Ind. Eng. Chem. Res. 2005, 44, 231-235
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Discrete Countercurrent Contacting: An Experimental Method for Developing Continuous Countercurrent Reactors Jeroen L. den Hollander,† Antonio Aversente, Marjon B. Diender,‡ Adrie J. J. Straathof,* and Luuk A. M. van der Wielen Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands
To facilitate experimental studies on countercurrent reactors, a discrete contacting mode was worked out experimentally and theoretically. Enzymatic hydrolysis of penicillin G to phenylacetic acid and 6-aminopenicillanic acid was carried out in biphasic aqueous organic systems without pH control. The two phases were countercurrently contacted in a discrete manner, so that equilibrium was reached in each stage. Sets of three and five shake flasks served to mimic equilibrium stages in the countercurrent setup. It was shown that discrete countercurrent contact leads to the same extent of improvement of the equilibrium conversion as continuous countercurrent contact does, when compared to the batch situation. Therefore, discrete experiments may be used to simplify the development of continuous countercurrent reactors. 1. Introduction Countercurrent reactors are contactors in which the reaction products are directly separated from the reactants and from each other by a continuous countercurrent movement of two phases (see Figure 1 for an example). This removal of products from the reaction zone may be useful for shifting equilibrium reactions to completion or for preventing uncontrolled loss or degradation of product.1,2 Moreover, product purification becomes easier; hence, the term “reactive chromatography” is also being used. Different combinations of phases can be used, for example, liquid/gas in reactive distillation,3 liquid/liquid in reactive extraction,4 and liquid/solid in reactive adsorption.5 In, the latter case, reactive simulated beds are being used. Relatively many studies on countercurrent reactors seem to be dealing with the theory rather than with experiments. The number of variables is so large that it will not be very fruitful to perform countercurrent experiments without proper understanding based on good theoretical models. However, there are several other experimental problems. (1) Dedicated equipment has to be used to obtain the calculated countercurrent flow rates at favorable holdups. (2) There may be additional hydrodynamic requirements, in particular when the catalyst is heterogeneous. (3) Preferably, the reactor should allow for sampling of both phases at several positions along the reactor axis, so that the predicted and experimental concentration profiles can be compared. (4) To reach the desired steady state, specific start-up procedures may have to be developed. All of these demands lead to relatively large investments in equipment, chemicals, and manpower, even for running just a laboratory-scale countercurrent reac* To whom correspondence should be addressed. Tel.: +3115-278 2330. Fax: +31-15-278 2355. E-mail: ajjstraathof@ tnw.tudelft.nl. † Present address: Univalid, Archimedesweg 17, NL-2333 CM Leiden, The Netherlands. ‡ Present address: Unilever Foods NA, 800 Sylvan Avenue, Englewood Cliffs, NJ 07632.
tor. The area of countercurrent reactors might develop faster if the model calculations could partly be tested using simpler experiments. The aim of the present study is to demonstrate a type of simple experiment that will be of particular value for equilibrium reactions that might be shifted to completion in a countercurrent reactor. To implement the countercurrent movement of the two phases, we suggest a discrete countercurrent contacting mode. A set of shake flasks can be used in which each flask corresponds to a single equilibrium stage. The two phases can be separated and manually transported to the neighboring shake flasks in order to simulate the countercurrent movement. In this way, samples can be taken from each phase in each equilibrium stage. Massand heat-transfer limitations and limiting reaction kinetics can be avoided, which could facilitate interpretation of the results in a continuous countercurrent system. This method differs from the classical “countercurrent distribution” method,6 which is a batch purification operation that does not reach a steady state. Before each equilibration step in our experiments, there will be feeding of the reactant into an intermediate stage. The concentrations in each stage after equilibration will be different, but after a number of equilibration steps, the concentrations will reach a steady state. In a very interesting but infrequently cited paper, Semenov et al.7 described an enzymatic peptide synthesis by an experimental method closely related to the one that we propose. A series of four batch reactors was used to simulate a countercurrent contact between two liquid phases. The reactants were not introduced in intermediate reactors, but one reactant was introduced in the first stage and one in the last stage. They found that this way of discrete countercurrent contacting was more effective than single-stage batch contacting. However, they did not elaborate on the topic and did not relate their work to continuous countercurrent contacting. We will carry out discrete countercurrent contacting for a reaction that has already been studied in a continuous countercurrent contactor.4 This reaction is
10.1021/ie0492395 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/07/2004
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Figure 1. Schematic representation of countercurrent PenG hydrolysis in biphasic systems. For an explanation, see the text.
the enzymatic hydrolysis of penicillin G (PenG) to 6-aminopenicillanic acid (APA) and phenylacetic acid (PAA). The discrete and continuous systems will be compared in order to show that the steady state of a discrete system can give a useful experimental representation of the steady state of a continuous system. APA, one of the hydrolysis products of PenG, is the main precursor for the production of semisynthetic β-lactam antibiotics, which are manufactured at a more than 20 000 ton/scale.8 In conventional processes for the production of APA, PenG is extracted from the fermentation broth using organic solvents at low pH. At these extraction conditions, PenG is in its acidic form (HPenG). After backextraction to the aqueous phase at higher pH, PenG is obtained as the potassium salt (KPenG). Subsequently, PenG is hydrolyzed at pH 7-8 in immobilized enzyme reactors to PAA and APA.9 These steps all require pH adjustment by the addition of a base, thereby leading to salt production. However, Diender et al.10 have shown that the PenG hydrolysis can also be performed without necessarily having salt production and yet obtaining a good yield. They carried out the reaction in a batch biphasic system of water and butyl acetate at low pH (3-5). At this low pH, PAA partitions mainly to the organic phase and is consequently separated from APA. A countercurrent process for PenG hydrolysis in a biphasic system is schematically shown in Figure 1. The PenG feed is introduced in a central stage of the column. In the aqueous phase, the enzymatic reaction occurs. PAA partitions to the organic phase at low pH, and APA prefers the aqueous phase. Because of the countercurrent movement of the two phases, the two products are separated in the column and reversal of hydrolysis is prevented. If the number of equilibrium stages is sufficient, PenG may be completely converted and two pure products may be obtained at two different exits. 2. Theory 2.1. Equilibrium Model. Diender et al.10 have presented a batch equilibrium model for the simultaneous reaction and partitioning of PenG, PAA, and APA in butyl acetate-water biphasic systems. With their model, it is possible to calculate the concentration of the reacting components in both the aqueous and organic phases. Because a reaction occurs, we need to set up stoichiometric balances instead of component mass balances. For PenG hydrolysis, there are two balances that follow from the stoichiometry of the hydrolysis reaction. These
are the balances for the β-lactam nucleus and the PAA side chain, respectively: org aq aq org aq nHPenG + nAPA ( + nPenG- + nHPenG + nAPA- + aq aq total total nAPA ( + nAPA+ ) nPenG,0 + nAPA,0 (1) aq aq org aq aq + norg nHPenG PAA + nPenG- + nHPenG + nPAA- + nPAA ) total total + nPAA,0 (2) nPenG,0
In eqs 1 and 2, ni is the number of moles of compound i in the organic or aqueous phase. Subscript 0 indicates the total number of moles of that compound in the initial situation. Because the organic phase molalities of the net uncharged compounds can be calculated from the partition coefficients and the aqueous phase molalities, aq aq there are nine unknown molalities: mPenG -, mHPenG, aq aq aq aq aq aq aq mPAA-, mPAA, mAPA-, mAPA(, mAPA+, mH+, and mOH-. In addition to eqs 1 and 2, five dissociation equilibria apply, of HPenGaq, PAAaq, APA+aq, APA(aq, and water, and the reaction equilibrium between PenG, APA, and PAA applies. The nineth equation needed to calculate the nine unknown molalities is the electroneutrality relation in the aqueous phase: aq aq aq aq aq mH + + mAPA+ + mK+ - mOH- - mPenG- aq aq mAPA - - mPAA- ) 0 (3)
The molality of the potassium ion is a known constant for each stage and depends on the feed composition. The system of nonlinear equations is solved using Powell’s hybrid method. This method is based on the NewtonRaphson method using a numerical approximation of the Jacobian matrix.11 Note that the proton concentration is also solved, and consequently the pH can be calculated. 2.2. Countercurrent Model. To understand the discrete countercurrent system, which is an experimental model of a continuous countercurrent system, mathematical modeling of discrete countercurrent systems will also be discussed. In a continuous countercurrent system of equilibrium stages, the concentrations at all stages can be solved simultaneously.12 The same could be done for a discrete system. However, this method does not yield information about the transient period before a steady state is reached. This transient period is the time it takes before a steady-state profile is attained in the system (comparable to the start-up time of conventional liquid-liquid extraction columns). Because we carry out the counter-
Ind. Eng. Chem. Res., Vol. 44, No. 1, 2005 233 Table 1. Details for Experiments 1 and 2 expt no.
flasks Nmax
equilib steps
water flowa (g)
1 2
5 3
6 6
20 20
BuAc flowb (g)
BuAc in the feedc (g)
20 10
10
feed KPenGd (mmol)
HPenGe (mmol)
PenGf (mmol/kg)
0.41
43.6 10.2
1.74
a The amount of water that enters flask 1. b The amount of BuAc that enters the last flask. c The amount of BuAc that enters the feed flask via the feed. d The number of millimoles of PenG added as (solid) KPenG to the feed flask. e The number of millimoles of PenG added as HPenG in BuAc to the feed flask. f The calculated amount of PenG added to the feed flask, based on the total mass of the biphasic system.
current process in a discontinuous mode, we are interested in the development of the profile in time in order to check whether the profile approached its steady-state profile. Therefore, we solve the set of equations for an equilibrium stage subsequently for all stages, for every equilibration step (dynamic solution of the system). The initial amount of a species (nj,0) for flask N is calculated from the molality of the species in the entering aqueous and organic phases from the previous equilibration step. These are the molalities in the organic phase in flask N + 1, the aqueous phase in flask N - 1, and eventually the feed stream in flask Nfeed. 3. Materials and Methods 3.1. Materials. Immobilized penicillin amidase (Assemblase, EC 3.5.1.11), the potassium salt of penicillin G (benzylpenicillanic acid, PenG), and 6-aminopenicillanic acid (APA) were kind gifts of DSM AntiInfectives (Delft, The Netherlands). Phenylacetic acid (PAA; >99%) was from Fluka (Seelze, Germany). Water was prepared with a Milli-Q water system (Millipore, Molsheim, France). Butyl acetate (BuAc; >99.7%) was from Aldrich. Water and BuAc were saturated with each other by mixing and stirring them for at least 1 h at 25 °C in a thermostated water bath (Julabo A3, Julabo Labortechnik, Seelbach, Germany). The two phases were separated in a separation funnel and stored for further use. The densities of the top and bottom phases were measured with a DMA 48 density meter (Anton Paar, Graz, Austria). The density meter was calibrated using air and water at 25 °C. The density of the lower aqueous phase was 996.8 kg‚m-3, and the density of the upper organic phase was 877.4 kg‚m-3. A stock solution of HPenG was prepared by adding KPenG to a biphasic system of water and butyl acetate (2:3 on a mass basis). By the addition of 0.3 M HCl until the pH was 2.6, HPenG was extracted into the organic phase upon intensive stirring of the system. The organic phase was separated in a separation funnel and stored at 4 °C for further use. The concentration of the HPenG stock solution in BuAc was 172 mmol/kg according to highperformance liquid chromatograph (HPLC) analysis. Lower concentrations of HPenG in saturated BuAc were obtained by diluting the stock solution by saturated BuAc. 3.2. Countercurrent Enzymatic Conversions. Multistage countercurrent contact of the two immiscible liquid phases was performed using a set of three or five shake flasks. The feed was composed of KPenG salt or HPenG in butyl acetate. The details are presented in Table 1. All experiments were done at 25 °C, and the aqueous phase contained 1 g of immobilized enzyme/20 g of aqueous phase. There will be no significant loss in enzyme activity at these conditions.13 The experimental procedure for the discontinuous countercurrent contact of the two liquid phases for a
set of three flasks is schematically visualized in Figure 2 and explained below. A similar approach is followed for a system of five shake flasks but is not described in detail. All flasks were thermostated in a water bath at 25 °C. Fresh (water-saturated) butyl acetate and fresh (butyl acetate saturated) water were introduced to the feed flask in the desired mass ratio, and enzyme was added. Subsequently, the feed (KPenG or HPenG) was introduced to this flask. The biphasic system was stirred for 30 min, which was assumed to be sufficient for more than 99% equilibration according to preliminary experiments. This was checked by observing the pH signal, which was stable in all cases, using a Metrohm electrode with a precision of 0.001 pH units. After settling of the phases, a sample of both the organic and aqueous phases was taken and filtered (0.2-µm Nylaflo nylon membrane filters; Gelman Sciences, Ann Arbor, MI) for analysis by HPLC as described before.4 After the aforementioned sampling in step 1, the organic phase was transported to flask 1 for step 2. A fresh amount of aqueous phase with enzyme was added to flask 1. A fresh amount of organic phase was added to flask 3, and the aqueous phase with enzyme from flask 2 was transported to flask 3. A new phase system and a new PenG feed were introduced to flask 2. All three flasks were equilibrated and sampled as described before. In the next steps (step 3 and further), a fresh amount of aqueous phase with enzyme was introduced to flask 1, the aqueous phase with enzyme of flask 1 was transported to flask 2, and the aqueous phase of flask 2 was transported to flask 3. A fresh amount of organic phase was introduced to flask 3, the organic phase from flask 3 was transported to flask 2, and the organic phase of flask 2 was transported to flask 1. PenG feed was introduced to flask 2, and all systems were equilibrated again. This procedure was periodically repeated. 4. Results and Discussion For experiment 1, the concentrations of PenG, APA, and PAA in the aqueous phase (Figure 3A) and in the organic phase (Figure 3B) illustrate that PAA is extracted to the organic phase and leaves the system mainly with the organic phase (flask 1). APA remains in the aqueous phase and leaves the system with this phase (flask 5). Because of this reaction and separation, a pH profile over the flasks is established, as shown in Figure 3C. In flask 1, the pH is 3.4, and in flask 5, the pH increases to 5.9. The hydrolysis reaction produces a proton.10 The released protons are partly taken up by PenG- or PAA-. The fraction of protons that is taken up by PenG- and PAA- depends on the pH. The formed neutral compounds partition to the organic phase. Because of the transport of HPenG and PAA with the organic phase, flask 1 is effectively acidified. Although potassium ions might form ion pairs with PenG- or
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Figure 2. Procedure for a three-flask experiment. The feed flask (2) is filled with enzyme and aqueous and organic phases, and PenG is fed. The system is intensively stirred for 30 min (step 1). After settling, the top and bottom phases are transported respectively to flasks 1 and 3. A fresh amount of water with enzyme is fed to flask 1, fresh BuAc is fed to flask 3, and a new feed is added to flask 2. All systems are equilibrated (step 2). This procedure is repeated for step 3 and further. For more details, see the text.
Figure 3. Measured values and model predictions for the five flasks in experiment 1 after six equilibration steps. For conditions, see Table 1. A: Molalities of APA, PenG, and PAA in the aqueous phase. B: Molalities of APA, PenG, and PAA in the organic phase. C: pH in the aqueous phase.
PAA- that can partition to the organic phase, it is assumed that this does not occur to a significant extent
in water-butyl acetate phase systems. Therefore, the potassium ions cannot be transported with the organic phase, and potassium is only present in flasks 2-5. Both the molalities of the compounds in the aqueous and organic phases and the pH can be predicted reasonably well using the presented mathematical model, as can be seen in Figure 3. The deviations may be partly attributed to the low chemical stability of PenG and APA at low pH. For this five-flask experiment, the mathematical model was checked only for its capability to predict the concentrations in the dynamic period during the first six equilibrium steps. Model calculations indicated that after six equilibrium steps the concentrations deviated 1-30% from their steadystate values. It would take 12 equilibrium steps before all concentrations would deviate by 0-4% from their steady-state value. For experiment 2, with three flasks, transient results are not shown. It was observed that the concentration of APA did not change anymore after approximately five equilibrium steps. The same was observed for the pH. The model predicts that, after six equilibrium steps, all concentrations deviate by less than 4% from their steady-state value. The largest deviation from the steady-state value is predicted for PenG in flask 3 (3.2%). On the basis of these results and model simulations, a steady state was assumed. Figure 4 shows a parity plot of the experimental molalities and the model predictions of experiment 2 and of a continuous experiment4 at virtually identical feed and flow conditions. It is clear that for the discrete system the experiment and theory agree again, except for an unexpected high experimental value of 6.0 mmol/ kg of PAA in the organic phase of the third vessel. The continuous results do not show this deviation. For the continuous reactor, the most significant deviation, which was discussed before,4 is the experimental value of 4.8 mM APA in the aqueous phase of the first vessel in the continuous setup.
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the kind gift of Assemblase. The Dutch School for Process Technology (OSPT) is thanked for partially financing the project. Literature Cited
Figure 4. Parity plots of the theoretical and experimental steadystate molalities of reactants in experiments and theories of the discrete experiment 2 of this study and of the continuous experiment III of ref 4. A particular marker may refer to PenG, APA, or PAA; in either the aqueous or organic phase; and in either flask 1, 2, or 3.
The pH is not shown in a parity plot because at these particular feed and flow conditions it was theoretically in the range 3.6-3.7 in any vessel for the discrete as well as for the continuous setup, and experimentally this was confirmed, except for one sample with pH 3.4. 5. Conclusions A series of shake flasks can be used conveniently to study, in a discrete mode, the performance of the same series of equilibrium stages in a continuous countercurrent reactor setup. Theoretically, one would expect almost the same degree of improvement of PenG hydrolysis to PAA and APA in discrete and in continuous countercurrent reactors, as compared to a batch reactor. Experimentally, this was indeed observed, with reasonable accuracy, according to the concentrations of the components and the pH at every equilibrium stage. The convenience of the discrete countercurrent experiments therefore may simplify the development of continuous countercurrent reactors. Acknowledgment Cor Ras is thanked for setting up the HPLC analysis for the antibiotics. DSM is gratefully acknowledged for
(1) Podgornik, A.; Tennikova, T. B. Chromatographic reactors based on biological activity. Adv. Biochem. Eng./Biotechnol. 2002, 76, 165. (2) Fricke, J.; Schmidt-Traub, H.; Kawase, M. Chromatographic reactors. Ullmann’s Encyclopedia of Industrial Chemistry, 6th ed.; 2000, electronic release. (3) Malone, M. F.; Doherty, M. F. Reactive distillation. Ind. Eng. Chem. Res. 2000, 39, 3953. (4) den Hollander, J. L.; Zomerdijk, M.; Straathof, A. J. J.; Van der Wielen, L. A. M. Continuous enzymatic penicillin G hydrolysis in countercurrent water-butyl acetate biphasic systems. Chem. Eng. Sci. 2002, 57, 1591. (5) Lode, F.; Houmard, M.; Migliorini, C.; Mazzotti, M.; Morbidelli, M. Continuous reactive chromatography. Chem. Eng. Sci. 2001, 56, 269. (6) Craig, L. C.; Hogeboom, G. H.; Carpenter, F. H.; du Vigneaud, V. Separation and characterization of some penicillins by the method of counter-current distribution. J. Biol. Chem. 1947, 168, 665. (7) Semenov, A. N.; Gatchok, A. P.; Titov, M. I.; Martinek, K. Enzymes in preparative organic synthesis. Chemical equilibrium in countercurrent biphasic water-organic systems. Biotechnol. Lett. 1989, 11, 875. (8) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations; Wiley-VCH: Weinheim, Germany, 2000. (9) Harrison, F. G.; Gibson, E. D. Approaches for reducing the manufacturing costs of 6-aminopenicillanic acid. Process Biochem. 1984, 19, 33. (10) Diender, M. B.; Straathof, A. J. J.; Van der Does, T.; Ras, C.; Heijnen, J. J. Equilibrium modelling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid precipitation. Biotechnol. Bioeng. 2002, 78, 395. (11) Powell, M. J. D. A hybrid method for nonlinear algebraic equations. In Numerical Methods for Nonlinear Algebraic Equations; Rabinowitz, P., Ed.; Gordon and Breach: London, 1970; p 87. (12) den Hollander, J. L.; Straathof, A. J. J.; Van der Wielen, L. A. M. Performance of fractionating reactors in the absence of rate limitations. J. Chem. Technol. Biotechnol. 2004, 79, 1025. (13) Ferreira, J. S.; Straathof, A. J. J.; Franco, T. T.; Van der Wielen, L. A. M. Activity and stability of immobilized penicillin amidase at low pH values. J. Mol. Catal. B: Enzym. 2004, 27, 29.
Received for review August 19, 2004 Revised manuscript received November 4, 2004 Accepted November 22, 2004 IE0492395
