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SEPARATIONS Process Integration of Separation of Amino Acids by a Temperature-Induced Aqueous Two-Phase System Mian Li,*,†,‡,§ Zi-Qiang Zhu,‡ and Alı´rio E. Rodrigues† Laboratory of Separation and Reaction Engineering, Faculty of Engineering, University of Porto, Rua dos Bragas, 4050-123 Porto, Portugal, and College of Chemical Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China
Random copolymers of ethylene oxide and propylene oxide (EOPO) and (hydroxypropyl)starch (PES) have been used in aqueous two-phase partitioning of amino acids. The partitioning behaviors of L-Ile and L-Val were investigated in EOPO/PES aqueous two-phase systems (ATPSs). The effects of the addition of salt and pH on the partitioning were studied. Fundamental data were employed for further scale-up to process 1000 mL of fermentation broth. An integrated bioseparation process was designed on the basis of the partitioning behavior of amino acids in the primary ATPSs and in the temperature-induced phase systems. L-Ile crystals were finally obtained through (1) selective partition in the EOPO/PES system, (2) recovery from the water phase during the temperature-induced phase separation of the EOPO phase, and (3) crystallization from the water phase by isoelectric precipitation. The mass of L-Ile crystals was 13.76 g with a purity of 97.17%. The overall recovery was 74.28%. Aqueous two-phase extraction combined with temperature-induced phase separation appears to be a promising alternative in the amino acid industry. 1. Introduction L-Isoleucine (L-Ile), like most other amino acids, is produced by fermentation processes. Because the broth is a dilute mixture of similar components, many steps are needed to reach the purity required for food and medical applications. In industry, the downstream processing of amino acids conventionally consists of several stages such as cell removal by centrifugation and filtration, ion exchange, and crystallization. The recovery and purification costs in these cases can reach up to 80% of the final product cost.1 It is, therefore, desirable that efficient and inexpensive methods be made available for the separation, concentration, and purification of amino acids from fermentation broth. Reverse micellar extraction and an immobilized liquid membrane have been used to separate amino acids.2,3 Among the new and promising strategies, aqueous two-phase systems (ATPS) have demonstrated significant potential for the successful separation of a wide variety of bioproducts under mild conditions on a small or medium scale.4,5 Recent advances in ATPS processes include utilizing environmentally benign polymers in biological, industrial, and environmental processes such as cloud-point extraction, micellar extraction, and extractions using thermoseparating polymers (J. Chro-
* To whom correspondence should be addressed. Tel: +319335-1241. Fax: +319-335-1415. E-mail: limian@engineering. uiowa.edu. † University of Porto. ‡ Zhejiang University. § Present address: Department of Chemical and Biochemical Engineering, The University of Iowa, Iowa City, IA 52242.
matogr., B 2000, 743). Because ATPS is not accompanied by a phase change, it is also considered as an energy-saving process. The most commonly used systems are poly(ethylene glycol) (PEG)/dextran or PEG/ salt systems. Kula6 stated that the relatively high consumption of chemicals to form the immiscible ATPS represents a disadvantage; thus, application on an industrial scale is prevented. In addition, the common problem encountered with ATPS is the difficulty in separating target biomolecules from the polymer solution. Because separation of the phase-forming polymers and biomolecules after the primary extraction is an important step in laboratory- and industrial-scale processes, expensive and time-consuming methods, such as ultrafiltration, electrophoresis, and chromatography, have been employed to separate bioproducts from polymer solutions. However, this separation is far from being satisfactory. To overcome the drawbacks, the use of the temperature-induced phase formation combined with an inexpensive ATPS has been employed to solve the problems of polymer removal and recycling.7-16 The ethylene oxide/propylene oxide random copolymer (EOPO), which is linear and nonionic, has a decreased solubility in water solution at higher temperatures. When heated over the lower critical solution temperature (LCST), it can be subjected to temperature-induced phase separation from the aqueous solution. A twophase system composed of an EOPO bottom phase and an aqueous top phase is consequently formed. The temperature at which this phenomenon occurs is known as the cloud point (CP) of the polymer. A 10% EOPO (1:1), for example, has a CP of 70 °C,16 while a 10% PEG (Mw ) 20 000) solution has a CP of 111.7 °C.7 The CP
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can be lowered either by increasing the PO/EO ratio or by increasing the amount of the added sodium sulfate concentrations. To date, studies have been focused on the purification of enzymes. There were, however, few studies in the application of an aqueous two-phase partition to the process of small molecular weight products. This is due to the traditional theories, such as the Brφnsted equation, which indicated that small molecules were partitioned more or less evenly between the two phases. However, work in our laboratory and others has shown some good results.17-22 For example, unequal partition of amino acids (3.7 for L-Phe and 0.13 for L-Glu) can be achieved in PEG/salt systems.22 Partitioning of amino acids in thermoseparation of polymer systems has also been investigated.16,23 In the present work, extractive purification of L-Ile whole broth based upon the partitioning of pure L-Ile in an EOPO/(hydroxypropyl)starch (PES) ATPS was studied. The aim was to separate L-Ile from other amino acids in the top EOPO phase. After extraction in the primary phase system, the top EOPOrich phase was removed and heated above the LCST. In this way L-Ile was partitioned to the water phase and further crystallized. The EOPO copolymer could be recovered and recycled for a second use.
L-Ile was produced for extraction from fermentation broth by Corynebacterium crenatum AS 1.998. Predetermined quantities of EOPO (1:1) and PES 100 stock solution were added directly to the fermentation broth (1000 mL). The entire mixture was stirred in a mechanically agitated container for 30 min. The phases were then allowed to settle for 2 h, which led to complete phase separation. 2.3. Temperature-Induced Phase Separation. After the primary phase extraction, the top EOPO copolymer phase was removed and put into a vessel. Sodium sulfate was added to the top phase to a concentration of 0.2 M. The container with this top phase was placed in a water bath at 48 °C for 30 min. After the new two-phase system had formed and separated into an aqueous top phase and an EOPO-rich bottom phase, the top phase was then removed, and both phases were analyzed to determine the amino acid partition. Partition of amino acids between the two phases is expressed by the partition coefficient, K, defined as the ratio between the concentrations of amino acids in the top and bottom phases in eq 1, where Ct and Cb are the
2. Experimental Section
concentrations of amino acid in the top and bottom phases, respectively. In addition, it is advantageous to calculate the distribution ratio, G, defined as in eq 2, where Vt and
2.1. Chemicals. EOPOs with two different molar ratios of EO to PO, 1:1 and 1:2, were produced by Zhejiang University Chemical Factory. They had average molecular weights, Mn, of 3500 and 3000, respectively. The polydispersity of EOPO samples was 1.11, which indicated that EOPO samples used in this study had a narrow molecular weight distribution. After the polymer was dissolved in water and dichloromethane was added, EOPO was extracted into the dichloromethane phase. The solution of polymer in dichloromethane was dried with magnesium sulfate. The solvent was evaporated under vacuum at 65 °C.8 PES 100 (Mn ) 10 000) and PES 200 (Mn ) 20 000) were from Carbamyl (Kristianstad, Sweden). L-Isoleucine (L-Ile) and L-valine (L-Val) were from Shanghai Chemical Reagent Corp. They were all of paper chromatography grade. All other reagents were of analytical grade. 2.2. Partitioning of Amino Acids in an ATPS. All polymer concentrations were of weight/weight percent. ATPSs of various compositions of EOPO/PES were prepared from stock solutions of EOPO [30% (w/w)] and PES [30% (w/w)]. A mixture was prepared by weighing out appropriate quantities of the stock polymer solutions. An amino acid stock solution (1 g) was added. The final phase systems had the following compositions: 10% (w/w) EOPO and 7-12% (w/w) PES, respectively, and about 14 mM amino acid. The pH was adjusted to be equal to the isoelectric point of each amino acid by the addition of a small amount of HCl or NaOH and was measured with a precision pH meter. The systems thus prepared were vortex mixed for 1 min, centrifuged at 1500 rpm for 10 min to speed up phase separation, and then allowed to equilibrate for 30 min. After the phase volumes were measured, both top and bottom phases were withdrawn separately for analysis of amino acid concentrations. Samples were run in tripilicate to determine values with 95% confidence intervals. The detailed description of the procedure has been given in a previous paper.16
K ) Ct/Cb
G ) KVt/Vb
(1)
(1)
Vb are the volumes of the top and bottom phases, respectively. G values give the ratios between the total amounts of amino acid in each phase. 2.4. Crystallization by Isoelectric Precipitation. The L-Ile extracted to the water phase was fed into an evaporative crystallizer. Supersaturation of L-Ile in the solution was achieved by vacuum evaporation. While the solution was stirred at 20-30 rpm, HCl was added dropwise to adjust the solution pH to 6.02 until precipitates began to form. Then the crystallizer stood undisturbed for 2 h for nucleation. It took another 4-6 h for L-Ile to form good crystals. After the crystals were filtered out and washed with fresh alcohol, they were dried on a vacuum desiccator and frozen for storage. The purity of L-Ile crystals was assayed by high-performance liquid chromatography (HPLC). 2.5. Analysis. Amino acids were identified and quantified24 by a Shimadzu HPLC LC-10 system (Shimadzu Corp., Kyoto, Japan) equipped with a Hypersil ODS (200 × 2.1 mm) column in combination with automated online precolumn derivatization and a fluorescence detector. The column was maintained at 53 °C. The mobile phase was 0.03 M sodium acetate and 0.1 M sodium acetate/acetonitrile (1:4). The flow rate was 0.225 mL/min. Sample aliquots (1 µL) were injected onto the column. 3. Results and Discussion 3.1. Partition of Pure Amino Acids. Figure 1 shows the results of the partitioning behavior of amino acids in EOPO (1:1)/PES100 ATPS at their isoelectric points. The hydrophobic character is considered to be the dominant factor in the present case because there is no electrostatic force contribution at the isoelectric
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Figure 1. Partition behavior of amino acids in EOPO (1:1)/PES 100 ATPSs. The concentration of EOPO (1:1) is 10% (w/w): (O) L-Ile; (0) L-Val.
Figure 2. Partition behavior of amino acids in EOPO (1:2)/PES 200 systems at 25 °C. The concentration of EOPO (1:2) is 10% (w/w): (O) L-Ile; (0) L-Val.
point nor are there any size, conformation, or biospecificity contributions to the partitioning.4 The basic equation for partitioning due to hydrophobicity can be expressed as in eq 3,25 where K is the partition coef-
RT ln K ) (R + ∆f)∆w2
(3)
ficient and ∆w2 is the PES concentration difference between the two phases, which is proportitional to the tie-line length. The solute hydrophobicity, ∆f, is defined as the value relative to that of glycine, whose hydrophobicity is taken to be zero. When this hydrophobicity assignment is made, ∆f becomes the free energy of transfer of an amino acid side chain. The phase constant R is a constant for a particular two-phase system related to the phase hydrophobicity difference. Thus, amino acid partitioning at its isoelectric point is dependent upon the phase hydrophobicity and its own relative hydrophobicity scale. The hydrophobicity scale of a single amino acid is Val < Ile;25,26 i.e., ∆f is 7357 J/mol for Ile and 5100 J/mol for Val.25R for EOPO (1:1)/PES 100 is estimated as -6300 J/mol.12 The top EOPO-rich phase is more hydrophobic in character than the bottom PESrich phase. Thus, L-Val partitioned preferably to the bottom phase, whereas L-Ile preferred the top phase, although there is a slight difference in structure between the two amino acids. In L-Ile fermentation broth, L-Val is one of the main impurities.27 This indicates that in a crude mixture of L-Ile and L-Val a desired amino acid can be separated on the basis of the difference of hydrophobicity. Figure 2 shows the effect of the molecular weight of PES on the partitioning of amino acids. Higher PES molecular weight resulted in lower partitioning coef-
Figure 3. Effect of pH on the partition behavior of L-Ile in EOPO (1:1)/PES 100 systems at 25 °C: (O) 10% (w/w) EOPO (1:1)/9% (w/w) PES 100 system; (0) 10% (w/w) EOPO (1:1)/10% (w/w) PES 100 system. Samples were run in tripilicate to determine values with 95% confidence intervals.
ficients. The K values were 1.71-3.0 for L-Ile and 0.33-0.67 for L-Val in EOPO (1:2)/PES 200 ATPS, which are lower than those in EOPO (1:1)/PES 100 ATPS. Although the recovery of the EOPO copolymer could be increased by using a higher molecular weight polymer for the bottom phase,28 the disadvantage was that PES 200 was much more viscous than PES 100. Therefore, PES 100 was chosen for the following study. 3.2. Effect of pH on L-Ile Partitioning. The effect of pH on the partitioning of L-Ile is illustrated in Figure 3. The partition coefficient took its maximum value near the isoelectric point of L-Ile (pH 6.02) and decreased toward more acidic or more alkaline pHs. In 10% EOPO (1:1)/9% PES100 ATPS, the maximum K value was 3.63 at pH 7.0, while in 10% EOPO (1:1)/10% PES100 ATPS, K was 3.39 at pH 7.5. The pI value of L-Ile is 6.02. At pH 7 or 7.5, it is negatively charged. The partitioning of charged L-Ile far from the isoelectric point may not be based on the charge effect alone. The increase and then decrease in the K value could be due to both hydrophobic interaction and net charge effect, which is a function of the polymer concentration and solution pH. 3.3. Effect of Salt on L-Ile Partitioning. It is wellknown that, in ATPS, among the most effective and frequently employed adjustments is the addition of salt.4 The addition of salt can provide more selective separations. However, while amino acids are charged materials, their partitioning appears only slightly affected by the addition of sodium sulfate in EOPO/PES ATPSs, as shown in Figure 4. The partitioning coefficient increased slightly from 3.28 at 0 M Na2SO4 to 3.42 at 0.3 M Na2SO4 in 10% EOPO (1:1)/9% PES100 ATPS. Although the effect of the salt on L-Ile partitioning was very little, the addition of sodium sulfate lowered CP by 23 °C for the EOPO (1:1) phase.16 3.4. Two-Step Extraction Using TemperatureInduced Aqueous Two-Phase Separation. After the partition behavior of individual amino acids in EOPO/ PES ATPS as well as the effect of pH and the addition of salt on amino acid partitioning was studied in order to obtain the optimal operation, the separation of L-Ile from the fermentation broth was scaled to 1000 mL using the data obtained from the 10 mL experiments. A similar process scheme using temperature-induced phase formation has been previously proposed.7 This scheme, which allows a mixture of amino acids to be separated and EOPO copolymer to be recycled, is shown in Figure 5.
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Figure 4. Effect of the addition of Na2SO4 on the partition behavior of L-Ile in EOPO (1:1)/PES 100 systems at 25 °C: (O) 10% (w/w) EOPO (1:1)/9% (w/w) PES 100 system; (0) 10% (w/w) EOPO (1:1)/10% (w/w) PES 100 system. Samples were run in tripilicate to determine values with 95% confidence intervals.
Figure 5. L-Ile extraction scheme using temperature-induced phase formation and recycling of the EOPO polymers and crystallization: 1, mixer; 2 and 3, separators; 4, evaporative crystallizer. Table 1. Composition of Amino Acids in the Fermentation Broth and in L-Ile Crystals amino acid
concn (g/L) in fermentation broth
L-Ile
18 1.5 1 1 2 2
L-Val L-Ala L-Ser L-Glu L-Thr
content in crystal (%)
L-Ile
97.17 1.94 0.64 0.00 0.13 0.10
EOPO (1:1) and PES 100 stock solutions were added directly to the fermentation broth (1000 mL) produced by C. crenatum AS 1.998.27 The pH was adjusted to 7.0. In the case of producing L-Ile, other amino acids were also produced although they were in low concentrations. The different amino acid components in the fermentation broth are shown in Table 1. The efficient recovery by partitioning depends on the volume ratio of the phases and partition coefficients of contaminating materials as well as on its own partition coefficient. To recover L-Ile with relatively low partition coefficient (3.44) to the top phase efficiently, a two-step extraction (the primary one and the back-extraction) was designed. In the first stage, after the solution was vigorously mixed and reached equilibrium, L-Ile was partitioned to the top EOPO-rich phase while L-Val favored the bottom phase in the primary ATPS. Cell debris and most of the proteins were also partitioned to the bottom phase. In the second stage, the top EOPO-rich phase was removed and sodium sulfate was added to a concentration of 0.2 M. After the temperature of this
system was increased to 48 °C over the CP (45 °C) for 30 min, a new two-phase system was achieved with a top aqueous phase and an EOPO-rich bottom phase, where the K values were about 9.5-17.7 for L-Ile and 0.18-0.29 for L-Val.16 These results are interesting in that they seem to appear contrary to the chemical behavior of these two amino acids. Because L-Ile is the more hydrophobic molecule, it would be expected to partition more strongly to the EOPO phase. However, according to experimental results, L-Ile separated into the water-rich phase, while L-Val remained in the EOPO-rich phase (the strongly hydrophobic phase). This is most likely due to the addition of Na2SO4 into EOPO/ water systems. Although the addition of Na2SO4 had less effect on L-Ile and L-Val partitioning in EOPO/PES ATPS, it may affect the partitioning in EOPO/water systems. Na+ has a strong repulsive interaction with the polymer and cannot enter the polymer phase easily, which directs the partitioning of hydrophobic amino acids to the water-rich phase. A similar observation has been reported, because the addition of NaClO4 as a counterion caused amino acids to be effectively transferred from one phase to another in UCON (EOPO polymer)/water two-phase systems.23 In the EOPO/water two-phase system, L-Ile was mostly recovered in the nonpolymer aqueous phase and can be further crystallized by isoelectric precipitation. L-Val and EOPO were in the bottom phase; thus, this EOPO copolymer could be recycled for a back-extraction of L-Ile with the original bottom phase, which still contained 9.3% L-Ile. Part of L-Ile that originally partitioned into the bottom phase could be transferred to the top EOPO-rich phase by the back-extraction step. Table 2 illustrates the efficiency of the recovery of L-Ile from the fermentation broth by a batchwise two-step extraction in a 10% (w/w) EOPO (1:1)/9% (w/w) PES 100 ATPS combined with temperature-induced phase separation. As shown in Table 2, nearly the same partitioning (3.44) was found, indicating that scaling up of the procedure did not change the parameters obtained under the laboratory (10 mL) partition experiment conditions (3.28). The results presented in Table 2 show that 89.63% of L-Ile was extracted into the top aqueous phase by aqueous two-phase separation combined with temperature-induced phase separation. Furthermore, a second back-extraction resulted in an additional recovery of 3.88%, and the total recovery could be increased up to 93.51%. In this case the EOPO copolymer was recycled once. In another case where the recycled EOPO copolymer and the fresh PES 100 made up a 10% (w/w) EOPO (1:1)/10% (w/w) PES 100 ATPS, 87.12% of L-Ile was extracted in the first extraction step and an additional 4.19% of the L-Ile was recovered in the second back-extraction, thus yielding a total recovery of 91.32%. The EOPO copolymer was totally recycled three times. It should be pointed out that the partition coefficient of L-ILE changed between the two stages from 3.44 to 0.80 because of the change of total phase compositions. In the first step, the total phase compositions were 10% EOPO (1:1)/9% PES 100, while the second one was 28.18% EOPO (1:1)/19.35% PES. This is because the recycled EOPO copolymer was mixed again with the original “bottom” phase to form a new ATPS for a second back-extraction of L-ILE. The latter one was much closer to the critical point in the phase diagram, resulting in a shorter tie-line length, which, in turn, led to lower partitioning of L-Ile. However, Persson and co-workers28
Ind. Eng. Chem. Res., Vol. 41, No. 2, 2002 255 Table 2. Two-Step Extraction of L-Ile by Aqueous Two-Phase Separation Combined with Temperature-Induced Phase Formation Using Recycled EOPO Copolymera
system
step
1. 10% EOPO (1:1)/ 9% PES 100 2. 10% EOPO (1:1)/ 10% PES 100
first extraction second back-extraction first extraction second back-extraction
aqueous two-phase extraction K (25 °C) G (25 °C) Vt/Vb 3.44 0.80 2.95 0.60
9.77 0.62 7.54 0.51
2.84 0.78 2.55 0.85
temperature-induced phase separation K (48 °C) G (48 °C) Vt/Vb 13.6 10.0 14.5 9.5
84.54 40.0 74.14 28.5
recovery (%)
total recovery (%)
89.63 3.88 87.12 4.19
93.51
6.22 4.00 5.18 3.0
91.32
a The primary phase systems were EOPO (1:1) and PES 100. K and G values at 25 °C were for the partition between EOPO (1:1)/PES 100 ATPS. K and G values at 48 °C were for the partition between the water and EOPO phases formed by the increase in temperature.
have demonstrated that the partition coefficients of lysozyme and total protein were not significantly changed during recycling of the EOPO copolymer. In their case fresh bottom phase polymer and some amount of fresh copolymer were added to the recycled copolymer; therefore, the total phase compositions after the thermoseparation would be the same as the initial concentration of the primary phase system. On the basis of the above findings, the principle of aqueous two-phase extraction is successfully applied to the extraction and purification of L-Ile from a fermentation broth. A higher degree of separation (93.51%) was thus obtained in 10% EOPO (1:1)/9% PES 100 ATPS. The recovery of EOPO copolymer could be up to 90% because of the presence of 3% (w/w) PES 100 and 10 mM sodium salt in the EOPO top phase.28 Another advantage was that the recovery of L-Ile into the water phase after the thermoseparation would make the further downstream process, crystallization, simple. The recycling of the EOPO copolymer was performed three times. However, after three times, emulsification between the phases was observed because of the buildup of L-Val and other impurities from the broth. The accumulation of impurities made the system greatly unstable. So, the EOPO copolymer needs to be purified,29 or fresh EOPO needs to be added to the system. 3.5. Crystallization by Isoelectric Precipitation. L-Ile was crystallized by isoelectric point precipitation. The solubility of amino acids depends on, among other things, the pH of the solution. At low pHs, amino acids have a net positive charge because the amine gains an extra proton. At high pHs, they have a net negative charge because the carboxyl loses its proton. The intermediate pH at which an amino acid molecule has a net charge of zero is called the isoelectric point (pI). At their pI value, amino acids have no net charge, thus leading to the reduced solubility because they are unable to interact with the medium and will then fall out of solution. The flowsheet for the crystallization is also shown in Figure 5. After the two-step extraction, L-Ile was mostly recovered in the aqueous phase and was fed into an evaporative crystallizer. Supersaturation of L-Ile in the solution was achieved by vacuum evaporation, where solvent was removed at 0 °C. L-Ile crystal was filtered out from the mother liquor. To prevent liquor from drying on the crystals, they were washed twice with fresh ethanol instead of water, because the solubility of L-Ile in water is higher than that of L-Ile in ethanol. The solid crystal product was finally dried on a vacuum desiccator. Table 1 shows the analytical results. The final mass of L-Ile was 13.76 g with a purity of 97.17%. The specific rotation (R20D) was +40.0°. The recovery yield for the
Table 3. Comparison of Several Separation Techniques for L-Ile purification method ion exchange copper salt salting out new ion exchange aqueous two-phase extraction combined with temperature-induced phase separation
purity (%)
recovery (%)
ref
94 98 94.1 98.5 97.17
51.2 50 60 60.8 74.28
30 31 32 33 this work
two-step extraction was 93.51% with a purity of 92.50%, and the yield for the crystallization step was 79.42%. The overall recovery was 74.28%. Table 3 shows a comparison of the traditional separation methods of L-Ile with the new one described in this paper. L-Ile is separated conventionally by means of ion exchange,30,33 copper salt,31 or salting out.32 Although all of these methods provided a purity of 94-98.5%, the recovery yield was relatively low. Aqueous two-phase extraction combined with temperature-induced phase separation, in contrast to other time- and energyconsuming procedures, resulted in a higher yield of 74.28% while maintaining a purity of 97.17%. The main point in using this kind of multistep procedure is that several amino acids can be separated in the same procedure. This simple and rapid method can be used to extract amino acids from the fermentation broth directly without any pretreatment of filtration. Because of the fact that the starch derivative is a low-cost substitute for dextran and the EOPO copolymer is easy to recycle, aqueous two-phase extraction combined with temperature-induced phase separation appears to be a technically feasible and environmentally benign alternative for the amino acid industry. 4. Conclusions The partition behavior of amino acids has been studied in EOPO/PES systems combined with the temperature-induced phase separation. The pH is an important influence on the partitioning of L-Ile. The K value reached its maximum of 3.63 at pH 7.0 in 10% EOPO (1:1)/9% PES100 ATPS. The process was scaled up to 1000 mL of fermentation broth where the parameters in the pilot-scale were almost consistent with those obtained under the laboratory (10 mL) partition experiments. Separation of L-Ile from the fermentation broth was achieved by the integration of aqueous-two phase extraction combined with temperature-induced phase separation and crystallization. L-Ile was first partitioned to the top EOPO phase in the primary ATPS. Then, the top phase was removed and its temperature increased above the CP of EOPO. This resulted in the formation
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of a new two-phase system with a top aqueous phase containing most of L-Ile and a bottom EOPO-rich phase, thus allowing the lower EOPO phase to be recycled for a second extraction. L-Ile was crystallized by isoelectric point precipitation. The mass of L-Ile crystals was 13.76 g with a purity of 97.17%. The overall yield was 74.28%. It demonstrates that aqueous two-phase extraction combined with temperature-induced phase separation may be considered as an economically attractive alternative for an extractive fermentation of amino acids on a large scale. Acknowledgment This work was supported by the National Natural Science Foundation of China. M.L. acknowledges the postdoctoral fellowship provided by Ministry of Science and Technology of Portugal to LSRE. Literature Cited (1) Thien, M. P.; Hatton, T. A.; Wang, D. I. C. A liquid emulsion membrane process for the separation of amino acids. Biotechnol. Bioeng. 1991, 35, 853-860. (2) Vera, J. H.; Rabie, H. R. Extraction of zwitterionic amino acids with reverse micelles in the presence of different ions. Ind. Eng. Chem. Res. 1996, 35, 3665-3672. (3) Adarkar, J. A.; Sawant, S. B.; Joshi, J. B.; Pangarkar, V. G. Extraction of amino acids using an immobilized liquid membrane. Biotechnol. Prog. 1997, 13 (4), 493-496. (4) Albertsson, P. A. Partition of Cell Particles and Macromolecules, 3rd ed.; John Wiley & Sons: New York, 1986. (5) Walter, H.; Brooks, D. Partition in Aqueous Two-Phase Systems: Theory, Methods, Uses and Applications in Biotechnology; Academic Press: Orlando, FL, 1985. (6) Kula, M. R. Trends and future prospects of aqueous twophase extraction. Bioseparation 1990, 1, 181-189. (7) Harris, P. A.; Karlstrom, G.; Tjerneld, F. Enzyme purification using temperature-induced phase formation. Bioseparation 1991, 1, 237-246. (8) Alred, P. A.; Tjerneld, F.; Kozlowski, A.; Harris, J. M. Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning. Bioseparation 1992, 2, 363-373. (9) Alred, P. A.; Tjerneld, F.; Modlin, R. F. Partitioning of ecdysteroids using temperature-induced phase separation. J. Chromatogr. 1993, 628, 205-214. (10) Alred, P. A.; Kozlowski, A.; Harris, J. M.; Tjerneld, F. Application of temperature-induced phase partitioning at ambient temperature for enzyme purification. J. Chromatogr. A 1994, 659, 289-298. (11) Modlin, R. F.; Alred, P. A.; Tjerneld, F. Utilization of temperature-induced phase separation for the purification of ecdysone and 20-hydroxyecdysone from spinach. J. Chromatogr. A 1994, 668, 229-236. (12) Li, M. Fundamental and Engineering Study on New Aqueous Two-Phase Systems: Partitioning, modeling, and technical aspects of erythromycin and amino acids. Ph.D. Dissertation, Zhejiang University, Hangzhou, China, 1996. (13) Berggren, K.; Johansson, H.-O.; Tjerneld, F. Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers. J. Chromatogr. A 1995, 718, 67-79. (14) Farkas, T.; Stalbrand, H.; Tjerneld, F. Partitioning of β-mannanase and R-galactosidase from Aspergillus niger in UCON/ Reppal aqueous two-phase systems and using temperatureinduced phase separation. Bioseparation 1996, 6, 147-157. (15) Li, M.; Zhu, Z. Q.; Wu, Y. T.; Lin, D. Q. Measurement of phase diagrams for new aqueous two-phase systems and prediction
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Received for review January 19, 2001 Revised manuscript received October 16, 2001 Accepted November 5, 2001 IE010060T
