Production and applications of microbial exopolysaccharides 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 tricarb 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 production are given be KH2PO. Glutamate FeSO4 7H2O MnSO47H2O Glucose H3BO4 MgsO4 7H2O CuSO4. 5H20 Cocl2. 6H2O CaCl26H2O 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 Mg, Co, phate(PO2)is the major anionic component Fumaric acid is a TCa cycle intermediate and may improve metabolic balance through the catabolic pathways and oxidation through the TCa cle. Peptone may improve growth through the provision of growth factors(amino acids, vitamins nucleotides) limiting The balance between carbon substrate and a growth limiting nutrient also influence ensure good yields of exopolysaccharide. However, very high cell density cultures 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 phorous have also been studied. The type of li ng nutrient has be 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 industrial Media used for laboratory studies of exopolysaccharide ction may vary producton considerably from industrial production media. In laboratory pure substrates med 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: cost of pure substrate product yield for the substrate the quality of the product required
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 high-quality 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 mos commonly used substrates for industrial production are: glucose from cane or beet starch or starch hydrolysates com syrup 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 com starch; cereal grain hydrolysates For industrial production, the nitrogen source may be a relatively cheap proteinaceous product, such as · yeast hydrolysate; distillers solubles casein hydrolysate · soybean meal 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 medium 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 salt for example, calcium, copper and iron, can have adverse effects on both the growth of the organism and the rheological properties of the exopolysaccharides
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 SAQ 7.3 A medium for the production of an exopolysaccharide in batch culture has the following component Glucose ucainic ac Ammonium chloride (limiting substrate) Yeast hydrolysate Potassium Phosphorous Magnesium sulphate 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 gg,what concentration of biomass would you expect if ammonia was added to a concentration of 25gr? 3)If the growth yield coefficient for magnesium for the organism grown in the medium described in 2)was 200 gg, what concentration of biomass would you expect if magnesium was added to this medium at a concentration of 0.2 1? 7.5 Fermentation steps The various steps involved in commercial production of exopolysaccharide are 1)Strain maintenance 2)Inoculum train. 3)Exopolysaccharide production(50-200 m) 4) Enzyme treatment to modify properties, eg filterability 5)Concentration via precipitation or ultrafiltration 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 othersare moculum lyophilised (freeze-dried), or frozen under nitrogen, or held at.C in 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
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