6-deoxy-L-galactose) and L-rhamnose n (6-deoxy-1-mannose). Use the information in Figure 7.1 to help you do this
Chapter 7 carboxylic COOH O OH<+hydroxyl in p-isomer position C-1 asymmetric carbon atom OH B-D-glucose a-D-galacturonic acid pyranose form, six-membered ring) a= position of the hydroxyl on C-2 in D-mannose b= position of the hydroxyl on 4 in D-galacto O OH L-glucose COOH H2N-CH CH3 CH,OH CH2 H→c一NH CH2 COOH COOH COOH serine L-glutamic acid COOH Cl HOH CH2 CH3 c CH2 COOH COOH SuCcInIc Figure 7.1 Structures of some of the components of microbial exopolysaccharides rare sugars Examples of rare sugars which have been found in some microbial polysaccharides are: Logit
196 Chapter 7 Figure 7.1 Structures of some of the components of microbial exopolysaccharides. rare sugars Examples of rare sugars which have been found in some microbial polysaccharides are: L-glucose; L-galactose;
Production and applications of microbial exopolysaccharides N-acetyl-D-glucosamine(an amino sugar); · D-glucuronic acid The presence of uronic acids in microbial exopolysaccharides results in their oolyanionic nature In addition to one or more sugars, exopolysaccharides from prokaryotes commonly contain pyruvate ketals and various ester-linked organic substituents. These are only rarely found in eukaryotic exopolysaccharides pyruvate Pyruvate ketals add to the anionic nature of the exopolysaccharide and are usually ketals present in stoichiometric ratios with the carbohydrate component. Pyruvate is normally attached to the neutral hexoses but may also be attached to uronic acids. In the absence of uronic acids, pyruvate alone contributes to the anionic nature of the acetate Acetate is the commonest ester-linked component of exopolysaccharides and does not contribute to their anionic nature. Less common ester- linked components, which may be found along with acetate in some exopolysaccharides, include propionate; e succinate . 3-hydroxybutanoate organic acids The presence of organic acid substituents in exopolysaccharides increases the pophilicity of the molecule. In addition, for some exopolysaccharides with relatively amino ada high organic acid contents their interaction with cations and with other polysaccharides may be influenced. Several amino acids have also been found in bacterial exopolysaccharides, including serine and L-glutamic acid(Figure 7.1 phosphate and Some microbial exopolysaccharides contain the inorganic substituents phosphate and sulphate sulphate. Phosphate has been found in exopolysaccharide from bacteria of medical importance, including Escherichia coli Sulphate is far less common than phosphate and has only been found in species of cyanobacteria. In addition to these inorganic components, which form part of the structure of some exopolysaccharides, all polyanionic polymers will bind a mixture of cations Exopolysaccharides are, therefore, purified in the salt form. The strength of binding of the various cations depend on the exopolysaccharide; some bind the divalent cations calcium, barium and strontium very strongly, whereas others prefer certain monovalent cations, eg Na
Production and applications of microbial exopolysaccharides 1 97 polyanionic nature pyrwate htals acetate organicacids amino acids phosphate and sulphate N-acetyl-D-glucosamine (an amino sugar); D-glucuronic acid; D-galacturonic acid. The presence of uronic acids in microbial exopolysaccharides results in their polyanionic nature. In addition to one or more sugars, exopolysaccharides from prokaryotes commonly contain pyruvate ketals and various ester-linked organic substituents. These are only rarely found in eukaryotic exopolysaccharides. Pyruvate ketals add to the anionic nature of the exopolysaccharide and are usually present in stoichiometric ratios with the carbohydrate component. Pyruvate is nody attached to the neutral hexoses but may also be attached to uronic acids. In the absence of uronic acids, pyruvate alone contributes to the anionic nature of the exopolysaccharide. Acetate is the commonest ester-linked component of exopolysaccharides and does not contribute to their anionic nature. Less common ester-linked components, which may be found along with acetate in some exopolysaccharides, include: propionate; glycerate; succinate; 3-hydroxybutanoate. The presence of organic acid substituents in exopolysaccharides increases the lipophilicity of the molecule. In addition, for some exopolysaccharides with relatively high organic acid contents, their interaction with cations and with other polysaccharides may be influenced. Several amino acids have also been found in bacterial exopolysaccharides, including serine and L-glutamic acid (Figure 7.1). Some microbial exopolysaccharides contain the inorganic substituents phosphate and sulphate. Phosphate has been found in exopolysaccharide from bacteria of medical importance, including Escherichia coli. Sulphate is far less common than phosphate and has only been found in species of cyanobacteria. In addition to these inorganic components, which form part of the structure of some exopolysaccharides, all polyanionic polymers will bind a mixture of cations. Exopolysaccharides are, therefore, purified in the salt form. The strength of binding of the various cations depend on the exopolysaccharide; some bind the divalent cations calcium, barium and strontium very strongly, whereas others prefer certain monovalent cations, eg Na
Chapter 7 SAQ 7.1 Identify each of the following statements as true or false If false give a reason for your response 1)D-xylose is a sugar commonly found in microbial polysaccharides 2) Pyruvate ketals contribute to the cationic nature of exopolysaccharides 3)The presence of acetate in exopolysaccharides increases their lipophilicity 4) Exopolysaccharides are not produced by yeasts and filamentous fungi 5)An exopolysaccharide containing a high content of D-ght ic acid will tend to bind cations 7.3 Exopolysaccharide structure As with all polysaccharides, microbial exopolysaccharides can be divided into homopolysaccharide and heteropolysaccha Homopolysaccharides contain only one type of monosaccharide, whereas heteropolysaccharides contain more than one Many are neutral glucans, being composed of the monosaccharide component D-glucose. 7.3.1 Homopolysaccharide types of homopolysaccharide are known(Figure e polyanionic homopolymers and, unlike the glucans, also contain acyl groups 1)Single linkage type. Several of these are neutral glucans, eg curdlan. Others Side chain type. Scleroglycan is typical and possesses tetrasaccharide repeating units due to a 1, 6p-D-glucosyl side-chain on every third main chain residue 3) Branched types. These are dextrans which are composed entirely of a-linked glucosyl residues. In some dextrans the linkage is almost entirely 1-6, but up to 50% of the glucose residues may be linked1→2,1→3or1→4
198 Chapter 7 Identify each of the following statements as true or false. If false give a reason for your response. 1) D-xylose is a sugar commonly found in microbial polysaccharides. 2) Pyruvate ketals contribute to the cationic nature of exopolysaccharides. 3) The presence of acetate in exopolysaccharides inmases their lipophilicity. 4) Exopolysaccharides are not produced by yeasts and filamentous fungi. 5) An exopolysaccharide containing a high content of D-glucuronic acid will tend to bind cations. 7.3 Exopolysaccharide structure As with all polysaccharides, microbial exopolysaccharides can be divided into homoplysaccharides and heteropolysaccharides. Homopolysaccharides contain only one type of monosaccharide, whereas heteropolysaccharides contain more than one. Many are neutral glucans, being composed of the monosaccharide component D-glucose. 7.3.1 Homopolysaccharides Three main types of homopolysaccharides are known (Figure 7.2). 1) Single linkage type. Several of these are neutral glucans, eg curdlan. Others are polyanionic homopolymers and, unlike the glucans, also contain acyl groups. 2) Side chain type. Weroglycan is typical and possesses tetrasaccharide repeatingunits due to a 1,6-~-Dglucosyl side-chain on every third main chain residue. 3) Branched types. These are dextrans which are composed entirely of a-linked glucosyl residues. In some dextrans the linkage is almost entirely 1 + 6, but up to 50% of the glucose residues may be linked 1 4 2,1+ 3 or 1 + 4
Production and applications of microbial exopolysaccharides CH,OH CH2OH CH,OH b)βDgu(1→3)-BDu(1→3)-Dgu(1→3)阝-Dgu B-D-glu OOOooO6 ooooooooooo。 igure 7. 2 Three main types of homopolysaccharide. a) Single linkage type, eg curdlan. b) Side chain type, eg scleroglycan. c)Branched chain type, eg dextran. The figure also show various ways of illustrating exopolysaccharide structure 7.3.2 Heteropolysaccharides repeat units Microbial heteropolysaccharides are almost entirely all composed of repeating units of between two and eight monosaccharides. The units often contain D-glucuronicacid and short side-chains of one to four residues are com everal different side-chains are found in some heteropolysaccharides. The structure of xanthan illustrates these points and is shown in Figure 7.3. A few bacterial alginates are exceptions
Production and applications of microbial exopolysaccharides 199 Figure 7.2 Three main types of hornopolysaccharides. a) Single linkage type, eg curdJan. b) Side chain type, eg scleroglycan. c) Branched chain type, eg dextran. The figure also show various ways of illustrating exopolysaccharide structure. 7.3.2 Heteropolysaccharides Microbial heteropolysaccharides are almost entirely all composed of repeating units of between two and eight monosaccharides. The units often contain D-glucuronic acid and short sidechains of one to four residues are common. Several different sidechains are found in some heternplysaccharide. The structure of xanthan illustrates these points and is shown in Figure 7.3. A few bacterid alginates are exceptions. repeat units
Chapter 7 B1 -G-G-G- presence or absence give rise to different repeat units( Pyr not present with 2x A G= D Ac=acetate 4, 6-2 pyr Pyr= pyruvate ooo6o9oa。9o9。0 sntermsolecuyar Figure 7. 3 The structure of xanthan illustrated in three ways(a, b, c). SAQ 7.2 Complete the illustration of the repeat unit in xanthan by adding bonds and ups to the molecule(similar to the illustration in Figure 7.2a). Refer to Figures 7.1 and 7. 3 for chemical structures ∏ Examine Figure 7.3 and give two reasons why xanthan has an anionic natur The anionic nature of xanthan arises from the presence of glucuronic adid and pyruvate
200 Chapter 7 v I Figure 7.3 The structure of xanthan illustrated in three ways (a, b, c). Complete the illustration of the repeat unit in xanthan by adding bonds and groups to the molecule (similar to the illustration in Figure 7.2a). Refer to Figures 7.1 and 7.3 for chemical structures. n Examine Figure 7.3 and give two reasons why xanthan has an anionic MM. The anionic nature of xanthan arises from the presence of glucuronic acid and pyruvate
Production and applications of microbial exopolysaccharides The unique physical properties of microbial exopolysaccharides(considered in Section s molecular 7.7), which determine their commercial importance, arises from their molecular informaton conformation. This, in turn, is determined by the primary structure and from associations between molecules in solution ond angles For most exopolysaccharides their shape is determined by the angle of bonds which governs the relative orientations of adjacent sugar residues in the chain. However, the range of relative orientations of adjacent sugar molecules is limited by stericinteractions between molecules along the chain ∏ Which group substituents influence inter-atomic electrostatic repulsion in a glyc The carbonyl( Coo)group, which carries a full charge, will have the most pronounced effect. Oxygen atoms of hydroxyl groups carry a partial negative charge and, therefore each oth helical Exopolysaccharides in solution have an ordered helical conformation, which may be conformation single, double or triple; forexample, xanthan forms a double or triple helix(Figure 7.3c) These are stabilised by intermolecular hydrogen bonds. The helical conformation makes the exopolysaccharide semirigid and the molecules can move large volumes of solution. These volumes overlap even at low concentrations of exopolysaccharide, giving rise to relatively high viscosities. intermolecular The intermolecular interactions stabilise the helices and greatly influence the properties racbons of exopolysaccharides in solution, ie solubility, viscosity and gel-formation. a strong interaction or good-fit between molecules will lead to insolubility, whereas poor interaction will lead to solubility of exopolysaccharides. The interactions between molecules is influenced by the presence of side-chains. For example, cellulose is insoluble but introduction of a three monosaccharide side- chain into the cellulose chain gives the soluble xanthan. Small changes in the structure of the side-chains can alter the molecular interactions and thus properties of the exopolysaccharide These changes may be brought about by choice of production organism; conditions of fermentation chemical modification(post fermentation); nzymatic modification(post fermentation) 7.4 Medium composition for exopolysaccharide production Many different types of carbon substrate can be converted by micro-organisms to exopolysaccharides, these include · carbohydrates; · amino acids
Production and applications of microbial exopolysaccharides 201 molecular mformatial bond angles held conformatian inbrmdecular inbractions The unique physical properties of microbial exopolysaccharides (considered in Section 7.7), which determine their commercial importance, arises from their molecular conformation. This, in turn, is determined by the primary structure and from associations between molecules in solution. For most exopolysaccharides their shape is determined by the angle of bonds which governs the relative orientations of adjacent sugar residues in the chain. However, the range of relative orientations of adjacent sugar molecules is limited by steric interactions between molecules along the chain. Which group substituents influence inter-atomic electrostatic repulsion in a n glycosyl chain? The carbonyl (COO-) group, which carries a full charge, will have the most pronounced effect. Oxygen atoms of hydroxyl pups carry a partial negative charge and, therefore, repel each other. Exopolysaccharides in solution have an ordered helical conformation, which may be single, double or triple; for example, xanthan forms a double or triple helix Figure 7.3~). These are stabilised by intermolecular hydrogen bonds. The helical confoxmation makes the exopolysaccharide semirigid and the molecules can move large volumes of solution. These volumes overlap even at low concentrations of exopolysaccharide, giving rise to relatively high viscosities. The intermolecular interactions stabilise the helices and greatly influence the properties of exopolysaccharides in solution, ie solubility, viscosity and gel-formation. A strong interaction or 'good-fit' between molecules will lead to insolubility, whereas poor interaction will lead to solubility of exopolysaccharides. The interactions between molecules is influenced by the presence of side-chains. For example, cellulose is insoluble but introduction of a three monosaccharide side-chain into the cellulose chain gives the soluble xanthan. Small changes in the structure of the side-chains can alter the molecular interactions and thus properties of the exopolysaccharide. These changes may be brought about by: choice of production organism; conditions of fermentation; chemical modification (post fermentation); enzymatic modification (post fermentation). 7.4 Medium composition for exopolysaccharide production Many different types of carbon substrate can be converted by micmrganisms to exopolysaccharides, these include: carbohydrates; a aminoacids;
Chapter 7 fatty acids; certain central metabolites(such as TCA cycle intermediates) carbohydrates Carbohydrates are the most widely utilised carbon substrate by exopolysaccharide producing micro-organisms and are used as substrate for commercial production. The structure of the exopolysaccharide is generally independent of the carbon substrate However, choice of carbon substrate can influence both the quantity produced and the extent of acylation of exopolysaccharides. The bacteria that produce dextran are unusually specific in their carbon substrate requirement for exopolysaccharide production: they synthesise dextran only when grown on sucrose and are apparently unable to synthesise the polymer when grown on other substrates, such as glucose ∏ Why do you think carbohydrates are the most widely utilised carbon substrate for commercial production? Carbohydrates are relatively cheap, available in large quantities and utilisable source of carbon and energy for most micro-organisms. These cons are particularly important for those exopolysaccharides produced on a Utilisable nitrogen sources for exopolysaccharide producing organisms include ammonium salts; · amino acid · nitrate dinitrogen(nitrogen gas) Ammonium salts or amino acids are by far the most commonly used nitrogen sources in production media. Nitrate is rarely used. Although most nitrogen fixing micro-organisms do produce exopolysaccharide, their growth and the quantity of the polymer produced is often improved if a fixed source of nitrogen, such as Nh, is cations Most micro-organisms require various cations for optimal growth, in particular K and Ca. Other cations(trace elements) are required in smaller quantities and, in some culture media, may be present as components of other ingredients Phosphate is the major anionic requirement of micro-organisms K has a role in substrate uptake and during efficient exopolysaccharide synthesis, adequate supplies of this ion is essential for ensuring sufficient intracellular carbon substrate is maintained. Other ions, such as phosphate and magnesium, have roles in the acylation of exopolysaccharides and influence their physical properties
202 Chapter 7 0 hydrocarbons; fattyacids; certain central metabolites (such as TCA cycle intermediates). Carbohydrates are the mast widely utilised &n substrate by exopolysamharide producing micro-organisms and are used as substrate for commercial production. The structure of the exopolysaccharide is generally independent of the carbon substrate. However, choice of carbon substrate can influence both the quantity produced and the extent of acylation of exopolysaccharides. The bacteria that produce dextran are unusually specific in their carbon substrate requirement for exopolysaccharide production: they synthesise dextran only when grown on sucrose and are apparently unable to synthesise the polymer when grown on other substrates, such as glucose. Why do you think carbohydrates are the most widely utilised carbon substrate n for commercial production? cartmhydrates Carbohydrates are relatively cheap, available in large quantities and are readily utilisable source of carbon and energy for most miao-organisms. These considerations are particularly important for those exopolysaccharides produd on a large (bulk chemical) scale. nitrogen sources Utilisable nitrogen sources for exopolysaccharide producing organisms include: ammoniumsalts; 0 aminoacids; nitrate; dinitrogen (nitrogen gas). Ammonium salts or amino acids are by far the most commonly used nitrogen sources in production media. Nitrate is rarely used. Although most nitrogen fixing micmrganisms do produce exopolysaccharide, their growth and the quantity of the polymer produced is often improved if a fixed source of nitrogen, such as M, is supplied. Most micro-organisms require various cations for optimal growth, in particular K', Mg", Fe2+ and Ca2+. Other cations (trace elements) are required in smaller quantities and, in some culture media, may be present as components of other inments. Phosphate is the major anionic requirement of micro-organisms. K+ has a role in substrate uptake and during efficient exopolysaccharide synthesis, adequate supplies of this ion is essential for ensuring sufficient intracellular carbon substrate is maintained. Other ions, such as phosphate and magnesium, have roles in the acylation of exopolysaccharides and influence their physical properties. cations