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Separation of isomaltose from high sugar concentrated enzyme reaction mixture by dealuminated β-zeolite
Separation and Purification Technology 38 (2004) 129–138
Separation of isomaltose from high sugar concentrated enzyme reaction mixture by dealuminated -zeolite S. Berensmeier, K. Buchholz∗ Department for Carbohydrates, Technical University Braunschweig, Langer Kamp 5, D-38106 Braunschweig, Germany Received in revised form 23 October 2003; accepted 31 October 2003
Abstract The adsorption of isomaltose from high sugar concentrated reaction mixture was systematically investigated using static experiments. The isotherm data for the single-components glucose, fructose, and isomaltose fit the Langmuir adsorption model well. The product isomaltose has much more affinity for the zeolite than the other carbohydrates, which allows in situ separation process. Isomaltose is separated in situ from the enzyme reaction mixture by dealuminated -zeolites (Si/Al = 75) to avoid further reactions involving it. © 2003 Published by Elsevier B.V. Keywords: Adsorption; -Zeolite; Fluid phase; Isomaltose
1. Introduction The prediction of adsorption behavior of mixtures is important for modelling and design of adsorption processes for separation and purification. The production of isomaltose by immobilized dextransucrase (DS) using glucosyl transfer from sucrose, the substrate, to glucose, the acceptor, was used to demonstrate this approach. Meanwhile by-product fructose is released in the medium. Using sucrose as a versatile substrate, a limited range of oligosaccharides, such as isomaltose, leucrose, panose, and others, can be obtained when appropriate acceptors like glucose, fructose or maltose are used in the enzyme reaction [1–5]. These and other ∗ Corresponding author. Tel.: +49-531-3917260; fax: +49-531-2344783. E-mail address: [email protected] (K. Buchholz).
1383-5866/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.seppur.2003.10.012
dextransucrase-catalyzed acceptor reactions have an interesting impact on industrial operations, because they offer new opportunities to synthesize defined oligosaccharides that up to now have been difficult to produce either by enzymatic or by chemical synthesis using nucleotide-activated sugars. However, mixtures of saccharides are obtained that require appropriate separation processes. As glucose is only an intermediate acceptor, dextran formation from the simple substrate reaction is not completely repressed. Under likely reaction conditions, such as 50 g l−1 sucrose and 500 g l−1 glucose, with a temperature of 30 ◦ C and pH 5.4, it is possible to shift the reaction pathway toward optimal isomaltose formation and selectivity. That means that the normal dextran formation from sucrose by dextransucrase will be largely repressed and high isomaltose end concentration will be reached. Based on the fact that isomaltose can also be glucosylated,
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Nomenclature ci ce ki Ki KiEx mad m0 qi qiEx qi,s qs Vl θ
initial concentration (g l−1 ) equilibrium concentration (g l−1 ) adsorption constant Henry constant or affinity parameter (ml g−1 ) excess Henry constant or affinity parameter (ml g−1 ) mass of adsorbent (g) mass of added water (g) equilibrium concentration of adsorbed component (mg g−1 ) excess equilibrium concentration of adsorbed component (mg g−1 ) maximal concentration of adsorbed component (mg g−1 ) void volume (mg g−1 ) volume of liquid-phase (ml) covered fraction of surface
an intermediate selective separation of the product from the high concentrated substrate solution in a continuously working reactor will be necessary to favor high product yields and degrees of purity. This will be accomplished using a multiphase reactor. The special feature of this kind of reactor is that it has three different phases. With regard to the bio-transformation, the reactor works as a fluidized/expanded biocatalyst bed. Sieves hold the immobilized enzyme inside the reactor. The second phase, the substrate solution (sucrose and glucose), is pumped from a separate reservoir to the bottom of the reactor. For isomaltose separation, the reactor also works like a tubular reactor with suspended zeolite fed together with the substrate solution, which selectively adsorbs the product. The product-loaded adsorbents are small enough to pass the sieves and are pumped out of the reactor together with unreacted substrate solution. In the downstream processing that follows, the adsorbed product shall desorbed after zeolite filtration and washing. Concerning the filtration the particle size of zeolite shall not be too small in order to avoid pressure problems. Based on the previous work of Buttersack et al. [6], the adsorption process for the downstream processing
of the product was chosen, since dealuminated zeolite of the -zeolite (BEA) type is a selective adsorbent for carbohydrates in aqueous solution. Heterogeneous catalysis connected closely with adsorption plays a significant role in modern industrial and environmental protection [7]. The future development of adsorption science represents a major opportunity for research and industry, since better adsorption processing may reduce downstream costs, as well as allowing regenerability of most of the used adsorbents and the potential to design and manufacture novel materials specific for a given need. Adsorption processes based on molecular sieving and selectivity are always reversible in theory and are usually reversible in practice. This allows the zeolite to be reused many times, cycling between the adsorption and desorption steps of actual processes. This emphasizes the considerable economic value of zeolites in adsorption applications. Industrial synthetic zeolites represent a variety of microporous crystalline Si/Al-oxide powders with particle dimensions in the micrometer range. The main traditional applications are cation exchange, adsorption, and catalysis, using extrudates or other forms of compacted zeolite powder. Significant innovation can improve mass and heat transfer to realize the ultimate integration of chemical conversion and product separation processes. Zeolites are characterized not only by high selectivity (selective separation mechanism), but also by the ability to separate substances based on differences in molecular size and shape (steric separation mechanism). Zeolite crystallographic lattices have uniform window sizes. Consequently, species with molecular diameters too large to pass through zeolite pores are effectively excluded. Commercial adsorption processes for separating liquid mixtures are accomplished by selective adsorption of certain substances from their mixtures. For those purposes, the adsorbent pore system is only slightly limited by diffusion; separation is caused mainly by selective adsorption that depends upon van der waals forces between the constituents of the liquid mixtures. A fundamental and important feature of industrial sorbents is their high porosity and usually high surface area as well as their specific sorption sites. That is why their most important characteristics are total pore volume (qs ), pore size distribution over the pore diameter, and specific surface area.
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An important tool in adsorption process design methodology is the measurement of adsorption isotherms. These isotherms have to be interpreted in terms suitable for theoretical models. This point is of importance since experimental techniques for the measurement of multi-component sorption data are generally very time-consuming and difficult to interpret. In this study single, binary, and multi-component adsorption experiments on dealuminated -zeolites have been systematically investigated in the face of a continuous integrated product separation in a multiphase enzyme reactor. Further investigations were done with regard to particle size and agglomeration of zeolite and desorption of products by ethanol.
perimentally accessible values as follows: qiEx =
2. Experimental set-up and theory
Dealuminated -zeolites of Si/Al = 12.5 and 150 were obtained from Zeolyst International (USA) and of Si/Al = 50 and 75 from Süd-Chemie AG (Munich, Germany). Both companies use different methods to synthesize dealuminated zeolites. Zeolyst International dealuminates after synthesis, whereas the Süd-Chemie AG adjusted during snthesis to the desired modulus value. All zeolites were treated with 0.2 M NaCl and the pH was adjusted to 5.4 with HCl. After heating for 2 h at 100 ◦ C, the zeolites were washed with water and dried at 100 ◦ C. After sieving (200 m) to delete aggregates, the zeolites were activated at 400 ◦ C for 4 h. Particle size analysis was performed by laser diffraction spectrometry both without and after ultrasonic treatment (Sympatec, Helos, Germany).
(ci − ce ) Vl Mi mad
(1)
where Vl Mi denotes the total amount of substances in the bulk and surface phases, and ci − ce is the difference between the concentrations of the ith component before and after adsorption (see Section 2.6). The absolute adsorption equilibrium constant is calculated with regard to the specific pore volume, which is obtained by measurement of the equilibrium between the zeolite and an aqueous solution of a substance (here sucrose) that cannot penetrate the pores by the following equation: qi =
2.1. Zeolites
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ci − (1 − qs (mad /m0 ))ce mad ((1/Vl ) + ((1/m0 )ce ))
(2)
The variation in solid-phase concentration of the sorbate, qi (mg g−1 of adsorbent) as a function of the solute equilibrium concentration ce (g l−1 ) in liquid is expressed by an isotherm. One of the basic relations often used for liquid adsorption is the Langmuir isotherm [8] which is given by the following equation: θ=
k i ce qi = qi,s 1 + k i ce
(3)
where θ is the fraction of surface covered by gas or liquid sorbate molecules and qi,s is the maximal amount of sorbate molecules adsorbed for full surface coverage, which is constant for a sorbate-adsorbent system at a given temperature. For ce → 0 the adsorption equilibrium constant Ki , an analogue to the Henry constant of gas-phase adsorption, is defined as: qi = Ki ce
(4)
Ki = ki qi,s
(5)
2.2. Batch experiments For equilibrium liquid-phase adsorption 1 g of dry zeolite was well mixed with 5 ml of the corresponding component in 25 mM Ca acetate, pH 5.4, by shaking for 244 h at 30 ◦ C, which is given by the optimal reaction conditions of the enzyme (see below). Analysis of concentration–time curves was not required. The adsorption excess qiEx of the ith component, related to the mass of adsorbent mad , can be expressed by ex-
It correlates the affinity between sorbate and adsorbent. The ratio of the two Ki values measures the selectivity and the possibility to separate different components. After some transformations of the Langmuir model, the following equations can be obtained for n-component liquid mixtures: θ=
qi k i ce = qi,s 1 + ki ce + j kj cj,e
(6)
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2.3. Enzyme preparations The enzymes were purchased by Critt Bio-Industries (Toulouse, France). Dextransucrase was produced by standard fed-batch culture with the constitutive mutant Leuconostoc mesenteroides NRRL B-512F with sucrose inducer [9,10]. The enzyme has a specific activity of 0.478 U mg−1 . 2.4. Immobilization method [11] All the immobilization steps were carried out at 4 ◦ C. The immobilization of dextransucrase from L. mesenteroides was carried out by ionotropic gel formation with sodium alginate. A mixture of 30% (w/w) silica flour and sand was added to the alginate before the gelation process to increase the bead density for application in a fluidized bed reactor. The mixture was pushed through a needle with an internal diameter of 0.55 mm. The drops fell into a gently stirred vessel containing 200 mM CaCl2 as the crosslinking agent. The beads with 1.5% (w/w) alginate and 1.4 g cm−3 density were stirred in the CaCl2 solution for a further 20 min to increase their mechanical stability. The bead diameter ranged from 1.8 to 2.2 mm. 2.5. Isomaltose formation The sucrose/glucose test solution was composed of 2523 mM glucose (500 g l−1 ) and 146 mM sucrose (50 g l−1 ) in a 25 mM calcium acetate buffer at pH 5.4. Exactly 18 ml of this solution was incubated with 2.8 g alginate beads (4 unit g−1 ) with and without 10 or 20% (v/v) -zeolite (Si/Al = 75). Samples were
taken after 0, 60, 120, 180, 240, 420, 600, 1200, and 1440 min. The samples were heated at around 100 ◦ C for 5 min to inactivate leaked enzyme. The sucrose and isomaltose content of the samples was analyzed by HPLC. 2.6. HPLC analysis Samples were analyzed by HPLC with an aminobound analytical column [Lichrospher-NH2 (5 m), 250 × 4 mm i.d., E. Merck/VWR, Darmstadt, Germany], with acetonitrile:water (80:20, v/v) eluent and a refractive index detector. In general, centrifugation (5 min at 12,000×g), dilution and membrane filtration (0.2 m) were sufficient for sample preparation.
3. Results 3.1. Choice of zeolite Based on the previous investigations of Buttersack et al. [6], dealuminated -zeolites with different Si/Al ratios were chosen for use. This kind of zeolite, an analogue of the natural zeolite Tschernichite, has a two-dimensional network of 12-membered ring channels (7.6 × 6.4 Å) with crossed 10-membered ring channels (5.5 × 6.5 Å) (Fig. 1). Buttersack et al. [6] showed that the excess adsorption equilibrium constant KEx at 25 ◦ C for Si/Al = 12.5 is much higher for the product isomaltose (KEx = 4 ml g−1 ) than for the three other components, glucose (KEx = 0.6 ml g−1 ), fructose (KEx = 0.8 ml g−1 ) and sucrose (KEx = −0.2 ml g−1 ), which
Fig. 1. Framework of -zeolite.
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is excluded from the pores (negative KEx ) in static experiments. The different disaccharide selectivities were explained by the molecular size and presence of the ␣-(1,2)--fructosyl subunit of sucrose, since other oligosaccharides with the same subunits like raffinose (KEx = −0.2 ml g−1 ), stachyose (KEx = −0.2 ml g−1 ), and melezitose (KEx = −0.2 ml g−1 ) are also excluded. However, they also demonstrated that exclusion is not in every case limited by large structures, such as isomaltose and gentiobiose (KEx = 7 ml g−1 ), which both consist of 1,6-linked glucosyl units that are rather flexible because of their three free rotating bonds [12,13]. However, sucrose has not this 1,6-glucosic bond and this may be the reason, why isomaltose is adsorbed and sucrose not. Adsorption of carbohydrates with hydroxyl groups on dealuminated zeolite should be dependent on the Si/Al ratio, which is relevant for hydrophobic or hydrophilic pore surfaces. Predictions of adsorption equilibriums are difficult to perform, since hydrophilic carbohydrates have also hydrophobic regions due to their CH groups [13]. Thus, both hydrophobic and hydrophilic interactions are responsible for adsorption on dealuminated zeolites [14–18]. The di- or oligosaccharides may rotate around their glycosidic bonds to find an optimal fit for their hydrophobic sites [14]. We did not obtain a linear correlation of increasing affinity (KiEx ) of fructose, glucose, sucrose and isomaltose versus increasing modulus (Si/Al) of the four different zeolites (Table 1) determined at initial sugar concentrations of 10 g l−1 . These low concentrations were chosen to obtain first estimations. However, sucrose could not enter the pores of any of these zeolites, as shown by the negative excess adsorption equilibrium constants qiEx and KiEx . Dextran, the second product, was not tested, because it did not leave
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Table 2 Void volume qs , which is determined by totally exclusion of sucrose of the pores
-Zeolite -Zeolite -Zeolite -Zeolite a
Si/Al
qs (mg g−1 )
12.5 25 75 150
231 202 158 119
± ± ± ±
3a 2 1 6
Data from [23,24].
the alginate beads during the enzyme reaction and could not contact the reaction medium. Isomaltose, the desired product, is adsorbed preferentially over the other sugars. The zeolite modulus 75 was best suited for further application, because it not only shows the highest affinity for isomaltose but also has low affinities for fructose and glucose. Isomaltose separation selectivity is improved with increasing differences Ki . The fact that there was no direct correlation between modulus and affinity can be tentatively explained by the different methods to synthesize dealuminated zeolites. Zeolites with moduli of 12.5 and 150 were dealuminated after synthesis, where the lattice structure could be partially destroyed and active surfaces could be lost, resulting in lower sugar affinities. In contrast, the zeolites with moduli of 50 and 75 were adjusted during synthesis to the desired modulus value, which leads to the expected increasing affinity with increasing modulus. This results let guess that this synthesis method is the better one for our purposes. For calculating the real liquid-phase loading, the pore volume of the dry zeolite must be known. This volume was obtained by measuring the totally excluded sucrose, since only buffer can fill the pores (Table 2). The pore volume decreases with increasing modulus similar to the zeolite Modenit [19].
Table 1 Excess adsorption equilibrium constants qiEx and KiEx of sugars on dealuminated -zeolites at 30 ◦ C (ci = 10 g l−1 , Vl /mad = 5 ml g−1 ) Si/Al = 12.5a qiEx (mg g−1 ) Fructose Glucose Isomaltose Sucrose a b
6.91 5.45 16.74 −1.75
Si/Al = 25b KiEx (ml g−1 ) 0.80 0.61 4.13 −0.17
Zeolites from Zeolyst International. Zeolites from Süd-Chemie AG.
qiEx (mg g−1 ) 5.01 2.83 17.49 −2.22
Si/Al = 75b KiEx (ml g−1 ) 0.55 0.31 2.52 −0.20
qiEx (mg g−1 ) 6.96 3.11 30.97 −1.70
Si/Al = 150a KiEx (ml g−1 ) 0.76 0.33 8.68 −0.15
qiEx (mg g−1 ) 3.83 0.73 19.05 −1.28
KiEx (ml g−1 ) 0.41 0.08 2.86 −0.12
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Fig. 2. Mean particle diameter of -zeolites with different moduli (Si/Al = 12.5, 25, 75, and 150) without and with ultrasound treatment (5, 10, and 15 min).
3.2. Particle size For their application in the enzyme reactor with immobilized enzyme particles, zeolite particles must be smaller than the latter but nevertheless not too small. The upper particle size is that of the alginate beads, to ensure that the zeolite particles can be separated by sieving. The lower particle size limit is such that filtration is possible, which is necessary following product desorption. The first condition is satisfied by all four zeolites: they are all bidisperse. But the Zeolyst zeolites (Si/Al = 12.5 and 150) are much smaller than those from Süd-Chemie (Si/Al = 25 and 75) (Fig. 2). Ultrasound treatment shows that the zeolites agglomerate (Fig. 3), which is important for their use in the reactor and the downstream process. But as well all types of zeolites are stable in aqueous solution and no slow dissolution of the zeolite could be observed. The zeolite with the Si/Al ratio of 75 has the best properties for practical application, with a 8–170 m particle size range, decreasing to 8–60 m after 15 min of ultrasound treatment (Fig. 3). The larger the particle size of the zeolite the better is the possibility to separate the zeolite from the not reacted substrate solution by filtration with minimized practical effort. 3.3. Single-component adsorption -Zeolite with Si/Al = 75 in batch experiments exhibits Langmuir adsorption behavior for the
Fig. 3. Correlation of ultrasound treatment and particle size of the -zeolite (Sl/Al = 75).
single-component systems isomaltose, fructose, and glucose (Fig. 4). The solid curves are Langmuir isotherms. Sucrose is not adsorbed by these types of zeolites. Isomaltose, the reaction product, has a significantly higher affinity for the zeolite than glucose or fructose, as deduced from the sharp increase of its adsorption capacity with its increasing solution concentration. Similar behavior also occurred with the zeolites of Si/Al = 12.5 and 25, whereas adsorption on -zeolite with Si/Al = 150 is better described by a Freundlich isotherm (data not shown). Additional should be
Fig. 4. Adsorption isotherms of single carbohydrates on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C. Solid lines: correlated Langmuir model (ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
S. Berensmeier, K. Buchholz / Separation and Purification Technology 38 (2004) 129–138 Table 3 Adsorption capacity qi,s and adsorption equilibrium constant Ki of sugars on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C
Fructose Glucose Isomaltose
qi,s (mg g−1 )
Ki (ml g−1 )
161 ± 13 112 ± 14 94 ± 1.4
1.05 ± 5 × 10−4 0.65 ± 3 × 10−4 14.91 ± 0.13
(ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
mentioned that isomaltose has a much lower affinity for the last three dealuminated zeolites. Adsorption capacity qi,s and adsorption equilibrium constant Ki (Table 3), which are deduced from the Langmuir isotherm (Eqs. (3)–(5)) confirm the affinities (KiEx ) between carbohydrates and zeolite (Table 1). Furthermore, a high Ki value does not correlate with a high total adsorption capacity qi,s .
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Table 4 Adsorption capacity qi,s and adsorption equilibrium constant Ki of isomaltose on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C Ki (ml g−1 ) Without glucose 115 g l−1 glucose 230 g l−1 glucose 350 g l−1 glucose
14.91 8.89 6.07 4.57
± ± ± ±
0.13 0.10 0.04 0.01
qi,s (mg g−1 ) 93.8 95.7 96.6 96.9
± ± ± ±
1.4 4.2 4.2 3.1
(ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ) at different glucose concentrations.
Few investigations have determined adsorption behavior of liquid saccharide mixtures on zeolites by static experiments [6,14,15], whereas dynamic methods like chromatographic separation are well established [20–22]. It is of particular importance to know how isomaltose adsorption behavior is influenced by high glucose concentrations in the reaction medium (500 g l−1 ), since glucose can also be adsorbed. Isomaltose adsorption with 115, 230 and
350 g l−1 glucose is depicted in Fig. 5, with curves showing calculated Langmuir isotherms (binary form of Eq. (6)) fitting the data well. These data indicate that increasing glucose concentration decreases the affinity of isomaltose for the adsorbent, whereas total adsorption capacities remain the same (Table 4). Ideal Langmuir behavior is confirmed by Fig. 6, which shows a linear ratio of Ki values for both sugars as a function of the ratios of adsorption amounts and equilibrium concentrations. Furthermore, glucose adsorption from the same binary mixture decreases with increasing isomaltose concentration (Fig. 7). This shows that high isomaltose formation rates and final concentrations as well as lower glucose concentrations will improve isomaltose separation from the product solution. In the next step mixed model solutions with sucrose, fructose, glucose and isomaltose were analyzed.
Fig. 5. Adsorption isotherms of isomaltose on dealuminated -zeolite (Si/Al = 75) with 115, 230 or 350 g l−1 glucose at 30 ◦ C. Solid line correlated Langmuir model for single-components (ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
Fig. 6. Relationship of adsorbed glucose and isomaltose concentrations to their equilibrium concentrations in solution. The straight line indicates simulated ideal bi-Langmuir behavior (Kglucose /Kisomaltose = 0.07; 30 ◦ C; Si/Al = 75).
3.4. Multi-component adsorption
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Fig. 7. Adsorption of glucose on dealuminated -zeolite (Si/Al = 75) in the presence of 5–200 g l−1 isomaltose (Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
Maximal concentrations under optimal reaction conditions were chosen to check if isomaltose adsorption will be favorable under the reaction conditions (Fig. 8). Glucose concentration was varied while holding the amount of sucrose, fructose, and isomaltose constant. Sucrose was excluded, as expected, when both fructose and glucose were combined. Isomaltose was adsorbed, although high concentration of glucose and fructose compete for adsorption sites on the zeolite. Fig. 9. (A) Acceptor reactions of immobilized dextransucrase with and without zeolite (B) adsorption capacity qi of isomaltose on zeolite during the acceptor reaction (0.6 U ml−1 ; 10% (v/v) alginate beads; 10% or 20% (v/v) zeolite; 50 g l−1 sucrose + 500 g l−1 glucose; 30 ◦ C).
3.5. Heterogeneous catalysis and adsorption
Fig. 8. Adsorbed amount qi of isomaltose, glucose and fructose from five standard multi-component solutions, all of which have the same isomaltose (ci = 20 g l−1 ), fructose (ci = 25 g l−1 ), and sucrose (ci = 50 g l−1 ) concentrations but varying glucose concentrations (ci = 100, . . . , 500 g l−1 ); (-zeolite (Si/Al = 75); Vl /mad = 5 ml g−1 ; qs = 158 mg g−1 ; 30 ◦ C).
To integrate the enzyme reaction and adsorption step for isomaltose formation and isolation, the enzyme-catalyzed acceptor reaction was combined with adsorption. If 10 or 20% (v/v) zeolites were added to the reaction under standard conditions, the isomaltose concentration should decrease compared to the same reaction without zeolites (Fig. 9A). These shifts correlate with time-dependent isomaltose adsorption. Adsorption capacities qi correlate fairly well with Langmuir parameters when 350 g l−1 glucose was added to isomaltose (Fig. 9B). The quality of fit should increase if the parameters
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Fig. 10. Excess adsorption qiEx of isomaltose, glucose and fructose on -zeolite (Si/Al = 75) in the presence of different concentrated ethanol–water solutions at room temperature.
had been determined with around 500 g l−1 glucose (Fig. 7). Sucrose concentrations were always higher than in the reaction without zeolites due to sucrose exclusion. To obtain complete product adsorption from the reaction mixture, further investigation will be required. The amount of zeolite must be adapted to the enzyme activity and a continuous exchange of loaded and unloaded zeolites must be achieved. Furthermore, an optimal zeolite residence time must be determined to guarantee optimal isomaltose and minimal glucose and fructose adsorption. 3.6. Desorption experiments To investigate desorption, adsorption experiments with different concentrations of non-polar ethanol in water were performed for all adsorbed sugars. Isomaltose, glucose and fructose, which were adsorbed without ethanol, were not adsorbed when only 5% (v/v) ethanol was present (Fig. 10). This result shows that the hydrophobic interaction decreases when the water solvent is replaced by ethanol, and that complete desorption is possible by elution with a water–ethanol mixture.
4. Conclusions Different synthesized dealuminated -zeolites with different moduli were investigated to determine their affinities for the isomaltose, glucose, sucrose and
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fructose in the reaction mixture. A expected correlation between moduli and affinity was impossible, because of the different synthesis methods. Data from static experiments with -zeolite (Si/Al = 75) correlated reasonably well with the Langmuir adsorption isotherm, and isotherm parameters (Ki and qi ) were calculated for single-components. Isotherms exhibit about a 15-fold higher affinity of isomaltose for the zeolite compared to glucose. Thus, an isomaltose adsorption on special dealuminated -zeolite is possible even when much higher glucose concentrations compete for adsorption sites. A bi-Langmuir model is suitable to characterize the isomaltose/glucose binary mixture. Isomaltose adsorption in an integrated system during the dextransucrase acceptor reaction is possible as well. Zeolite separation from the reaction mixture by filtration is given by the optimal particle size and the product desorption is possible by elution with 5% (v/v) ethanol.
Acknowledgements This research was supported by the German Research Foundation (DFG) by means of the Collaborative Research Center 578 ‘Development of Biotechnological Processes by Integrating Genetic and Engineering Methods’. Material support by Hayashibara Biochemical Laboratories Inc. and helpful discussion by Mr. C. Buttersack are gratefully acknowledged.
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[15] C. Buttersack, W. Wach, K. Buchholz, Adsorption of glucose and fructose containing disaccharides on different faujasites, Stud. Surf. Sci. Calatal. 84 (1994) 1363–1371. [16] W. Wach, C. Buttersack, K. Buchholz, Special zeolites for saccharide chromatography, in: Abstracts Pharmaceutical Engineering and Food Processing, ACHEMA, 1994. [17] W. Wach, Wechselwirkungen von Kohlenhydraten mit Zeolithen vom Faujasit-Typ, Dissertation, 1994. [18] C. Buttersack, H. Rudolph, J. Mahrholz, K. Buchholz, High specific interaction of polymers with the pores of hydrophobic zeolites, Langmuir—ACS J. Surf. Colloids 12 (13) (1996) 3101–3106. [19] C. Moreau, R. Durand, S. Razigade, J. Duhamet, P. Faugeras, P. Rivalier, P. Ros, G. Avignon, Dehydration of fructose to 5-hydroxymethylfurfural over H-modenites (A3487), Appl. Catal. A Gen. 145 (1996) 211–224. [20] R.V. Jasra, Liquid-phase sorption of higher alkanes and alkenes in zeolites NaZSM-5 at 10, 30 and 50 ◦ C, Separ. Sci. Technol. 32 (9) (1997) 1571–1588. [21] C.B. Ching, K. Hidajat, M.N. Rathor, Chromatographic evaluation of sorption and diffusion characteristics of glucose, maltose and maltotriose in silica gels, J. Chromatogr. A 463 (1989) 261–270. [22] P. Vrátný, J. Coupek, S. Vozka, Z. Hostomska, Accelerated reversed-phase chromatography of carbohydrate oligomers, J. Chromatogr. A 254 (1983) 143–155. [23] I. Fornefett, Adsorption und Chromatographie von Kohlenhydraten und anderen Hydroxyverbindungen an Zeolithen, Dissertation, 1999. [24] C. Buttersack, I. Fornefett, K. Buchholz, Separation of carbohydrates from aqueous solution by selective adsorption on apolar zeolites, in: DECHEMA-Jahrestagung. 1996, Wiesbaden.
Separation of isomaltose from high sugar concentrated enzyme reaction mixture by dealuminated -zeolite S. Berensmeier, K. Buchholz∗ Department for Carbohydrates, Technical University Braunschweig, Langer Kamp 5, D-38106 Braunschweig, Germany Received in revised form 23 October 2003; accepted 31 October 2003
Abstract The adsorption of isomaltose from high sugar concentrated reaction mixture was systematically investigated using static experiments. The isotherm data for the single-components glucose, fructose, and isomaltose fit the Langmuir adsorption model well. The product isomaltose has much more affinity for the zeolite than the other carbohydrates, which allows in situ separation process. Isomaltose is separated in situ from the enzyme reaction mixture by dealuminated -zeolites (Si/Al = 75) to avoid further reactions involving it. © 2003 Published by Elsevier B.V. Keywords: Adsorption; -Zeolite; Fluid phase; Isomaltose
1. Introduction The prediction of adsorption behavior of mixtures is important for modelling and design of adsorption processes for separation and purification. The production of isomaltose by immobilized dextransucrase (DS) using glucosyl transfer from sucrose, the substrate, to glucose, the acceptor, was used to demonstrate this approach. Meanwhile by-product fructose is released in the medium. Using sucrose as a versatile substrate, a limited range of oligosaccharides, such as isomaltose, leucrose, panose, and others, can be obtained when appropriate acceptors like glucose, fructose or maltose are used in the enzyme reaction [1–5]. These and other ∗ Corresponding author. Tel.: +49-531-3917260; fax: +49-531-2344783. E-mail address: [email protected] (K. Buchholz).
1383-5866/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/j.seppur.2003.10.012
dextransucrase-catalyzed acceptor reactions have an interesting impact on industrial operations, because they offer new opportunities to synthesize defined oligosaccharides that up to now have been difficult to produce either by enzymatic or by chemical synthesis using nucleotide-activated sugars. However, mixtures of saccharides are obtained that require appropriate separation processes. As glucose is only an intermediate acceptor, dextran formation from the simple substrate reaction is not completely repressed. Under likely reaction conditions, such as 50 g l−1 sucrose and 500 g l−1 glucose, with a temperature of 30 ◦ C and pH 5.4, it is possible to shift the reaction pathway toward optimal isomaltose formation and selectivity. That means that the normal dextran formation from sucrose by dextransucrase will be largely repressed and high isomaltose end concentration will be reached. Based on the fact that isomaltose can also be glucosylated,
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Nomenclature ci ce ki Ki KiEx mad m0 qi qiEx qi,s qs Vl θ
initial concentration (g l−1 ) equilibrium concentration (g l−1 ) adsorption constant Henry constant or affinity parameter (ml g−1 ) excess Henry constant or affinity parameter (ml g−1 ) mass of adsorbent (g) mass of added water (g) equilibrium concentration of adsorbed component (mg g−1 ) excess equilibrium concentration of adsorbed component (mg g−1 ) maximal concentration of adsorbed component (mg g−1 ) void volume (mg g−1 ) volume of liquid-phase (ml) covered fraction of surface
an intermediate selective separation of the product from the high concentrated substrate solution in a continuously working reactor will be necessary to favor high product yields and degrees of purity. This will be accomplished using a multiphase reactor. The special feature of this kind of reactor is that it has three different phases. With regard to the bio-transformation, the reactor works as a fluidized/expanded biocatalyst bed. Sieves hold the immobilized enzyme inside the reactor. The second phase, the substrate solution (sucrose and glucose), is pumped from a separate reservoir to the bottom of the reactor. For isomaltose separation, the reactor also works like a tubular reactor with suspended zeolite fed together with the substrate solution, which selectively adsorbs the product. The product-loaded adsorbents are small enough to pass the sieves and are pumped out of the reactor together with unreacted substrate solution. In the downstream processing that follows, the adsorbed product shall desorbed after zeolite filtration and washing. Concerning the filtration the particle size of zeolite shall not be too small in order to avoid pressure problems. Based on the previous work of Buttersack et al. [6], the adsorption process for the downstream processing
of the product was chosen, since dealuminated zeolite of the -zeolite (BEA) type is a selective adsorbent for carbohydrates in aqueous solution. Heterogeneous catalysis connected closely with adsorption plays a significant role in modern industrial and environmental protection [7]. The future development of adsorption science represents a major opportunity for research and industry, since better adsorption processing may reduce downstream costs, as well as allowing regenerability of most of the used adsorbents and the potential to design and manufacture novel materials specific for a given need. Adsorption processes based on molecular sieving and selectivity are always reversible in theory and are usually reversible in practice. This allows the zeolite to be reused many times, cycling between the adsorption and desorption steps of actual processes. This emphasizes the considerable economic value of zeolites in adsorption applications. Industrial synthetic zeolites represent a variety of microporous crystalline Si/Al-oxide powders with particle dimensions in the micrometer range. The main traditional applications are cation exchange, adsorption, and catalysis, using extrudates or other forms of compacted zeolite powder. Significant innovation can improve mass and heat transfer to realize the ultimate integration of chemical conversion and product separation processes. Zeolites are characterized not only by high selectivity (selective separation mechanism), but also by the ability to separate substances based on differences in molecular size and shape (steric separation mechanism). Zeolite crystallographic lattices have uniform window sizes. Consequently, species with molecular diameters too large to pass through zeolite pores are effectively excluded. Commercial adsorption processes for separating liquid mixtures are accomplished by selective adsorption of certain substances from their mixtures. For those purposes, the adsorbent pore system is only slightly limited by diffusion; separation is caused mainly by selective adsorption that depends upon van der waals forces between the constituents of the liquid mixtures. A fundamental and important feature of industrial sorbents is their high porosity and usually high surface area as well as their specific sorption sites. That is why their most important characteristics are total pore volume (qs ), pore size distribution over the pore diameter, and specific surface area.
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An important tool in adsorption process design methodology is the measurement of adsorption isotherms. These isotherms have to be interpreted in terms suitable for theoretical models. This point is of importance since experimental techniques for the measurement of multi-component sorption data are generally very time-consuming and difficult to interpret. In this study single, binary, and multi-component adsorption experiments on dealuminated -zeolites have been systematically investigated in the face of a continuous integrated product separation in a multiphase enzyme reactor. Further investigations were done with regard to particle size and agglomeration of zeolite and desorption of products by ethanol.
perimentally accessible values as follows: qiEx =
2. Experimental set-up and theory
Dealuminated -zeolites of Si/Al = 12.5 and 150 were obtained from Zeolyst International (USA) and of Si/Al = 50 and 75 from Süd-Chemie AG (Munich, Germany). Both companies use different methods to synthesize dealuminated zeolites. Zeolyst International dealuminates after synthesis, whereas the Süd-Chemie AG adjusted during snthesis to the desired modulus value. All zeolites were treated with 0.2 M NaCl and the pH was adjusted to 5.4 with HCl. After heating for 2 h at 100 ◦ C, the zeolites were washed with water and dried at 100 ◦ C. After sieving (200 m) to delete aggregates, the zeolites were activated at 400 ◦ C for 4 h. Particle size analysis was performed by laser diffraction spectrometry both without and after ultrasonic treatment (Sympatec, Helos, Germany).
(ci − ce ) Vl Mi mad
(1)
where Vl Mi denotes the total amount of substances in the bulk and surface phases, and ci − ce is the difference between the concentrations of the ith component before and after adsorption (see Section 2.6). The absolute adsorption equilibrium constant is calculated with regard to the specific pore volume, which is obtained by measurement of the equilibrium between the zeolite and an aqueous solution of a substance (here sucrose) that cannot penetrate the pores by the following equation: qi =
2.1. Zeolites
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ci − (1 − qs (mad /m0 ))ce mad ((1/Vl ) + ((1/m0 )ce ))
(2)
The variation in solid-phase concentration of the sorbate, qi (mg g−1 of adsorbent) as a function of the solute equilibrium concentration ce (g l−1 ) in liquid is expressed by an isotherm. One of the basic relations often used for liquid adsorption is the Langmuir isotherm [8] which is given by the following equation: θ=
k i ce qi = qi,s 1 + k i ce
(3)
where θ is the fraction of surface covered by gas or liquid sorbate molecules and qi,s is the maximal amount of sorbate molecules adsorbed for full surface coverage, which is constant for a sorbate-adsorbent system at a given temperature. For ce → 0 the adsorption equilibrium constant Ki , an analogue to the Henry constant of gas-phase adsorption, is defined as: qi = Ki ce
(4)
Ki = ki qi,s
(5)
2.2. Batch experiments For equilibrium liquid-phase adsorption 1 g of dry zeolite was well mixed with 5 ml of the corresponding component in 25 mM Ca acetate, pH 5.4, by shaking for 244 h at 30 ◦ C, which is given by the optimal reaction conditions of the enzyme (see below). Analysis of concentration–time curves was not required. The adsorption excess qiEx of the ith component, related to the mass of adsorbent mad , can be expressed by ex-
It correlates the affinity between sorbate and adsorbent. The ratio of the two Ki values measures the selectivity and the possibility to separate different components. After some transformations of the Langmuir model, the following equations can be obtained for n-component liquid mixtures: θ=
qi k i ce = qi,s 1 + ki ce + j kj cj,e
(6)
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2.3. Enzyme preparations The enzymes were purchased by Critt Bio-Industries (Toulouse, France). Dextransucrase was produced by standard fed-batch culture with the constitutive mutant Leuconostoc mesenteroides NRRL B-512F with sucrose inducer [9,10]. The enzyme has a specific activity of 0.478 U mg−1 . 2.4. Immobilization method [11] All the immobilization steps were carried out at 4 ◦ C. The immobilization of dextransucrase from L. mesenteroides was carried out by ionotropic gel formation with sodium alginate. A mixture of 30% (w/w) silica flour and sand was added to the alginate before the gelation process to increase the bead density for application in a fluidized bed reactor. The mixture was pushed through a needle with an internal diameter of 0.55 mm. The drops fell into a gently stirred vessel containing 200 mM CaCl2 as the crosslinking agent. The beads with 1.5% (w/w) alginate and 1.4 g cm−3 density were stirred in the CaCl2 solution for a further 20 min to increase their mechanical stability. The bead diameter ranged from 1.8 to 2.2 mm. 2.5. Isomaltose formation The sucrose/glucose test solution was composed of 2523 mM glucose (500 g l−1 ) and 146 mM sucrose (50 g l−1 ) in a 25 mM calcium acetate buffer at pH 5.4. Exactly 18 ml of this solution was incubated with 2.8 g alginate beads (4 unit g−1 ) with and without 10 or 20% (v/v) -zeolite (Si/Al = 75). Samples were
taken after 0, 60, 120, 180, 240, 420, 600, 1200, and 1440 min. The samples were heated at around 100 ◦ C for 5 min to inactivate leaked enzyme. The sucrose and isomaltose content of the samples was analyzed by HPLC. 2.6. HPLC analysis Samples were analyzed by HPLC with an aminobound analytical column [Lichrospher-NH2 (5 m), 250 × 4 mm i.d., E. Merck/VWR, Darmstadt, Germany], with acetonitrile:water (80:20, v/v) eluent and a refractive index detector. In general, centrifugation (5 min at 12,000×g), dilution and membrane filtration (0.2 m) were sufficient for sample preparation.
3. Results 3.1. Choice of zeolite Based on the previous investigations of Buttersack et al. [6], dealuminated -zeolites with different Si/Al ratios were chosen for use. This kind of zeolite, an analogue of the natural zeolite Tschernichite, has a two-dimensional network of 12-membered ring channels (7.6 × 6.4 Å) with crossed 10-membered ring channels (5.5 × 6.5 Å) (Fig. 1). Buttersack et al. [6] showed that the excess adsorption equilibrium constant KEx at 25 ◦ C for Si/Al = 12.5 is much higher for the product isomaltose (KEx = 4 ml g−1 ) than for the three other components, glucose (KEx = 0.6 ml g−1 ), fructose (KEx = 0.8 ml g−1 ) and sucrose (KEx = −0.2 ml g−1 ), which
Fig. 1. Framework of -zeolite.
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is excluded from the pores (negative KEx ) in static experiments. The different disaccharide selectivities were explained by the molecular size and presence of the ␣-(1,2)--fructosyl subunit of sucrose, since other oligosaccharides with the same subunits like raffinose (KEx = −0.2 ml g−1 ), stachyose (KEx = −0.2 ml g−1 ), and melezitose (KEx = −0.2 ml g−1 ) are also excluded. However, they also demonstrated that exclusion is not in every case limited by large structures, such as isomaltose and gentiobiose (KEx = 7 ml g−1 ), which both consist of 1,6-linked glucosyl units that are rather flexible because of their three free rotating bonds [12,13]. However, sucrose has not this 1,6-glucosic bond and this may be the reason, why isomaltose is adsorbed and sucrose not. Adsorption of carbohydrates with hydroxyl groups on dealuminated zeolite should be dependent on the Si/Al ratio, which is relevant for hydrophobic or hydrophilic pore surfaces. Predictions of adsorption equilibriums are difficult to perform, since hydrophilic carbohydrates have also hydrophobic regions due to their CH groups [13]. Thus, both hydrophobic and hydrophilic interactions are responsible for adsorption on dealuminated zeolites [14–18]. The di- or oligosaccharides may rotate around their glycosidic bonds to find an optimal fit for their hydrophobic sites [14]. We did not obtain a linear correlation of increasing affinity (KiEx ) of fructose, glucose, sucrose and isomaltose versus increasing modulus (Si/Al) of the four different zeolites (Table 1) determined at initial sugar concentrations of 10 g l−1 . These low concentrations were chosen to obtain first estimations. However, sucrose could not enter the pores of any of these zeolites, as shown by the negative excess adsorption equilibrium constants qiEx and KiEx . Dextran, the second product, was not tested, because it did not leave
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Table 2 Void volume qs , which is determined by totally exclusion of sucrose of the pores
-Zeolite -Zeolite -Zeolite -Zeolite a
Si/Al
qs (mg g−1 )
12.5 25 75 150
231 202 158 119
± ± ± ±
3a 2 1 6
Data from [23,24].
the alginate beads during the enzyme reaction and could not contact the reaction medium. Isomaltose, the desired product, is adsorbed preferentially over the other sugars. The zeolite modulus 75 was best suited for further application, because it not only shows the highest affinity for isomaltose but also has low affinities for fructose and glucose. Isomaltose separation selectivity is improved with increasing differences Ki . The fact that there was no direct correlation between modulus and affinity can be tentatively explained by the different methods to synthesize dealuminated zeolites. Zeolites with moduli of 12.5 and 150 were dealuminated after synthesis, where the lattice structure could be partially destroyed and active surfaces could be lost, resulting in lower sugar affinities. In contrast, the zeolites with moduli of 50 and 75 were adjusted during synthesis to the desired modulus value, which leads to the expected increasing affinity with increasing modulus. This results let guess that this synthesis method is the better one for our purposes. For calculating the real liquid-phase loading, the pore volume of the dry zeolite must be known. This volume was obtained by measuring the totally excluded sucrose, since only buffer can fill the pores (Table 2). The pore volume decreases with increasing modulus similar to the zeolite Modenit [19].
Table 1 Excess adsorption equilibrium constants qiEx and KiEx of sugars on dealuminated -zeolites at 30 ◦ C (ci = 10 g l−1 , Vl /mad = 5 ml g−1 ) Si/Al = 12.5a qiEx (mg g−1 ) Fructose Glucose Isomaltose Sucrose a b
6.91 5.45 16.74 −1.75
Si/Al = 25b KiEx (ml g−1 ) 0.80 0.61 4.13 −0.17
Zeolites from Zeolyst International. Zeolites from Süd-Chemie AG.
qiEx (mg g−1 ) 5.01 2.83 17.49 −2.22
Si/Al = 75b KiEx (ml g−1 ) 0.55 0.31 2.52 −0.20
qiEx (mg g−1 ) 6.96 3.11 30.97 −1.70
Si/Al = 150a KiEx (ml g−1 ) 0.76 0.33 8.68 −0.15
qiEx (mg g−1 ) 3.83 0.73 19.05 −1.28
KiEx (ml g−1 ) 0.41 0.08 2.86 −0.12
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Fig. 2. Mean particle diameter of -zeolites with different moduli (Si/Al = 12.5, 25, 75, and 150) without and with ultrasound treatment (5, 10, and 15 min).
3.2. Particle size For their application in the enzyme reactor with immobilized enzyme particles, zeolite particles must be smaller than the latter but nevertheless not too small. The upper particle size is that of the alginate beads, to ensure that the zeolite particles can be separated by sieving. The lower particle size limit is such that filtration is possible, which is necessary following product desorption. The first condition is satisfied by all four zeolites: they are all bidisperse. But the Zeolyst zeolites (Si/Al = 12.5 and 150) are much smaller than those from Süd-Chemie (Si/Al = 25 and 75) (Fig. 2). Ultrasound treatment shows that the zeolites agglomerate (Fig. 3), which is important for their use in the reactor and the downstream process. But as well all types of zeolites are stable in aqueous solution and no slow dissolution of the zeolite could be observed. The zeolite with the Si/Al ratio of 75 has the best properties for practical application, with a 8–170 m particle size range, decreasing to 8–60 m after 15 min of ultrasound treatment (Fig. 3). The larger the particle size of the zeolite the better is the possibility to separate the zeolite from the not reacted substrate solution by filtration with minimized practical effort. 3.3. Single-component adsorption -Zeolite with Si/Al = 75 in batch experiments exhibits Langmuir adsorption behavior for the
Fig. 3. Correlation of ultrasound treatment and particle size of the -zeolite (Sl/Al = 75).
single-component systems isomaltose, fructose, and glucose (Fig. 4). The solid curves are Langmuir isotherms. Sucrose is not adsorbed by these types of zeolites. Isomaltose, the reaction product, has a significantly higher affinity for the zeolite than glucose or fructose, as deduced from the sharp increase of its adsorption capacity with its increasing solution concentration. Similar behavior also occurred with the zeolites of Si/Al = 12.5 and 25, whereas adsorption on -zeolite with Si/Al = 150 is better described by a Freundlich isotherm (data not shown). Additional should be
Fig. 4. Adsorption isotherms of single carbohydrates on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C. Solid lines: correlated Langmuir model (ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
S. Berensmeier, K. Buchholz / Separation and Purification Technology 38 (2004) 129–138 Table 3 Adsorption capacity qi,s and adsorption equilibrium constant Ki of sugars on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C
Fructose Glucose Isomaltose
qi,s (mg g−1 )
Ki (ml g−1 )
161 ± 13 112 ± 14 94 ± 1.4
1.05 ± 5 × 10−4 0.65 ± 3 × 10−4 14.91 ± 0.13
(ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
mentioned that isomaltose has a much lower affinity for the last three dealuminated zeolites. Adsorption capacity qi,s and adsorption equilibrium constant Ki (Table 3), which are deduced from the Langmuir isotherm (Eqs. (3)–(5)) confirm the affinities (KiEx ) between carbohydrates and zeolite (Table 1). Furthermore, a high Ki value does not correlate with a high total adsorption capacity qi,s .
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Table 4 Adsorption capacity qi,s and adsorption equilibrium constant Ki of isomaltose on dealuminated -zeolite (Si/Al = 75) at 30 ◦ C Ki (ml g−1 ) Without glucose 115 g l−1 glucose 230 g l−1 glucose 350 g l−1 glucose
14.91 8.89 6.07 4.57
± ± ± ±
0.13 0.10 0.04 0.01
qi,s (mg g−1 ) 93.8 95.7 96.6 96.9
± ± ± ±
1.4 4.2 4.2 3.1
(ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ) at different glucose concentrations.
Few investigations have determined adsorption behavior of liquid saccharide mixtures on zeolites by static experiments [6,14,15], whereas dynamic methods like chromatographic separation are well established [20–22]. It is of particular importance to know how isomaltose adsorption behavior is influenced by high glucose concentrations in the reaction medium (500 g l−1 ), since glucose can also be adsorbed. Isomaltose adsorption with 115, 230 and
350 g l−1 glucose is depicted in Fig. 5, with curves showing calculated Langmuir isotherms (binary form of Eq. (6)) fitting the data well. These data indicate that increasing glucose concentration decreases the affinity of isomaltose for the adsorbent, whereas total adsorption capacities remain the same (Table 4). Ideal Langmuir behavior is confirmed by Fig. 6, which shows a linear ratio of Ki values for both sugars as a function of the ratios of adsorption amounts and equilibrium concentrations. Furthermore, glucose adsorption from the same binary mixture decreases with increasing isomaltose concentration (Fig. 7). This shows that high isomaltose formation rates and final concentrations as well as lower glucose concentrations will improve isomaltose separation from the product solution. In the next step mixed model solutions with sucrose, fructose, glucose and isomaltose were analyzed.
Fig. 5. Adsorption isotherms of isomaltose on dealuminated -zeolite (Si/Al = 75) with 115, 230 or 350 g l−1 glucose at 30 ◦ C. Solid line correlated Langmuir model for single-components (ci = 0, . . . , 200 g l−1 , Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
Fig. 6. Relationship of adsorbed glucose and isomaltose concentrations to their equilibrium concentrations in solution. The straight line indicates simulated ideal bi-Langmuir behavior (Kglucose /Kisomaltose = 0.07; 30 ◦ C; Si/Al = 75).
3.4. Multi-component adsorption
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Fig. 7. Adsorption of glucose on dealuminated -zeolite (Si/Al = 75) in the presence of 5–200 g l−1 isomaltose (Vl /mad = 5 ml g−1 , qs = 158 mg g−1 ).
Maximal concentrations under optimal reaction conditions were chosen to check if isomaltose adsorption will be favorable under the reaction conditions (Fig. 8). Glucose concentration was varied while holding the amount of sucrose, fructose, and isomaltose constant. Sucrose was excluded, as expected, when both fructose and glucose were combined. Isomaltose was adsorbed, although high concentration of glucose and fructose compete for adsorption sites on the zeolite. Fig. 9. (A) Acceptor reactions of immobilized dextransucrase with and without zeolite (B) adsorption capacity qi of isomaltose on zeolite during the acceptor reaction (0.6 U ml−1 ; 10% (v/v) alginate beads; 10% or 20% (v/v) zeolite; 50 g l−1 sucrose + 500 g l−1 glucose; 30 ◦ C).
3.5. Heterogeneous catalysis and adsorption
Fig. 8. Adsorbed amount qi of isomaltose, glucose and fructose from five standard multi-component solutions, all of which have the same isomaltose (ci = 20 g l−1 ), fructose (ci = 25 g l−1 ), and sucrose (ci = 50 g l−1 ) concentrations but varying glucose concentrations (ci = 100, . . . , 500 g l−1 ); (-zeolite (Si/Al = 75); Vl /mad = 5 ml g−1 ; qs = 158 mg g−1 ; 30 ◦ C).
To integrate the enzyme reaction and adsorption step for isomaltose formation and isolation, the enzyme-catalyzed acceptor reaction was combined with adsorption. If 10 or 20% (v/v) zeolites were added to the reaction under standard conditions, the isomaltose concentration should decrease compared to the same reaction without zeolites (Fig. 9A). These shifts correlate with time-dependent isomaltose adsorption. Adsorption capacities qi correlate fairly well with Langmuir parameters when 350 g l−1 glucose was added to isomaltose (Fig. 9B). The quality of fit should increase if the parameters
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Fig. 10. Excess adsorption qiEx of isomaltose, glucose and fructose on -zeolite (Si/Al = 75) in the presence of different concentrated ethanol–water solutions at room temperature.
had been determined with around 500 g l−1 glucose (Fig. 7). Sucrose concentrations were always higher than in the reaction without zeolites due to sucrose exclusion. To obtain complete product adsorption from the reaction mixture, further investigation will be required. The amount of zeolite must be adapted to the enzyme activity and a continuous exchange of loaded and unloaded zeolites must be achieved. Furthermore, an optimal zeolite residence time must be determined to guarantee optimal isomaltose and minimal glucose and fructose adsorption. 3.6. Desorption experiments To investigate desorption, adsorption experiments with different concentrations of non-polar ethanol in water were performed for all adsorbed sugars. Isomaltose, glucose and fructose, which were adsorbed without ethanol, were not adsorbed when only 5% (v/v) ethanol was present (Fig. 10). This result shows that the hydrophobic interaction decreases when the water solvent is replaced by ethanol, and that complete desorption is possible by elution with a water–ethanol mixture.
4. Conclusions Different synthesized dealuminated -zeolites with different moduli were investigated to determine their affinities for the isomaltose, glucose, sucrose and
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fructose in the reaction mixture. A expected correlation between moduli and affinity was impossible, because of the different synthesis methods. Data from static experiments with -zeolite (Si/Al = 75) correlated reasonably well with the Langmuir adsorption isotherm, and isotherm parameters (Ki and qi ) were calculated for single-components. Isotherms exhibit about a 15-fold higher affinity of isomaltose for the zeolite compared to glucose. Thus, an isomaltose adsorption on special dealuminated -zeolite is possible even when much higher glucose concentrations compete for adsorption sites. A bi-Langmuir model is suitable to characterize the isomaltose/glucose binary mixture. Isomaltose adsorption in an integrated system during the dextransucrase acceptor reaction is possible as well. Zeolite separation from the reaction mixture by filtration is given by the optimal particle size and the product desorption is possible by elution with 5% (v/v) ethanol.
Acknowledgements This research was supported by the German Research Foundation (DFG) by means of the Collaborative Research Center 578 ‘Development of Biotechnological Processes by Integrating Genetic and Engineering Methods’. Material support by Hayashibara Biochemical Laboratories Inc. and helpful discussion by Mr. C. Buttersack are gratefully acknowledged.
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