Ion Exchange frederick j. Dechow 1.0 INTRODUCTION In 1850 Thompson reported the first ion exchange applications which used naturally occurring clays. However, ion exchange resins have only been used in biochemical and fermentation product recovery since the 1930s / In these early studies, biochemicals such as adenosine triphos phate, 4 alcohols, 5I alkaloids, 6) amino acids, 7 growth regulators, 3I hor- mones,penicillin[ol and vitamin B12 i were purified using ion exchange resins Ion exchange applications intensified following the work of Moore and Stein, [2 which showed that very complex mixtures of biochemicals, in this case amino acids and amino acid residues could be isolated from each other using the ion exchange resin as a column chromatographic separator. In biotechnology applications today, ion exchangers are important in preparing water of the necessary quality to enhance the desired microorganism activity during fermentation. Downstream of the fermentation, ion exchange resins may be used to convert, isolate, purify or concentrate the desired product or by-products. This chapter discusses ion exchange resins and their use in commercial fermentation and protein purification operations 382
Ion Exchange Frederick J. Dechow 1.0 INTRODUCTION In 1850 Thompson['] reported the first ion exchange applications which used naturally occurring clays. However, ion exchange resins have only been used in biochemical and fermentation product recovery since the 1930'~.[~1[~] In these early studies, biochemicals such as adenosine triphosphate,i4] alcohols,[5] alkaloids,[6] amino acids,['] growth regulators,[*] hormone~,[~] penicillin[10] and vitamin B12["1 were purified using ion exchange resins. Ion exchange applications intensified following the work of Moore and Stein,[12] which showed that very complex mixtures of biochemicals, in this case, amino acids and amino acid residues could be isolated from each other using the ion exchange resin as a column chromatographic separator. In biotechnology applications today, ion exchangers are important in preparing water of the necessary quality to enhance the desired microorganism activity during fermentation. Downstream of the fermentation, ion exchange resins may be used to convert, isolate, purify or concentrate the desired product or by-products. This chapter discusses ion exchange resins and their use in commercial fermentation and protein purification operations. 382
Ton Exchange 383 1.1 lon Exchange processes Processes involving ion exchange resins usually make use of ion interchange with theresin. Examples of these processes are demineralization conversion,purification and concentration. Chromatographic processes with ion exchange resins merely make use of the ionic environment that the resins provide in separating solutes Demineralization is the process in which the salts in the feed stream are removed by passing the stream through a cation exchange column in the hydrogen ion form, followed by an anion exchange column in the hydroxide or"free-base"form. Water is the most common feed stream in demineraliza tion. It may also be necessary to remove the salts from a feed stream before fermentation High metallic ion concentrations and high total salt content in the carbohydrate feed has been found to decrease the yield in citric acid fermentation. 3] These ions can be removed by passing the carbohydrate olution through cation and anion exchange resin beds. The salts required for optimum microorganism activity can be added in the desired concentration prior to fermentation Conversion or metathesis is a process in which salts of acids are converted to the corresponding free acids by reaction with the hydrogen form of a strong acid cation resin. One such example would be the conversion of calcium citrate to citric acid The terms may also be used to describe a process in which the acid salt is converted to a different salt of that acid by interaction with a ion exchang resin regenerated to the desired ionic form Many fermentation products may be purified by adsorbing them on ior exchange resins to separate them from the rest of the fermentation broth Once the resin is loaded, the product is eluted from the column for further purification or crystallization Adsorbing lysine on ion exchange resin is probably the most widely used industrial method of purifying lysine. The fermented broth is adjusted to pH20 with hydrochloric acid and then passed through a column of strong acid cation resin in the NH form. Dilute aqueous ammonia may be used to elute the lysine from the resin, [4 gordienkoll5)has reported that treating the resin with a citrate buffer solution ofpH3 2 and rinsing with distilled water before elution results in an 83-90% yield of lysine, with a purity of 93-96%
Ion Exchange 383 1.1 Ion Exchange Processes Processes involving ion exchange resins usually make use of ion interchange with the resin. Examples ofthese processes are demineralization, conversion, purification and concentration. Chromatographic processes with ion exchange resins merely make use of the ionic environment that the resins provide in separating solutes. Demineralization is the process in which the salts in the feed stream are removed by passing the stream through a cation exchange column in the hydrogen ion form, followed by an anion exchange column in the hydroxide or “free-base” form. Water is the most common feed stream in demineralization. It may also be necessary to remove the salts from a feed stream before fermentation. High metallic ion concentrations and high total salt content in the carbohydrate feed has been found to decrease the yield in citric acid fermentati~n.[’~] These ions can be removed by passing the carbohydrate solution through cation and anion exchange resin beds. The salts required for optimum microorganism activity can be added in the desired concentration prior to fermentation. Conversion or metathesis is a process in which salts of acids are converted to the corresponding free acids by reaction with the hydrogen form of a strong acid cation resin. One such example would be the conversion of calcium citrate to citric acid. The terms may also be used to describe a process in which the acid salt is converted to a different salt of that acid by interaction with a ion exchange resin regenerated to the desired ionic form. Many fermentation products may be purified by adsorbing them on ion exchange resins to separate them from the rest of the fermentation broth. Once the resin is loaded, the product is eluted from the column for further purification or crystallization. Adsorbing lysine on ion exchange resin is probably the most widely used industrial method of purifying lysine. The fermented broth is adjusted to pH 2.0 with hydrochloric acid and then passed through a column of strong acid cation resin in the NH; form. Dilute aqueous ammonia may be used to elute the lysine from the resin.[14] Gordienko[151 has reported that treating the resin with a citrate buffer solution ofpH 3.2 and rinsing with distilled water before elution results in an 83-90% yield of lysine, with a purity of 93-96%
384 Fermentation and Biochemical Engineering Handbook lon exchange can be used to concentrate valuable or toxic products of fermentation reactions in a manner similar to purification, the difference between the two processes is in the lower concentration of the desired product in the feed solution of concentration processes Shiratoll6] reported the concentration process for the antibiotic tubercidan produced from fermented rice grain using the microorganism, Streptomyces tubercidicus. Macroporous strong acid cation resin was used to concentrate the antibiotic from 700 ug/ml in the fermentation broth to 13 mg/ml when eluted with 0.25 N HCl. The yield of the antibiotic was about 1.2 Chromatographic Separation In most ion exchange operations, an ion in solution is replaced with an ion from the resin and the former solution ion remains with the resin, In contrast, ion exchange chromatography uses the ion exchange resin as an adsorption or separation media, which provides an ionic environment, allowing two or more solutes in the feed stream to be separated. The feed solution is added to the chromatographic column filled with the separation beads and is eluted with solvent, often water in the case of fermentation products. The resin beads selectively slow some solutes while others are eluted down the column(Fig. 1). As the solutes move down the column, they separate and their individual purity increases. Eventually, the solutes appear at different times at the column outlet where each can be drawn off separately Chromatographic separations can be classed according to four types depending on the type of materials being separated: affinity difference, exclusion, size exclusion and ion retardation chromatography. These types of separations may be described in terms of the distribution of the material to be separated between the phases involved Figure 2 shows a representation ofthe resin-solvent-solute components of a column chromatographic system. The column is filled with resin beads of the solid stationary phase packed together with the voids between the beads filled with solvent. The phases of interest are(i)the liquid phase between the resin beads, (ii)the liquid phase held within the resin beads and (iii)the solid phase of the polymeric matrix of the resin beads. When the feed solution is placed in contact with the hydrated resin in the chromatographic column, the solutes distribute themselves between the liquid inside the resin and that between the resin beads. The distribution for component i is defined by the distribution coefficient, K
384 Fermentation and Biochemical Engineering Handbook Ion exchange can be used to concentrate valuable or toxic products of fermentation reactions in a manner similar to purification. The difference between the two processes is in the lower concentration ofthe desired product in the feed solution of concentration processes. Shirato[l6] reported the concentration process for the antibiotic tubercidan produced from fermented rice grain using the microorganism, Streptomyces tubercidicus. Macroporous strong acid cation resin was used to concentrate the antibiotic from 700 pg/d in the fermentation broth to 13 mg/d when eluted with 0.25 N HCl. The yield of the antibiotic was about 83%. 1.2 Chromatographic Separation In most ion exchange operations, an ion in solution is replaced with an ion from the resin and the former solution ion remains with the resin. In contrast, ion exchange chromatography uses the ion exchange resin as an adsorption or separation media, which provides an ionic environment, allowing two or more solutes in the feed stream to be separated. The feed solution is added to the chromatographic column filled with the separation beads and is eluted with solvent, often water in the case of fermentation products. The resin beads selectively slow some solutes while others are eluted down the column (Fig. 1). As the solutes move down the column, they separate and their individual purity increases. Eventually, the solutes appear at different times at the column outlet where each can be drawn off separately. Chromatographic separations can be classed according to four types depending on the type of materials being separated: affinity difference, ion exclusion, size exclusion and ion retardation chromatography. These types of separations may be described in terms of the distribution of the materials to be separated between the phases involved. Figure 2 shows a representation ofthe resin-solvent-solute components of a column chromatographic system. The column is filled with resin beads ofthe solid stationary phase packed together with the voids between the beads filled with solvent. The phases of interest are (i) the liquid phase between the resin beads, (ii) the liquid phase held within the resin beads and (iii) the solid phase of the polymeric matrix of the resin beads. When the feed solution is placed in contact with the hydrated resin in the chromatographic column, the solutes distribute themselves between the liquid inside the resin and that between the resin beads. The distribution for component i is defined by the distribution coefficient, Kd,:
where Cr is the concentration of component i in the liquid within the resin bead and Cai is the concentration of component i in the interstitial liquid. The distribution coefficient for a given ion or molecule will depend upon that component's structure and concentration, the type and ionic form of the resin and the other components in the feed solution. The distribution coefficients for several organic compounds are given in Table 1. 7 Desorbent Added Added Solute Mixture 幽隙 c s Occurs Fast omen Component Removed Removed From Colun Figure 1. The steps of chromatographic separation are: addition of the mixed solutes to the column, elution to effect separations, and removal of the separated solute
Ion Exchange 385 where Cri is the concentration of component i in the liquid within the resin bead and C,, is the concentration of component i in the interstitial liquid. The distribution coefficient for a given ion or molecule will depend upon that component’s structure and concentration, the type and ionic form of the resin and the other components in the feed solution. The distribution coefficients for several organic compounds are given in Table 1 .[171 Solutes Addad 4 Solute { Mixture - Desorbent Added 4 4- sol Utes Added To col URll Separation s1 ow Occurs Fast Colrponent Component Removed FlWl col uan Removed Fron COluSn Figure 1. The steps ofchromatographic separation are: addition ofthe mixed solutes to the column, elution to effect separations, and removal of the separated solutes
386 Fermentation and Biochemical Engineering Handbook Resin Bead Interstitial Liquid Liquid in Rest r Figure 2. Representation of the three phases involved in chromatographic separatic The ratio of individual distribution coefficients is often used as a measure of the possibility of separating two solutes and is called the separation factor, a, or relative retention factor Kdy/k From Table l, the separation factors for acetone-formaldehyde separabil- ity are 0. 49, 0.98 and 1.54 for Dowex 50WX8(H"), Dowex 1X8(CI)and Dowex 1X8(So4)resins, respectively. For comparison purposes, it may be necessary to use the inverse of a, so that the values would be 2.03 and 1.02 for Dowex 50wX8(H)and Dowex 1X8(CI), respectively. When a is less han l, the solute in the numerator will exit the columnfirst. Whenais greater than l, the solute in the denominator will exit the column first
386 Fermentation and Biochemical Engineering Handbook -Resin Bead -Interstitial Llquld /iquid In @sin (v,, (Vq) Figure 2. Representation of the three phases involved in chromatographic separation. The ratio of individual distribution coefficients is often used as a measure of the possibility of separating two solutes and is called the separation factor, a, or relative retention factor. From Table 1 , the separation Mors for acetone-formaldehyde separability are 0.49, 0.98 and 1.54 for Dowex 50WX8 (H'), Dowex 1X8(C1-) and Dowex 1X8(SOi2) resins, respectively. For comparison purposes, it may be necessary to use the inverse of a, so that the values would be 2.03 and 1.02 for Dowex SOWX8(H') and Dowex 1X8(Cl-), respectively. When a is less than 1 , the solute in the numerator will exit the column first. When a is greater than 1, the solute in the denominator will exit the column first