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 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 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. 196 Chapter 7 Figure 7.1 Structures of some of the components of microbial exopolysaccharides. Examples of rare sugars which have been found in some microbial polysaccharides are: rare sugars L-glucose; L-galactose; 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'. 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 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 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 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; 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 Production and applications of microbial exopolysaccharides 203 organic mmpomnfs Exopolysaccharide production may be improved by the provision of various organic components, other than the main carbon and energy source. These can improve growth of the production organism (growth factors) and/or directly enhance the synthesis of exopolysaccharide. Additions that improve polymer yield include tricarboxylic acid (TCA) cycle intermediates, which are thought to improve metabolic balance between carbon flow from carbohydrate substrate through the catabolic pathways and oxidation through the TCA cycle. The components of a defined mineral salts medium for exopolysaccharide n production are given below: KH2P04 Glutamate FeS04.7H20 MnS04.7H20 KI Glucose H3804 MgS04.7Ha N4M004 cUso4.5H20 CoCI2.6Ha CaC12.6H20 What is the nitrogen source in the medium? List the cations provided in the medium. What is the main anionic component? How might the addition of fumaric acid and peptone (vegetable infusion) improve the medium? The nitrogen source in the medium is the amino acid glutamate. There are several cations: K’ Mn2+, Cn2+, Zn2+, Mg2+, Co2+, Fe2+, Ca” Mo”. Phosphate (PO:) is the mapr anionic component. Fumaric acid is a TCA cycle intermediate and may improve metabolic balance through the catabolic pathways and oxidation through the TCA cycle. Peptone may improve growth through the provision of growth factors (amino acids, vitamins, nucleotides). The balance between carbon substrate and a growth limiting nutrient also influences polysaccharide production. Carbon substrate must be provided in adequate amounts to ensure good yields of exopolysaccharide. However, very high cell density culturrs are not necessary during fermentation. It is, therefore, usual to control cell density by limitation of a nutrient other than carbon. Nitrogen has traditionally been used as the limiting nutrient although others, such as sulphur, potassium, magnesium and phosphorous have also been studied. The type of limiting nutrient has been shown to influence both the yield and composition of exopolysaccharide. In the case of potassium limitation, yields are often low because of the involvement of potassium in nutrient uptake. In the case of xanthan, limitation of sulphur, magnesium or phosphate is thought to influence acylation and thus the physical properties of the exopolysaccharide. Media used for laboratory studies of exopolysaccharide production may vary considerably from industrial production media. In laboratory studies pure substrates such as glucose, sucrose and glycerol, can be used to determine exopolysaccharide yields. In industrial production the main factors that influence the decision as to which substrate to use are: limiting nutrient industrial Pm*dm media cost of pure substrate; product yield for the substrate; the quality of the product required. 204 Chapter 7 For a highquality product, the substrate itself must be relatively pure to minimise carryover of impurities to the final product. Most industrial process micro-organisms produce optimal yields of exopolysaccharides from carbohydrate and the most commonly used substrates for industrial production are: 0 glucose from cane or beet; sucrose; 0 starch or starch hydrolysates; comsymp. Cruder and thus cheaper substrates may be used if less pure products are acceptable. Some of these are waste products from other industries, for example: dry milled corn starch; 0 cereal grain hydrolysates; 0 whey. For industrial production, the nitmgen source may be a relatively cheap proteinaceous product, such as: yeast hydrolysate; distillers solubles; 0 casein hydrolysate; 0 soybeanmeal. Local availability as well as cost may well determine the choice of the nitrogen source. Industrial production media must also contain sources of potassium, phosphorous and magnesium. Trace elements may also have to be added. The water used for dum preparation will be from the public water supply or other readily available source. The quality of the water is carefully monitored because the presence of certain metal salts, for example, calcium, copper and iron, can have adverse effects on both the growth of the organism and the rheological properties of the exopolysaccharides. Production and applications of microbial exopolysaccharides 205 A medium for the production of an exopolysaccharide in batch culture has the following components: Glucose Succinic acid Ammonium chloride (limiting substrate) Yeast hydrolysate Potassium Phosphorous Magnesium sulphate Iron Calcium Trace elements (Cu, Zn, Ni, Mo, Mn, Co). 1) What are the roles of each component? 2) If the growth yield coefficient for ammonia was 10 g g-', what concentration of biomass would you expect if ammonia was added to a concentration of 3) If the growth yield coefficient for mapesium for the organism grown in the medium described in 2) was 200 g g- , what concentration of biomass would you expect if magnesium was added to this medium at a concentration of 0.2 2.5 g 1-'? g 1-'? 7.5 Fermentation sreps The various steps involved in commercial production of exopolysaccharide are: 1) Strain maintenance. 2) Inoculum train. 3) Exopolysaccharide production (5(F200 m3). 4) Enzyme treatment to modify properties, eg filterability. 5) Concentration via precipitation or ultrafiltration. 6) Storage and packaging. Biocide may be added after steps four or five. 7.5.1 Strain maintenance Microbial strains must be maintained in such a way that they do not lose their desirable characteristics. Some strains are maintained by regular subculturing, whereas others are lyophilised (freeze-dried), or frozen under nitrogen, or held at -80°C m a freezer. To ensure that a standard inoculum can be obtained on demand, great care is taken to ensure that the stored cultures are pure and the viability is known. Standard inoculum 206 Chapter 7 7.5.2 Inoculum train The medium used to produce the inoculum should be designed for rapid growth of the production organism without exopolysaccharide production. Production of the latter in the inoculum train can give rise to highly viscous cultures that are difficult to transfer from one vessel to another. Optimisation of biomass production would require a large inoculum, comprising 10% of each inoculum stage. However, this involves many transfers which increases the risk of contamination. How many transfers are required for inoculation of a 200 m3 fermentor, using a 10% inoculum train? The initial transfer is the inoculation of a 200 ml shake flask with a lyophilised culture. n The correct answer is seven. In practice, the number of inoculum steps does not usually exceed four to reduce the risk of contamination. This also reduces capital investment and production costs since fewer transfers require fewer vessels for development of the inoculum. Contamination of the production vessel leads to serious financial penalties and each step in the inoculum train is monitored for contamination. To reduce the risk of contamination during sampling it is usual to take a sample from the residue left in each vessel after its contents have been transferred to the next reactor. Since these contamination checks are retrospective, a heavy reliance is placed on the growth characteristics of the production organism. Kinetic variables such as growth rate and oxygen consumption rate are also used to assess the quality of the inoculum. 7.5.3 Fermentation conditions The optimum pH for synthesis of exopolysaccharides is normally between 6.0 and 7.5. For xanthan the optimum is pH 7.0 and below pH 5.0 synthesis is severely depressed. n fermentation? congmkatim checks p~ Why do you think it is particularly important to control the pH of a xanthan Xanthan itself is an acidic product which, in the absence of pH control, would reduce the pH of the fermentation. Temperature can influence the characteristics of the product. For example, the succionoglycan from Agrobaderium rudiubader grown at 30 C has a lower viscosity than that grown at a temperature of 35'C, although the final concentration is similar. The amount of oxygen in an aerobic fermentation is used in a few seconds by actively growing cultures and most aerobic microbial industrial processes are oxygen limited. This problem is particularly acute for exopolysaccharide production on a large scale. Figure 7.4 demonstrates the dramatic effect of increasing viscosity on oxygen transfer. Clearly in a system that produces a highly viscous compound (xanthan at 3% (w/v) has a viscosity in excess of l0,OOO cp) oxygen transfer severely reduces the product concentration that can be achieved in the medium. viscosity and oxygen transfer Production and applications of microbial exopolysaccharides 207 Figure 7.4 The effed of viscosity on oxygen transfer rates. Adapted from Biochemical and Biotechnology Handbook. B Atkinson and F Mavituna, (Eds) 1991, Stockton press. 7.5.4 Production of xanthan gum From a commercial point of view, xanthan gum is the most important microbial exopolysaccharide currently being manufactured. Therefore, we shall consider the fermentation of this product by Xrmthomonas cmnpestris in some detail. The wild-type strain of X. cmnpestris requires complex nutrients, such as yeast extract and several vitamins, to achieve adequate growth and yield of exopolysaccharide. Give reasons why a requirement for 1) yeast extract and 2) vitamins is undesirable n for commercial production of exopolysaccharide. 1) Complex nutrients, such as yeast extract, are variable in composition and consequently it is difficult to maintain process reproducibility within the narrow window required to produce a product of consistent quality. complex nutdents 2) Vitamins are expensive. These limitations have been overcome by the development of strains able to grow well in a minimal salts medium in the absence of complex nutrients and vitamins. The carbon source is usually glucose and a complex nitrogen source, such as glutamate, can replace ammonia. The components of a defined mineral salts medium for xanthan production are n given below: 208 Chapter 7 KH2PO4 Glutamate MnSQ.M20 KI H3604 MgS04.7H20 C~S04.5H20 CoC12.6H20 ZnS04.M20 FeSQ.M20 Glucose Nm004 CaCI2.6Ha 1) List those ingredients which are added at concentrations below 1 mg I-' (microelements). 2) Which inments are added at the following concentrations? g 1" 25 -40 3.5 - 7.0 0.68 0.4 0.01 2 0.01 1 Our response: 1) The micm-elements are: 2) g1-1 Glucose 25 - 40 Glutamate 3.5 - 7.0 KH2P04 0.68 MgS04.7H20 0.4 CaCI2.2H20 0.01 2 FeSO4. 7H20 0.01 1 submerged fermentation - xan*an The characteristics of a typical submerged fermentation for xanthan are shown in Figure 7.5. Nitrogen exhaustion limits the cell density to around 4 g dry weight 1-'. The growth phase is complete in about 25 hours and exopolysaccharide production ceases soon after. 'Ihis reflects the high specific exopolysaccharide production rate during growth of the organisms. The sugar concentration and the viscosity of the fermentation is monitored at regular intervals and the production is considered to be complete when all the glucose is exhausted and the desired viscosity has been reached. Production and applications of microbial exopotysaccharides 209 Figure 7.5 Production of xanthan gum in batch culture using X. canpesfris. Bacterial dry weight (0); xanthan gum (H); residual glucose (A); residual glutamate (A). Adapted from Microbial exopolysaccharide, Yenton etalpp 21 7-261. In biornaterials; Novel Materials from Biological Sources, D Byrom (Ed), MacMillan Academic Professional Ltd, 1991. 7.5.4 Production of succinoglycan Typical batch production of succinoglycan by a Psatdomonas sp is shown in Fip 7.6. Figure 7.6 Production of succinoglycan in batch culture using a Pseudomonas sp. Bacterial dry weight (a); succinoglycan concentration (B); residual glucose (A); residual ammonia (A). Adapted from as Figure 7.5. 21 0 Chapter 7 In the sucanoglycan fermentation, exopolysaccharide production commences after cessation of growth. During the 80 hour production phase of the fermentation, there is a linear increase in product concentration and a linear decrease in residual glucose. n and variables for the fermentation: 1) Yp, the yield coefficient for product (kg polysaccharide (kg glucose)-'); 2) qp, the specific rate of polysaccharide production (kg polysaccharide h-' (kg dry weight cells)-'); 3) productivity (kg polysaccharide m" h-'); assume that the fermentor was harvested at t =lOOh and the set-up time for preparation of the fermentor was an additional 24h. Yp = 12'5 kg xanthan m3 produced= 0.5 kg polysaccharide (kg glucose)-' Use the data presented in Figure 7.6 to calculate the following kinetic parameters 1) 25 kg glucose mT3 consumed 12.0 g m3 ( 83h ] slope of polysaccharide accumulation line 2) - biomass concentration 2.5 qr = = 0.058 kg polysaccharide h" (kg dry wt cells I-' 3) duct concentration at end of fermentation total fermentation time Productivity = p - w = 0.10 kg polysaccharide m-3 h-I. 124 h Refer to Figures 7.5 and 7.7. 1) Explain briefly why the rate of xanthan production increases during the production phase while that of succinoglycan remains constant. limiting nutrient in the succinoglycan fermentation. 2) Explain why it would be inadvisable to use the carbon source as growth 3) What limits growth in the xanthan fermentation? 4) What limits succinoglycan production and why is it necessary to do so? 5) In the succinoglycan fermentation, what is the growth yield coefficient for ammonia? (in units of g g"; moIecular weight of ammonia = 17). Production and applications of microbial exopolysaccharides 21 1 polar organic solvents diStillaIi0t.l PrOPyleW Oxide methylethyl- ketone 7.6 Product recovery There are two main methods for the recovery of exopolysaccharides from fermentation liquors: 0 solvent precipitation followed by spray drying; 0 ultrafiltration. The method used is governed by the market application of the exopolysaccharide. In general, the food industry has a requirement for a dry powder, whereas for several other applications, such as enhanced oil recovery, a liquid product is required and the ultrafiltration concentrate is preferred. 7.6.1 Solvent precipitation Polar organic solvents readily precipitate exopolysaccharides from solution. The solvents commonly used are acetone, methanol, ethanol and propan-2-01. Cation concentration of the fermentation liquor influences the amount of solvent required for efficient product recovery. In the case of propan-2-01, increasing the cation concentration can lead to a four-fold reduction in the volume of solvent required to precipitate xanthan gum. Salts such as calcium nitrate and potassium chloride are added to fermentation broths for this purpose. Further reduction in the volume of solvent required can be achieved by heat treatment of the exopolysaccharide at 100-13OoC for 1-15 minutes before solvent precipitation. The solvent ca'n be recovered by distillation and the precipitated product is freed from excess liquid by centrifugation or pressing. Continuous drying by vacuum or with an inert gas, to reduce the dangers associated with the flammable organic solvents, gives the dry polymer. This is finally milled to the required mesh size, which forms an off-white powder. The powdered exopolysaccharide must be stored in a cool, dry atmosphere to prevent adsorbtion of moisture which would lead to biological degradation of the product. The dry powder remains stable if stored at temperatures below 25°C. For food and pharmaceutical applications, the microbial count must be reduced to less than 10,OOO viable cells per g exopolysaccharide. Treatment with propylene oxide gas has been used for reducing the number of viable cells in xanthan powders. The patented process involves propylene oxide treatment for 3 h in a tumbling reactor. There is an initial evacuation step before propylene oxide exposure. After treatment, evacuation and tumbling are alternated and if necessary the reactor is flushed with sterile nitrogen gas to reduce the residual propylene oxide level below the Food and Drug Administration permitted maximum (300 mg kg-'). The treated polysaccharide is then packaged aseptically. The main disadvantage of precipitation with a polar (water-soluble) solvent is the need for a costly distillation stage to recover the relatively large volumes of solvent used. Another disadvantage is the precipitation of proteins, salts and, in some cases, pigments which reduces the purity and leads to discoloration of the product. To overcome these problems, precipitation using less polar solvents, such as methylethylketone, has been proposed. Only 23% (w/v) methylethylketone is sufficient to saturate the aqueous phase and precipitate exopolysaccharides quantitatively. 21 2 membranes advantages of UhfiltratiOn anlimiaobid agents Chapter 7 What are the advantages of having two distinct phases (aqueous/organic) during n precipitation? 1) It is relatively easy to recover organic solvents. 2) Pigments arc extracted into the organic phase resulting in the production of a pm white precipitate. 7.6.2 Ultrafiltration The alternative large scale recovery method to precipitation is ultrafiltration. For concentration of viscous exopolysaccharides, ultrafiltration is only effective for pseudoplastic polymers (shearing reduces effective viscosity; see section 7.7). Thus, pseudoplastic xanthan gum can be concentrated to a viscosity of around 30,000 centipoise by ultrafiltration, whereas other polysaccharides which are less pseudoplastic, are concentrated only to a fraction of this viscosity and have proportionally lower flux rates. Xanthan gum is routinely concentrated 5 to 10-fold by ultrafiltration. Commercially available plate- and frame- type ultrafiltration equipment are used for exopolysaccharide concentration. The membranes are polysulphone or polyvinylidine fluoride with molecular weight cut-off between 2060,000. There is a relatively low energy requirement (1-2 kWh m-3> for pumping the fluid through the filtration unit at the desired pressure. Pressure difference across the membrane is of the order 2-14 atmospheres. Ultrafiltration has the advantage that there is removal of low molecuiar weight fermentation products and medium components during concentration of the exopolysaccharide. In addition, biological degradation is minimised because fluid is held only for a short time during the filtration process. Other advantages lie in the fact that there is no requirement for solvent recovery and the process is carried out at ambient (not elevated) temperature. The concentrate derived from ultrafiltration is usually a thick colourless gel containin and biological degradation. The type of antimicrobial agent used depends on the particular application for the exopolysaccharide. For example, the nature of the antimicrobial agent is less critical for industrial applications, such as enhanced oil recovery, than for use in cosmetics. Make your own list of advantages and disadvantages of the two main methods of recovery of exopolysaccharides from fermentation liquors, based on the information provided in this Section (7.6). about 4-896 solids. This must contain an antimicrobial agent to inhibit microbial gro WtE n Production and applications of microbial exopolysaccharides 21 3 List the following stages involved in recovery of exopolysaccharides by solvent precipitation and subsequent packaging in an appropriate order. 1) Addition of solvent. 2) Centrifugation. 3) Vacuum drying. 4) Aseptic packaging. 5) Propylene oxide treatment. 6) Cool dry storage. 7) Heattreatment. 8) Addition of cations. 9) Distillation. 10) Sterile nitrogen gas treatment. 11) Milling. 7.7 Physical properties Microbial exopolysaccharides are widely used in industry as viscosifiers and as gehg agents. In this section we will consider, in general, the rheology of exopolysaccharides in solution and their ability to form gels. Specific properties of individual mimbial exopolysaccharides and applications which exploit these characteristics are considered later in this chapter. 7.7.1 Viscosity Many microbial polysaccharides show pseudoplastic flow, also known as shear thinning. When solutions of these polysaccharides are sheared, the molecules align in the shear field and the effective viscosity is reduced. This reduction of viscosity is not a consequence of degradation (unless the shear rate exceeds 105 s”) since the viscosity recovers immediately when the shear rate is decreased. This combination of viscous and elastic behaviour, known as viscoelasticity, distinguishes microbial viscosifiers from solutions of other thickeners. Examples of microbial viscosifiers are: 0 xanthangum; 0 succinoglycan; welan; 0 scleroglucan. pseudopiastic ROW Vi-la9tiCitY 21 4 Chapter 7 The viscosity of a solution of microbial exopolysaccharide must, therefore, be defined as a function of the shear rate (see Figure 7.7). shar rate , I Figure 7.7 Viscosity verses shear rate profiles (polymer concentration, 2.25 kg ITI-~). Adapted from as Figure 7.5. We can see that although the rate of flow may be very low in very viscous solutions, there is no yield stress, ie a stress that must be exceeded for flow to commence. However, some exopolysaccharides do display yield stress characteristics. In practice, the pseudoplastic flow behaviour and elasticity are important characteristics. Try to think of useful properties that these characteristics would confer on exopolysaccharides. High viscosity at low shear rate is essential to inhibit particle sedimentation or to give good cling of a film of exopolysaccharide, whereas reduced viscosity under conditions of high shear rate maintains good pumping and spraying characteristics. 7.7.2 Gel formation One of the most striking and useful properties of exopolysaccharides is that they can form gels at relatively low concentration (typically around 1%). Gels are distinct from viscous solutions that flow readily and are used widely in the food industry and in some personal care products. The mechanism of gel formation depends on the type of microbial exopolysaccharide: polymers that require the presence of ions for gel formation, eg alginate and gellan; polymers that form gels without the involvement of ions, eg curdlan; polymers that require the presence of another polysaccharide, eg xanthan. Yie~s~~ n mechanismS Production and applications of microbial exopoiysaccharides 21 5 Curdlan gel formation is heat dependent. This polysaccharide is not soluble in water, but when an aqueous suspension is heated it becomes clear at about 54°C. Further heating leads to gel fonnation. The gels are stable over a wide range of pH (3 to 95) and do not melt at temperatures below 100°C. Curdlan gels are formed by "sslinking, involving conformational ordering of the exopolysaccharide molecules to give a triple helical structure. curd~an triple helix disordered state ordered state (triple helix) alsbab bndhg Of ca2' Alginate forms gels by the selective co-operative binding of divalent cations. Cross-linking of alginate gels involves the binding of Caz+ within alginate ribbons of polyguluronate sequences, giving rise to the so-called 'egg-box' model. The egg-box arrangement ends when the polyguluronate sequence gives way to polymannurOnic acid or mixed sequences. The physical properties of the alginate gels thus depend on the ratio of D-mannuronic acid and L-@uronic acid. xan*a Xanthan does not in itself form gels, despite the strong intermolecular interactions which occur in solution. However, some of the rheological properties of xanthan have 21 6 Chapter 7 been explained by non-covalent association of xanthan with relatively small amounts of other polysaccharides to form three-dimensional networks (weak gels). 7.7.3 Stability Microbial polysaccharides in solution lose their ordered conformation on heating. The temperature at which the polymer 'melts' to a disordered state is known as the melting temperature (Td and is determined by a variety of factors: polymer structure; 0 nature and concentration of ions in solution; nature and concentration of miscible solvent (if present). At low ionic strengths, T, increases exponentially with ion activity. The effect of high concentrations of salts or miscible solvents depends on the influence they have on hydrogen-bonding and may increase or decrease T,. In the case of xanthan gum, the value of T, can be adjusted from ambient to over 200°C by the addition of appropriate salts. Table 7.2 presents T, values for some industrial viscosifiers. Polymer ApproximateT,,, ("C) Xanthan 120 Succinoglycan 70 Scleroglucan 150 Welan 150 Table 7.2 Appropriate Tm values for rnicrobially derived viscoifiers in hard tap water (or sea waters). At temperatures below T, there is relatively little change in viscosity of microbial exopolysaccharides (Figure 7.8). meld- temperature ionic strength Figure 7.8 The effect of temperature on viscosity. Adapted from as Figure 7.5. Production and applications of microbial exopolysaccharides viscosity above Tm 21 7 At temperatures above T, the change in viscosity with temperature depends on the type and concentration of polymer in solution. At low polymer concentrations, the more flexible disordered conformation reduces both the viscosity and the pseudoplastic behaviour. However, at concentrations exceedmg a few percent the viscosity of some polymers will show a small increase compared to their viscosity below T,. Conversely, some exopolysaccharides such as succinoglycan show a virtual loss of all viscosity at temperatures above T, (Figure 7.8). On cooling to below T,,, some or all of the viscosity observed before heating will be regained, or gelling will occur. At temperatures above TW chemical and enzymatic degradation of microbial exopolysaccharides is enhanced. The apparent enhanced stability of microbial exopolysaccharides in their ordered confirmation is thought to be due to the glycosidic bonds in the backbone of the polymer which raises the activation energy. This restricted movement would also restrict access of enzymes and chemicals to the backbone. degradation 1) Complete the following statements: a) ~ flow is also known as shear thinning. b) behaviour describes recovery of viscosity with reduction in shear conformation of a microbial exopolysaccharide is lost at d) Chemical and enzymatic degradation of microbial exopolysaccharides is 2) F,xplain why brittle gels are obtained from alginate with a high guluronic acid content and more flexible gels from alginate if the D-mannuronic acid content is high. rate. c) The temperatures at or above its temperature. enhanced at temperatures Tm. 7.8 Biosynthesis dl membrane Exopolysaccharides, in almost every case, are synthesised at the cell membrane and then exported from the cell. The only exceptions that have been recorded to date are the homopolysaccharides levan and dextran which are synthesised extracellularly. The building blocks for the exopolysaccharides are usually sugar nucleotide diphosphates with some monophosphates. The sugar nucleotide diphosphates provide energy for the synthesis of the oligosaccharides and are readily interconverted (Figure 7.9). The energy released from the sugar nucleotide diphosphates (32 kJ per mole) is greater than that released by sugar monophosphates (20 kJ per mole) and provides up to 70% of the energy requirement for the synthesis. su!W nudeobde ph0sphates 21 a Chapter 7 Figure 7.9 Interconversion of sugar phosphates and sugar nucleotide phosphates. Adapted from "Biotechnology of microbial exopolysaccharides". I W Sutherland, Cambridge University Press, 1990. Other groups are added to modify the basic structure of the polysaccharide. These commonly include acetate, pyruvate, succinate, 3-hydroxy butanoate, phosphates and, in cyanobacteria, sulphate. Acetyl CoA is the source of acetyl groups in xanthan synthesis and is likely to be the source for most other exopolysaccharides. Methyl groups are derived from methionine or S-adenosylmethionine, as has been demonstrated for cell wall polysaccharides. There has as yet been no definitive study of the mechanism for introducing amino acids or inorganic substituents. It is probable that by anology with peptidoglycan (bacterial cell wall structural polysaccharide) synthesis, amino acids are added to the sugar nucleotides by a specific transferase requiring ATP and Mn' and the phosphate groups are derived from the sugar nucleotides. Heteropolysaccharide biosynthesis involves four stages: 0 synthesis of the sugar nucleotide diphosphates; 0 assembly of the repeat unit on a CF, lipid carrier (undecaprenyl phosphate); pyruvylation and addition of other substituents; 0 transfer of the polysaccharide to the new subunit. The completed exopolysaccharide is then transported to the cell surface through membrane adhesion zones. The synthesis of xanthan has been studied in some detail and is depicted in Figure 7.10. aceQ"CoA wansferases heteropolysacch arides Production and applications of microbial exopolysaccharides 21 9 Figure 7.10 Simplified scheme for the biosynthesis of xanthan. Adapted from Exopolysaccharides in Plant Bacterial Interactions. J A Leight and D L Coplin. Annu Rev Microbial 1992 46 pp 307-46 (see text). Glycosytransferases I to V add glucose, glucose, mannose, glucummic acid, mannose respectively. Pyruvate and acetate are added and the growing xanthan molecule is transferred to a single repeat unit on a second lipid carrier. The acetylation of xanthan is variable and this is probably explained by other processes requiring acetyl &A having priority when the intracellular concentrations are low. This process continues until the macromolecule is formed. The polymerase is relatively unspecific and will polymerise 'imperfectly formed' units synthesised by the gumK and I mutants resulting, in polytrimeric and polytetrameric gums respectively; the genes involved in xanthan synthesis are considered further in sections 7.8.1 and 7.8.2. Homopolysaccharides are synthesised by relatively few specific enzymes and are not constructed from subunits. The commercially important homopolymer dextran is synthesised extracellularly by the enzyme dextransucrase. In Leucmsfoc msenteroides the enzyme is induced by the substrate sucrose. This is cleaved to release free fructose and link the glucose to the reducing end of the acceptor dextran chain, which is bound to the enzyme. The product from this bacterium is composed almost exclusively of cpid canier Polyme~~ hompolysaccha rides 220 Chapter 7 1,6-a-linked glucose with 1,2, 1,3 and 1,4 linkages as minor components. The mechanism for branching is unclear but them is no specific branching emym or requirement for sugar nucleotides. Dextran synthesised by other bacterial species differs in the extent of branching. Alginates are unusual in that although they are heteropolysahrides they are synthesised as homopolymers in the cell. Final epimerisation from mannurdc acid to glucuronic acid then occurs extracellularly. Using a simple illustration show how the completed pentasaccharide subunits of xanthan are assembled into the finished polymer. Using the word list below fill in the blanks in the following paragraph concerning exopol ysaccharide biosynthesis. Exopolysaccharides are synthesised at the ell membrane and then exported. They are formed predominantly from sugar nucleotide required for polymer synthesis. They are constructed on carrier which are then added to the growing exopolysaccharide molecule. This process quires several specific systems to transfer each sugar nucleotide diphosphate to the subunit etc. are synthesised by relatively few specific enzymes and are not constructed &om subunits. Word list: bisphosphates, lipid, subunits, intracellularly, enzyme, homopolysaccharides, energy. , the cleavage of which provides much of the molecules. Heteropolysaccharides are made as 7.8.1 Genetics of exopolysaccharide synthesis A common feature is the arrangement of genes responsible for the control and regulation of synthesis of specific exopolysaccharides in Qjht gene clusters or 'cassettes'. This is exemplified by the arrangement in Xrmthomonas camp~tris which is used to produce the commen5aUy important xanthan gum. Mutation studies have revealed a number of strains that produce xanthan with alterations in the pattern of acetylation and pyruvylation, as well as mutants which show increases in yield, rates of production and composition of the repeat subunit. The genes are clustered in a 16kb DNA sequence containing 12 genes, gumB to M (Figure 7.11). P~d~~ Production and applications of microbial exopolysaccharides 221 Figure 7.1 1 Restriction map of Xanthomonas carrpestris xanthan gene cluster. Adwed from R W Vanderslice et a/. Genetic engineering of polysaccharide structure in Xanthomonas mrpestris. In Biomedical and Biotechnological Advances in Industrial Polysaccharides, 1989, Gordon and Breach N Y. These genes code for all the transferase activities, three polymerases and a gene (gumJ) which appears to control the export of the polysaccharide. Mutations in pmJ are lethal but blocking xanthan synthesis suppresses the lethality. This indicates that mutants deficient in pmJ cannot export the xanthan, which accumulates and kills the cells. Polytetramer and polytrimer products are synthesised by mutants unable to add the side chain terminal f3-mannosyl @mI) and the terminal disaccharide f3-mannosyl ggluclfronic acids (gumK) respectively. Genetic analysis has shown that there is only one promoter and no internal termination sequences have been found, indicating that this gene cluster is one very large operon. There is a second large gene cluster, about 35kb, also involved in the synthesis of xanthan. The precise function is unknown but it is thought to encode for proteins involved in the synthesis of sugar nucleotide diphosphates. Cell lysates from mutant strains of X. campestris were incubated with radiolabelled UDP[''C] glucose or GDP[I4C] mannose, the other sugar nucleotide substrates being unlabelled. The reaction mixture was then divided into lipid and soluble fractions. Where would you expect the radiolabel to be found and what product, if any, would you expect from strains with deficiencies in the following genes? GurnD, transferase I GumM, transferase II GumK, transferase IV gun genes large operon Briefly outline the mapr organisational features of genes encoding the enzymes responsible for exopolysaccharide synthesis, using X. cmnpestris as the example. 222 Chapter 7 7.8.2 Regulation of exopolysaccharide synthesis Several of the important exopolysaccharides are produced by bacteria that exploit plants either as pathogens (X. campestris) or symbionts (Rhizobium spp). Exopolysaccharide production is often essential for plant/bacterial interaction and in X. campestris is co-ordinately regulated with other pathogenicity factors. Non-pathogenic mutants defective in extracellular protease and polygalacturonic acid lyase also have very low amounts of exopolysaccharide. Seven positive regulatory genes have been identified (MA-F regulation of pathogenicity factors) that act mrdinately on the degradative enzymes, exopolysaccharides and pathogenicity. This system consists of an environmental sensor protein located in the membrane which detects changes in the external conditions and an effector protein in the cytoplasm which transmits this signal to the regulatory genes. The rpf system is balanced by a negative regulatory system; mutants defective in this chromosomal locus are pathogenic with elevated levels of exopolysaccharide and extracellular enzymes, whilst multiple functional copies have a repressive effect AII additional more specific regulation of exopolysaccharides has also been identified, involving a two component effector/sensor system, and there is also a general regulatory mechanism of which at least one component is the catabolite activating protein. Mutants deficient in catabolite activating protein have reduced amounts of exopol ysaccharide. The synthesis of the exopolysaccharide colanic acid in E. coli is limited by the availability of an activator protein (rcsA) which acts on the exopolysaccharide gene cassette (cps loci). This is normally rapidly degraded by a protease (lon protease). A two component effector/sensor system also regulates the cps loci. The sensor component (rsK), is likely to be a phosphorylase/kinase that activates the effector protein (rscB). This in turn activates the cps genes either as a rcsB dimer or as rcsB-rcsA. RcsC is itself negatively regulated by rcsD apparently through a modification of signal perception. Complete the following diagram by identifying the components to give a generalised scheme for the regulation of exopolysaccharide synthesis based on the examples from X. cmnpestris and E. coli. p”h”zz sensorpmbm negalive WJ~tOry vsbm cata~i acaivati* pmbh cotanic acid Vgme Component. activated complex catabolite activator protein effector protein pathogenicity regulating genes activator protein degrading enzyme sensor Production and applications of microbial exopolysaccharides 223 powders and concentrates xanthan 7.9 Applications of microbial exopolysaccharides The unique properties of exopolysaccharides are exploited in a wide range of industrial applications. They are sold either as powders or as concentrates (approx 8% w/v). Although more expensive to transport, concentrates offer several advantages: they can be pumped; any lumps formed during make up of the solid are avoided; the viscosity at a given concentration is higher than from solids. Xanthan has some unique properties and high activity at low concentrations and is commercially the most important exopolysaccharide. Food products account for approximately 60% of xanthan use, 15% is accounted for by toothpaste, textiles and crop protection products, 10% in the oil industry and the remainder in miscellaneous industrial/ consumer applications. 7.9.1 Food industry applications The properties of exopolysaccharides utilised in the food industry are presented in Table 7.3. Alteration of the food texture by thickening or gelling is one of the more important uses. This in turn affects less easily defined parameters that are nevertheless crucial in food stuffs, such as 'mouth feel'. The different properties of exopolysaccharides mean that a number of diffemt gel types are available for use in the food industry. I Property Food Exopolysaccharide adhesive icings, glazes binding agent pet foods coating confectionery emulsifying agent salad dressing gellan gellan, alginates xanthan xanthan, alginates film formation sausage case pullulan, elsinan gelling agent pastry, filling, jelly gellan, curdlan inhibit crystal formation frozen food, pastilles alginate, xanthan stabiliser ice cream alginate, gellan, xanthan syneresis inhibitor cheeses xanthan synergistic gels synthetic meat gels xanthan thickening agent jams, sauces xanthan, gellan, alginate foam stabiliser beer, dough xanthan, alginate' Table 7.3 Examples of the use of exopolysaccharides in the food industry. Alginate used in the propylene glycol form; this makes it less susceptible to precipitation by acid and can be used in food and beverages at pH < 3.0. 224 Chapter 7 Alginates form non-thennoreversible gels and are useful when shape retention on heating is desired. Alginates form gels with calcium ions that are bound between sequences of polyguluronyl residues. The ion binding, gel strength and sensitivity for calcium, barium and strontium ions (which are more strongly bound than other cations) increases with increasing guluronosyl content but is reduced if the mannuronic acid residues are heavily acetylated. Gellan is stable to heat and gives a very clear, thermoreversible gel which sets at lower concentrations and more rapidly than most other polysaccharides. Gellan is a fermentation product of Pseudomonas elodea comprised of glucose, rhamnose and glucuronic acid residues with 34.5% 0-acetyl groups. The native exopolysaccharide does not gel but removal of the acetyl groups by heating at pH 10 results in the low acetyl form which gives clear gels in the presence of divalent cations (Mg2+). The properties of this exopolysaccharide make it suitable for use in glazes, jellies and icings. Gellan also has superior flavour release characteristics than most other polysaccharides used in gels. Before microbial exopolysaccharides can be used in foodstuffs they must be evaluated against an industrial standard to ensure that product quality is maintained or improved. The tight deadlines imposed on industry mean that product evaluation must be effective and logical to ensure a rapid result. Therefore, clear objectives and criteria are set against which new products are tested. The criteria that must be satisfied for quick setting jelly desserts are as follows: 1) One stage make up from powder - the product (jelly dessert) is already multicomponent, the introduction of any further stage is undesirable. 2) The powder must be easily dispersible. If vigorous stirring is required, aeration of the product may occur reducing its clarity. Also as hot water is used, difficult systems to mix can be dangerous for the user. 3) Setting time. Must be within 60 minutes. 4) Texture. Must not be dramatically different to gelatine since the nature of the product will change unacceptably. 5) Flavour release properties. Must be similar or superior to the currently used compounds (gelatine). From Table 7.4 determine which of the gel-systems is the most suitable for n inclusion in quick-set jellies. alginates gelIan evduaticm Production and applications of microbial exopotysaccharides 225 Stages Powder Setting Texture Flavour Retake dispersal time mkase COSt carrageenan 1 moderate A0 +++ +4-t 5 carrageenan 1 moderate >60 ++++ + 4 gellan 1 moderate <<<60 + ++++ 20 gelatine 1 moderate >>60 + + 2 (CWS) pectin 2 moderate <60 + + 5 + LBG alginate 1 vigorous <60 + + 6 xanthan+ 2 moderate <60 + + 6 LBG Table 7.4 Comparative features of gel-systems evaluated for use in quick setting jelly products. CWS = cold water soluble, LBG = locust bean gum. Pectin and xanthan/locust bean gum fail on the first criterion as they would only work if a two stage make up p'ocess was used. Alginate is too difficult to mix, and cold water soluble gelatine takes an excessive time to set. The remaining three all show variations from the standard gelatine. Carrageenan and the carrageenan/locust bean gum mixture only just meet the criteria for setting time and gellan imparts a poorer texture. However, gellan scores heavily in the other tests: very rapid setting, excellent flavour release and can be used at lower concentrations than the others. The ovemding consideration is of course the price. Gellan is currently ten times the price of gelatine, nevertheless the advantages gained by using gellan justify the extra cost in certain products. Less polysaccharide can also be used in products by taking advantage of the synergistic gelling of xanthan/galactomannan mixtures which forms thermoreversible gels at lower concentrations than if each is used separately. Another promising exopolysaccharide that may come to replace some of the traditional setting agents is curdlan. This is an a-13 linked glucan made by AZcaZigenes faecal& which retains its shape in cooked food and only needs temperatures of between 55 and 80°C for preparation. n What do you think this might be? Flavour compounds and other heat labile constituents will not be lost as extensively in the preparation, thus improving the product and reducing the cost of production. Another feature of this particular exopolysaccharide is that gel strength depends upon the temperature used. It is constant between 60-8OoC, increasing in strength from 80-100°C and finally changing structure from a single to a triple stranded helix at temperatures over 12OoC. This makes it particularly well suited for use as a molecular sieve, immobilised enzyme support and a binding agent. Different polysaccharides change the perception of flavour, thus xanthan is superior to gum guar in the perception of sweetness. Mixtures of xanthan and locust bean gum have improved flavour release and texture when used in pies and pat& compared to starch. Many foods are emulsions, examples being soups, sauces and spreads. Exopolysaccharides are used to stabilise these emulsions and prevent the phases from This property of curdlan has an important consequence for use in food stuffs. 226 Chapter 7 separating when dried foods are reconstituted. Xanthan is particularly suited for this purpose as it is stable over a wide pH range and can accommodate the low pH’s found in several relishes and dressings. Alginates are less useful at lower pH values but conversion to the propylene glycol form makes it less susceptible to precipitation by acid or metal ions and extends its range of applications. 7.9.2 Other industrial uses Xanthan gum is the most important exopolysaccharide used commerdally, its unique properties making it ideal for an extraordinary range of applications Table 7.5. usage Physlcal properties required explosives (package gels) compatibillty with Ca(N03)2; water resistance (for dynamite) fire figMing flowable pesticides hydraulic fracturing jet printing laundry chemicals liquid fertilisers and herbicides liquid feed supplements oildrilling muds paper finishing thixotropic paints water clarification (or extraction) foam stabilisation suspension and drift control viscosity and cross-linking suspension of starch suspension suspension shear-thinning and viscosity control suspension of clay coatings stabiliser flocculant Table 7.5 Industrial applications of xanthan. Xanthan is used as a drilling fluid, either as a mixture with the traditional bentonite clays or alone, as a clear ’mud’. In this application, xanthan acts as a lubricant and removes cut material. Important factors are the ability to pump (pseudoplastic flow), suspend particles plus its resistance to relatively high temperature, pH and its salt compatibility. Succinoglycan has also been used, having the advantage that it interacts less with CaBr2, which is used to increase the density in drilling fluids. There has been some success in using xanthan to improve oil recovery. The water used can often push through the more viscous oil, therefore failing to drive the oil before it out of the well. Increasing the viscosity of the flushing liquid with xanthan can reduce this problem and thus enhance oil recovery. Cross-linking of xanthan with chromium ions has also been used to seal very permeable rocks - so called thief zones - where oil would otherwise be lost. The use of xanthan also has the advantage that the gel can be removed with oxidising agents such as sodium hypochlorite. The temperature limit for this application is 80°C. There are some problems encountered when using xanthan, however, and biocides are often included to prevent microbial degradation during the initial dilution and injection phase. Free radical scavengers are also added to reduce chemical attack and maintain the exopolysaccharide structure at higher temperatures. Furthermore, despite the prolonged stability of xanthan at temperatures of 95°C the temperatures in some oil wells can reach 150°C. drilling nuids inproved oil r-eV Production and applications of microbial exopolysaccharides 227 mmsan elastic modulus thickening agents purification of bmolecules immObilised cells or enzymes emulsan emulsifying agent Several new exopolysaccharides such as welan and rhamsan produced by AZcalingenes spp may supercede xanthan for some industrial applications. These are based on the same repeat tetrasaccharide backbone of glucose, glucuronic acid, glucose and rhamnose but differ in the substituents: rhamsan has a disaccharide side chain and welan a monosaccharide. Both are stable at high temperature and have excellent pseudoplastic properties. The higher elastic modulus (a measure of structure in solution) of rhamsan suggests that it should be superior to xanthan as a stabiliser. Rhamsan also has improved salt compatibility and is used in fertiliser suspension (high polyphosphates) and explosives (high ammonium nitrate). From Figure 7.8 and Table 7.2, which of the alternative gums do you think is the n most suitable for use in oil field applications? One of the most important criteria for choosing gums for use in oil recovery etc is their stability to extended periods of high temperature. Although succinoglycan has a high viscosity, this deteriorates at high temperatures over extended periods. Welan and xanthan are used in oil well drilling fluids. Welan is particularly useful in this application since it has high viscosity which is maintained at an almost constant level at 121 OC for extended periods and can tolerate temperatures as high as 149OC. Alginates and xanthan are used as thickening agents in the printing industry to control the spread of the dye through fabrics and hold it in place until the dye is fixed. These exopolysaccharides are compatible with a variety of dyes and are also easily washed off. Xanthan and succinoglycan, amongst other exopolysaccharides, are included in paint formulations to stabilise suspensions of the pigment and also confer superior spraying and pumping characteristics. 7.9.3 Enzyme technology applications The extraction of biological material often requires a relatively gentle method to avoid conditions that would otherwise cause losses through precipitation and denaturation. Aqueous two-phase systems with very high water content (>80%) and low interfacial tension provide such a method. They are extremely useful for the separation of enzymes, nucleic acids and even cells. Polyethylene glycol (PEG), dextran, water mixtures will form separate phases determined by polymer concentration, molecular weight of the polymers and temperature. Purification of biomolmles can be enhanced by the attachment of affinity ligands to PEG (upper phase) which accumulates the target molecule whilst the contaminating molecules partition into the dextran phase. This system can also be used for the simultaneous production and purification of bioproducts from cells or enzymes, for instance to purify a-amylase from B. subtilis. In a PEG/dextran system the cells partition into the lower phase with 80%+ of the enzyme product in the upper phase. Polysaccharides can also be used to immobilise cells or enzymes, permitting the reuse of the catalyst and continuous flow systems. Alginates have the advantage that gel formation occurs under mild conditions, therefore cells remain viable and enzymes am not denatured but calcium gradually leaches out and the gel dissolves. Gellan or other combinations may prove superior for this application. A strain of Acinefubacter calmucetius produces an unusual polysaccharide called emulsan. It is a complex polymer comprising about 15% fatty acyl esters and 20% protein. This structure enables it to act as an emulsifying agent, stabilising hydrocarbon/water emulsions at very low concentrations (0.1-1.0%). This properly, 228 Chapter 7 coupled with its low toxicity makes it ideal as a cleaning agent and it is incorporated into hand-cleaners to remove compounds used in paints, plastics and in ink manufacture. Other possible industrial uses include cleaning of storage tanks, reducing crude oil viscosity, dispersing slurries and pigments. 7.9.4 Medical applications In medical applications some important biological properties - immunogenic, anti-tumour and anti-viral - can be exploited, as well as the established functional properties based on rheology and gel formation. dextrans sephadex encapsulabd drugs anti-viral activi CurdIan vaccines Dextrans are particularly useful and are employed as a plasma substitute. A concentration of about 6% dextran (50,000-100,000 relative molecular weight) has equivalent viscosity and colloid-osmotic properties to blood plasma. Dextran can also be used as non-irritant absorbent wound dressings, an application also suited to alginate gels. Dextran can be produced in a range of molecular weights and crossed-linked or substituted with a variety of functional groups. These products (Sephadex) are routinely used in the purification of proteins and pharmaceutical and other medically important compounds. Exopolysaccharides are used in lotions and gel formation is exploited in encapsulated drugs. The latter ap lication also takes advantage of the mouth feel and flavour Many polysaccharides of eukaryotic origin show non-speafic anti-viral activity and this property may be shared by some of the exopolysaccharides. The structural requirements for activity are not immediately evident as the polysaccharides exhibiting this activity are very diverse. Curdlan possesses anti-tumour activity similar to that shown by fungal g-JI-glucans, a property which appears to be related to the ability to form triple helices. There are a number of practical problems involved with using polysaccharides as vaccines as there are frequently too my different chemotypes for it to be practicable to prepare a vaccine. In some cases a limited number of serotypes are the dominant cause of infection and it may then be possible to produce vaccines. A mapr problem is the poor immune response elicited by polysaccharide antigens, which may in some cases be improved by chemical modification. This is the case for vaccines for IjimzopkZw influenzae type b (a causative agent of meningitis), where the antigenicity of the polysaccharide can be increased by coupling to proteins. neutrality, qualities a P so vital for the food industry. Production and applications of microbial exopolysaccharides 229 Use the information given in section 7.9 to complete the 'Important Physical Properties' column of the following table. Exopoiysaccharide Application Important Physical welan enchanced oil recovery emulsan cleaning agent dextrans plasma substitute curdlan anti-tumor agent polyethylene glycol/ purification of biological dextran water mixtures materials alginates immobilising enzymes gellan Properties use in glazes, jellies and icings 230 Chapter 7 Summary and objectives In this chapter we have seen that microbial exopolysaccharides have a wide range of applications, from fine medical to large scale industrial. They are composed mainly of carbohydrate and their molecular conformation is determined by primary structure and associations between molecules in solution. This in turn detennines their physical properties and ultimately their commercial importance. Properties that have been exploited commercially include viscoelasticity, thermostability and gel formation. Exopolysaccharides are generally produced by submerged fermentation with high carbon conversion efficiency, although the yields from oxygen are inherently poor. High viscosities of fermentation liquors can adversely affect mixing, oxygen transfer and subsequent downstream processing. A common feature of genes responsible for the control and regulation of synthesis of exopolysaccharides is their arrangement tight gene clusters; most is known for X. campesfris and E. coli. Genetic manipulation of exopolysaccharide producing organisms has not, as yet, been exploited commercially. Now that you have completed this chapter you should be able to: describe broadly the chemical composition and structure of microbial exopolysaccharides and explain how these influence their physical properties; relate applications of named microbial exopolysaccharides to their physical properties; describe media and fermentation conditions for microbial exopolysaccharides, with particular reference to xanthan and succinogl ycan; list benefits and limitations of solvent precipitation and ultrafiltration as methods of recovery of exopolysaccharides; describe the biosynthesis and regulation of microbial exopolysaccharides with particular reference to xanthan and mlanic acid.