J . Chem. Tech. Biotechnol. 1992, 55, 191-199
A Study on the Removal of Urea from Aqueous Solution with Immobilized Urease and Electr odialysis Ting-Chia Huang* & Dong-Hwang Chen Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70 101 (Received 5 February 1992; revised version received I 5 April 1992; accepted 26 May 1992)
Abstract : A five-compartment electrodialyzer with immobilized urease was developed for the removal of urea from aqueous solution. The immobilized urease, supported on polyurethane foam, was placed in the central (dilute) compartment, where urea was hydrolyzed and the products NH; and COg-/HCO, were removed simultaneously by electrodialysis. The system was studied both under constant current and under constant voltage. The effects of urea concentration and applied current or voltage on the removal of urea and ammonium ions from the dilute solution were investigated. The variations of the pH of dilute solution, the current or voltage of system, and current efficiency were also examined during reactionelectrodialysis. The removal of urea by enzymic reaction was not affected significantly by the applied electric field. The current efficiencies for removing ammonium ions from dilute solution were mostly within 40-80’41, and the removal percentage of ammonium ions was dependent on current density and current efficiency.
Key words: membrane reactor-separator, immobilized urease, electrodialysis, urea-removal electrodialyzer, urea hydrolysis, polyurethane foam.
1 INTRODUCTION
NOTATION
Membrane separation has been used in various chemical, biochemical, a n d biomedical processes.’-4 Recent interest has centered on the combination of membrane separation with b i o ~ a t a l y s i s . ~T.h~e so-called reactor-separator in which simultaneous catalysis and removal o f products from the resulting reaction mixtures has particular attractions.‘. Ton exchange membrane electrodialysis has been utilized effectively in the d e s a l i n a t i ~ n ~a n. ~d concentrationln of sea water, the removal o r recovery of heavy metal ions a n d toxic compounds in waste water,’’-l5 desalination in the food a n d pharmaceutical industries,3. 1 6 . 1 7 the separation of amino acids and proteins,”. ’* the isolation of trace components from blood plasma,’ and the production of acids and b a s e s 3 The electrodialyzer was formerly used mostly as a separation unit with few reports of its combination with biocatalysts in a reactor-separator. Notable examples were the urea-
Effective area of membrane (cm2) Current efficiency (%) Faraday’s constant (coulomb mol-’) Current density (mA cm-2) Current (mA) Mole flux of ammonium ions (mmolcm-2 min-’) Ammonium ion concentration (mmol dm-3) Ammonium ion concentration in the concentrate solution (mmol dm-’) Time (min) Voltage (volts) Volume of concentrate solution (dms)
’
* To whom correspondence should be addressed. 191 J . Chem. Tech. Biotechnol. 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain 13-2
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Fig. 1. Urea-removal electrodialyzer and flow scheme. A : Anion exchange membrane (Selemion AMV); C : cation exchange membrane (Selemion CMV); P: five-head tubing pump; T: teflon frame; 1 : immobilized enzyme PU foam; 2 : P U foam; 3 : PVC spacer; 4:rubber gasket; 5: graphite (anode); 6 : stainless steel (cathode); 7 : platinum wire; 8 : adhesive foam sheet; 9: dilute solution (urea solution); 10: concentrate solution; 11 : electrode rinse; 12: constant-temperature water bath; 13: stir bar; 14: variable area
flowmeter.
removal dialyzer and electrodialyzer containing a comfermentation broths and the development of the dialyzate posite of immobilized urease membrane and anion regeneration system for artificial kidneys. Of course, it is exchange membrane.'g~20 However, it is now recognized also valuable to other similar chemical, biochemical, and that the product of urea hydrolysis by urease is biomedical processes. ammonium carbamate rather than ammonium carbona t e . 2 1 . 2 z Although ammonium carbamate rapidly breaks 2 EXPERIMENTAL down into ammonium ~ a r b o n a t e ,it' ~was found that the urea-removal dialyzer and electrodialyzer reported previously cannot remove ammonium-nitrogen from the 2.1 Materials feed solution of urea completely due to the backflow of carbamate ions across the anion exchange membrane.1g~20 Urease from Jack beans (Type 111) was purchased from In this study, a reactor-separator combining imSigma Chemical Co. (St Louis, Missouri). Urea and mobilized urease and electrodialysis was developed for bovine serum albumin were guaranteed reagents of E. Merck (Darmstadt). Glutaraldehyde (50 YOw/v in water) the removal of urea from aqueous solution. Polyurethane was the product of Fluka Chemie AG, Switzerland. (PU) foam was used as the support of immobilized Except those used for preparing PU foam, all other urease because it has a favorable hydrodynamic property owing to its quasi-spherical membrane structure.23 chemicals were extra pure grade or guaranteed reagents Urease was immobilized on the surface of the reticulated commercially available. PU foam with bovine serum albumin and glutaraldehyde. The flexible polyether-based PU foam used as the The performance of the reactor-separator at various urea support of immobilized urease was prepared by the oneconcentrations and at constant current or constant shot method.24All the raw materials: PC3010 (polyethervoltage was studied. Using the device, all ammoniumtype polyol), TDI-80 (toluene diisocyanate, 2,4-TDI/2,6nitrogen in the form of ammonium carbamate and TDI = 80/20), T9 (stannous octoate), DABCO-33 ammonium carbonate were removed from the urea(diazabicyclooctane, 33 % in dipropylglycol), L-580 containing feed solution. This newly developed urea(silicone oil), and methylene chloride, were those used in industry. The bulk density and porosity of the produced removal electrodialyzer is particularly important to both the development of the device for removing urea from PU foam were about 30 kg m-3 and 0.96 (v/v), re-
A study opt the removal of urea
193
TABLE 1 Experimental Conditions for Urea-Removal Electrodialyzer (A) Under constant current
Initial urea
Current
concentration (mmol dm-3)
(MA)
7 10
Weight of immobiIized urease (mg)
15
1.59 2.18 2.20
10 15 20
1.87 1.79 1.84
20 25 30 (B) Under constant voltage
1.97 1.88 1.23
5
10
50
Initial urea concentration (mmol d w 3 )
10
Voltage (volts)
Weight of immobilized urease (mg)
10
2.69 2.17 2.39
15
20
spectively. Prior to the immobilization of urease, PU foam was washed with water and cut into thin sheets of 5 cm in diameter with a thickness of 1 mm using an electrically-heated hot wire. Reagent-grade water produced by Milli-Q SP UltraPure-Water Purification System of Nihon Millipore Ltd, Tokyo, was used throughout this work.
2.2 Immobilization of urease on PU foam The enzyme solution for immobilization was prepared according to the method originally developed by Broun et ~ 2 1 by . ~ ~dissolving 0.6 g bovine serum albumin and 0.02g urease in 10 cm3 of 0.02 mol dm-3 potassium phosphate buffer solution containing 0.25 % (w/v) glutaraldehyde (pH 7.0) at 4°C. Urease was immobilized as follows: PU foam was first immersed in the enzyme solution and squeezed by a glass rod to remove any air trapped in the foam. The PU foam was then removed and brushed gently on the upper rim of the container in order to remove the excess solution. Finally it was air dried at 25°C. After drying (about 10 h), the PU foam was washed with 0.02 mol dm-3 cool potassium phosphate buffer solution (pH 7.0) until the washing solutions were free of glutaraldehyde and uncrosslinked urease and bovine serum albumin. Then the immobilized enzyme was dried and stored at 4°C prior to use. The thickness of the resulting enzyme film on PU foam was measured to be 7-15 pm microscopically. The
percentage crosslinking of enzyme film supported on PU foam was defined as the ratio of the dry weight of enzyme film after and before removal of the uncrosslinked portion. Since the crosslinking percentages were found to be above 90% for almost all the immobilized enzymes, the relative weight percentages of urease and bovine serum albumin in enzyme film were considered to be close to those in the free enzyme solution. Therefore, ignoring the weight of glutaraldehyde, the amount of urease immobilized was estimated to be : (weight of enzyme film) x (weight percentage of urease relative to the sum of urease and bovine serum albumin in the free enzyme solution). 2.3 Apparatus
The five-compartment electrodialyzer used in this work comprised three anion exchange membranes and a cation exchange membrane, forming one dilute compartment, two concentrate compartments and two electrode compartments (Fig. 1). The ion exchange membranes were Selemion AMV and CMV, from the Asahi Glass Co., Ltd, Japan. The electrodialyzer frames were made of teflon. The immobilized enzyme PU foam was placed in the central (dilute) compartment between an anion exchange membrane and a cation exchange membrane to form a fixed bed-like compartment through which urea solution flowed. On both sides of the central compartment two concentrate compartments received the products of urea hydrolysis, CO:-/HCO; and possibly NH, COO- on the anodic side and NH: on the cathodic side. Each concentrate compartment had a PU foam sheet (5 cm in diameter and 1 mm thick) to keep the same flow conditions as in the dilute compartment. The PU foam sheets were held in place with two polyvinyl chloride spacers (5 cm in diameter) against its two sides to keep the PU foam flat and to decrease the aqueous film resistances on the ion exchange membranes and on the PU foams. Both the anodic and the cathodic electrode compartments were separated from concentrate compartments by anion exchange membranes. For each electrode compartment, three rubber gaskets of 2 mm thick were interposed between two frames to allow the platinum wires connected to graphite anode or stainless steel cathode extend out of the compartment. Each of the dilute and the concentrate compartments has a diameter of 5 cm and a width of 5 mm. The effective area of each membrane and of each electrode were 20 cm2. 2.4 Experimental methods The flow scheme for the reaction-separation of urea in five-compartment electrodialyzer is illustrated in Fig. 1. The solutions in dilute and concentrate compartments initially were pure water and 0.005 mol dm-3 NaCI, respectively. The electrode rinse was 0.5 mol dm-3 NaCl. The electrode rinse, dilute solution, and concentrate
T.-C. Huang, D . - H . Chen
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Fig. 2. Variation of ammonium ion concentration with time: -, dilute solution; ----,concentrate solution. (A) Under constant current; initial urea concentration: 5 mmol dm-3; (0): I = 7 mA, immobilized urease 1.59 mg; (0): I = 10 mA, immobilized urease 2.18 mg; I = 15 mA, immobilized urease 2.20 mg. (B) Under constant current; initial urea concentration: 10 mmol dm-3;(0): I = 10 mA, immobilized urease 1.87 mg; (0): I = 15 mA, immobilized urease 1.79 mg; I = 20 mA, immobilized urease 1.84 mg. (C) Under constant current; initial urea concentration: 50 mmol dm-3; (0): I = 20 mA, immobilized urease 1.97 mg; (0): I = 25 mA, immobilized urease 1.88 mg; (A): I = 30 mA, immobilized urease 1.23 mg. (D) Under constant voltage; initial urea concentration: 10 mmol dm-3;(0): V = 10 volts, immobilized urease 2.69 mg; ( 0 ) V : = 15 volts, immobilized urease 2.17 mg; V = 20 volts, immobilized urease 2.39 mg.
(a):
(a):
(a):
solutions, kept in the same constant temperature bath, were pumped into the electrodialyzer from their solution bottles and were circulated by a five-head MasterFlex tubing pump (Cole-Parmer) at a flow rate of 60 cm3 min-’. When the flow of solutions reached steady state, the concentrated urea solution was added to the dilute solution to start the urease-catalyzed hydrolysis of urea. The electric field was applied after 10 min in order to avoid the water splitting because the dilute solution initially was free of salt. The volumes of the ureacontaining dilute solution, concentrate solution, and electrode rinse initially were 100, 200, and 200 cm3, respectively. The temperature was kept at 25°C. At each pre-set time interval, 0.1 cm3 dilute solution and 0.1 cm3 concentrate solution were removed for the analysis of ammonium ion concentrations. The system was studied under various urea concentrations and constant currents or constant voltages. The experimental conditions are listed in Table 1. The power was supplied by an ELBA D6012 dual-digital-power
supply, from which the applied voltage also could be read. The current was measured by an Escort EDM-2116 digital multimeter. The ammonium ion concentration was measured by the phenol-nitroprusside colorimetric method.2s The pH value of the dilute solution was monitored continuously by TOA AUT-211 pH STAT.
2.5
Calculation of current efficiency
Although the present reactor-separator was developed to remove all ammonium-nitrogen in the form of ammonium carbonate and ammonium carbamate, we found that all carbamate ions had been decomposed into ammonium and bicarbonate ions before they were transported into the anion exchange membrane in our other work with the same device.27 In that work, two concentrate solution streams were not mixed, and their ammonium-nitrogen concentrations were analyzed separately. This phenomenon was attributed to the fact that carbamate ions were produced not near enough to the
195
A study on the removal of urea
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(mfn) Fig. 3. Removal percentage of ammonium ions and retention percentage of urea : -, ammonium-removal ; ----, urea-retention. I = 7 mA, immobilized urease 1.59 mg; (0): I = 10 mA, (A) Under constant current; initial urea concentration : 5 mmol dm-3; (0): I = 15 mA, immobilized urease 2.20 mg. (B) Under constant current; initial urea concentration: immobilized urease 2.18 mg; (A): 10 mmol dm-3; (0): I = 10 mA, immobilized urease 1.87 mg; (0): I = 15 mA, immobilized urease 1.79 mg; (A): I = 20 mA, I = 20 mA, immobilized immobilized urease 1.84 mg. (C) Under constant current; initial urea concentration: 50 mmol dm-3; (0): urease 1.97 mg; (0): I = 25 mA, immobilized urease 1.88 mg; (A): I = 30 mA. immobilized urease 1.23 mg. (D) Under constant voltage; initial urea concentration: 10 mmol dm-3; (0): V = 10 volts, immobilized urease 2.69 mg; (0): V = 15 volts, immobilized urease 2.17 mg; (A): V = 20 volts, immobilized urease 2.39 mg.
surface of the anion exchange membrane a n d their halflife was too short. Therefore, the current efficiency (CE) for removing ammonium ions can be defined as C E (YO)= 100 N F / i
(1)
where i is current density (= Z/An,), A, is the effective area of membrane, F is Faraday’s constant, N is the mole flux of ammonium ions a n d can be represented as N
= ( y , / A n , )d[NH;],./dt
(2)
where y, is the total volume of the concentrate solutions, “Hi],, is the ammonium ion concentration in the concentrate solution, and t is the time. In this work, the value of d[NHi],./dt a t each time was obtained by differentiating the polynomial equation best fitting the time course of ammonium ion concentration. Since the current a t each time was measured, the current efficiency could be calculated according to eqns ( I ) and (2).
3 RESULTS AND DISCUSSION 3.1 Reaction-separation of urea by combining immobilized urease and electrodialysis The variations of ammonium ion concentrations in the dilute and the concentrate solutions during reactionelectrodialysis under various urea concentrations a n d constant currents o r constant voltages are shown in Fig. 2 . F o r each case, it was obviously observed that the concentration of ammonium ions in the concentrate solution increased both with time a n d with increasing current density. The concentration of ammonium ions in the dilute solution initially increased with time, and then decreased after reaching a maximum concentration under sufficiently high current density or after sufficiently long operation time. The maximum ammonium ion concentration was lower a n d occurred earlier under higher constant current or voltage. This is due to the
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variation of the difference between the hydrolysis rate of urea by immobilized urease and the removal rate of ammonium ions by electrodialysis. According to Fig. 2, both the removal of ammonium ions from the dilute solution and the retention of urea in the dilute solution were calculated. The results are shown in Fig. 3. It can be seen clearly that in each case the removal of ammonium ions increased both with time and with increasing current density. Also, ammonium ions were removed from the dilute solution almost completely by using a high current density or by operating for a sufficiently long time. Figure 3 also shows that the retention of urea in the dilute solution was not significantly affected by the intensity of the electric field. For the cases with the same initial urea concentration, the slight differences in urea retention at different current density could be attributed to the differences of enzyme activity. This indicated that the rate of urea hydrolysis by immobilized urease was not affected by the application of electric field. Furthermore, Fig. 3 shows that the retention of urea in the
dilute solution increased with increasing the initial urea concentration in spite of the fact that the amount of urea hydrolyzed by urease was increased. This is because the hydrolysis of urea by immobilized urease essentially followed a reaction scheme of the Michaelis-Menten equation.
3.2 Variation of pH of dilute solution with time The variations of pH of dilute solution during reactionelectrodialysis are shown in Fig. 4.For each case, it was found that the pH of dilute solution increased rapidly up to a constant value of about 9 within a few minutes. The constant pH value for the case under larger current density was smaller than that under lower current density. This is due to the buffer action of ammonium carbonate and the fact that the concentration of ammonium ions in the dilute solution was lower under larger current density as shown in Fig. 2. It was also found that the pH of dilute solution decreased gradually down to below 7 with time when
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operating under too large a current density. This is not due to the removal of ammonium ions because the same ammonium ion concentration was enough to maintain the pH at about 9 under lower current density. This phenomenon should be attributed to the fact that the current density exceeded the limiting current density of the system and resulted in water splitting. The products of water splitting, H+ and OH-, participated in the competitive transport and the reactions of the ionic species in the dilute solution, and hence resulted in the decrease of pH.
3.3 Variations of current or voltage with time
The variations of the voltages at constant currents and the currents at constant voltages during reactionelectrodialysis are shown in Fig. 5. Under lower constant current, the voltage applied to the system was decreased slightly during reactionelectrodialysis because more ammonium ions were
produced and the overall electric resistance of the system was hence reduced. Under larger constant current, the voltage applied to the system increased rapidly after operating for a period of time. This is due to the fact that high voltage was needed for the decomposition of water into H' and OH- ions in order to maintain the constant current when ammonium ions in the dilute solution were removed to a quite low concentration level. Under constant voltage of 10 volts, the current increased with time. Under constant voltages of 15 and 20 volts, the current initially increased with time, and then decreased after reaching a maximum value. These phenomena are obviously due to the variations of the ammonium ion concentrations in the dilute and the concentrate solutions, which resulted in the variation of the overall electric resistance of the system.
3.4 Variation of current efficiency with time According to eqns (1) and (2), the variations of current efficiencies with time for removing ammonium ions from
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4 CONCLUSIONS A urea-removal electrodialyzer with immobilized urease was developed. The system was studied both under constant current and under constant voltage. The effects of urea concentration and applied current density on the removal of urea and ammonium ions from the dilute solution were investigated. The variations of the pH of dilute solution, the current or voltage of system, and current efficiency during reaction-electrodialysis were
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the dilute solution were calculated and are shown in Fig. 6. The current efficiencies for various cases were found to vary from 20 to loo%, and mainly within 40-80%. It was also found that the current efficiency decreased with time after reaching a maximum value under large current density. The reduction of current efficiency is due to the H' and OH- ions resulting from water splitting beginning to participate in the current-carrying process.
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also examined. Although the system was not finally formulated because of its complexity, the experimental results showed that it is feasible to remove urea and ammonium ions from aqueous solution simultaneously using the newly developed reactor-separator. ACKNOWLEDGEMENT
This work was performed under the auspices of the National Science Council of the Republic of China, under contract number NSC 80-0402-E006-05, to which the authors wish to express their thanks. REFERENCES Hwang, S. T. & Kammermeyer, K., Membrunrs in Separations. John Wiley & Sons, New York, 1975. 2. Meares, P., Membrune Sepurution Processes. Elsevier, Amsterdam. 1976. 1.
A study on the renzocial of urea 3. Bungay, P. M., Lonsdale, H. K. & de Pinho, M. N., Synthetic Membranes : Science, Engineering and Applications. NATO, AS1 Series, Vol. 181, Reidel, 1986. 4. McGregor, W. C., Membrane Separations in Biotechnology. Marcel Dekker, New York and Basel, 1986. 5. Michaels, A. S. & Matson, S. L., Membranes in biotechnology: state of the art. DesaZination, 53 (1985) 231-58. 6. Barker, S. A. & Burns, R. F., Reactor separators incorporating membrane bound enzymes. Chem. & Ind., (1973) 801-2. 7. Matson, S. L. & Quinn, J. A., Membrane reactors in bioprocessing. Biocltem. Eng., 6 ( I 986) 1 5 2 4 5 . 8. Itoi, S., Ionic membrane electrodialyzer for marine use. Desalination, 2 (1 967) 378-86. 9. Seto, T., Ehara, L., Komori, R., Yamaguchi, A. & Miwa, T., Seawater desalination by electrodialysis. Desalination, 25 (1978) 1-7. 10. Yamane, R., Ichikawa, M., Mizutani, Y. & Onoue, Y., Concentrated brine production from sea water by electrodialysis using ion exchange membranes. Ind. Eng. Chem. Process Des. Dev., 8 (1 969) 159-65. 11. Korngold, E., Kock, K. & Strathmann, H., Electrodialysis in advanced waste water treatment. Desalination, 24 (1978) 129-39. 12. Itoi, S., Nakamura, I. & Kawahara, T., Electrodialytic recovery process of metal finishing waste water. Desalination, 32 (1 980) 393-9. 13. Nott, B. R., Electrodialysis for recovering acid and caustic from ion-exchange regeneration wastes. Ind. Eng. Chem. Prod. Res. Deu., 20 (1981) 170-7. 14. Huang, T. C., Yu, I. Y. & Lin, S. B., Ionic mass transfer rate of CuSO, in electrodialysis. Chem. Eng. Sci., 38 (1983) 1871-6. 15. Huang, T. C. & Juang, R. S., Recovery of sulfuric acid with multi-compartment electrodialysis. Ind. Eng. Chem. Process Des. Dev., 25 (1986) 53742. 16. Zang, J. A., Moshy, R. J. & Smith, R. N., Electrodialysis
199 in food processing. Chem. Eng. Progr. Syrn. Ser., 62 (1966) 105-10.
17. Leitz, F. B. & Eisenmann, J. L., Electrodialysis as a separation process. AIChE Syrn. Ser., 77 (1981) 204-12. 18. Hara, Y., The separation of amino acids with an ionexchange membrane. Bull. Chem. Soc. Jpn, 36 (1963) 1373-6. 19. Tanny, G. B., Kedem, 0. & Bohak, Z . , Coupling of transport and enzymic reaction in a membrane composed of an anion exchanger and immobilized urease. J . Membrane Sci., 4 (1979) 363-77. 20. Ohshima, Y., Shirane, K. & Funakubo, H., Fundamental studies on a urea-removal electrodialyzer for wearable artificial kidney. Ann. Rep. Eng. Res. Inst., Fac. Eng., Univ. Tokyo, 39 ( I 980) 8 1-6. 21. Blakeley, R. L., Hinds, J. A., Webb, E. C. & Zerner, B., Jack Beans urease (EC 3.5.1.5). Determination of a carbamoyl-transfer reaction and inhibition by hydroxamic acids. Biochemistry, 8 (1969) 1991-2000. 22. Jespersen, N. D., A thermochemical study of the hydrolysis of urea by urease. J . Am. Chem. Soc., 97 (1975) 1662-7. 23. Braun, T., Navratil, J. D. & Farag, A. B., Polyurethane Foam Sorbents in Separation Science. CRC, Boca Raton, Florida, 1985. 24. Oertel, G., Polyurethane Handbook. Carl Hanser Verlag, Munich, 1985. 25. Broun, G., Thomas, D., Gellf, G., Domurado, D., Berjonneau, A. M . & Guillon, C., New methods for binding enzyme molecules into a water-insoluble matrix : properties after insolubilization. Biotechnol. Bioeng., 15 (1973) 359-75. 26. Chaney, A. L. & Marbach, E. P., Modified reagents for determination of urea and ammonia. Clin. Chem., 8 (1962) 130-2. 27. Huang, T. C. & Chen, D. H., Coupling of urea hydrolysis and ammonium removal in an electrodialyzer with immobilized urease. Chem. Eng. Commun. (in press).
A Study on the Removal of Urea from Aqueous Solution with Immobilized Urease and Electr odialysis Ting-Chia Huang* & Dong-Hwang Chen Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan 70 101 (Received 5 February 1992; revised version received I 5 April 1992; accepted 26 May 1992)
Abstract : A five-compartment electrodialyzer with immobilized urease was developed for the removal of urea from aqueous solution. The immobilized urease, supported on polyurethane foam, was placed in the central (dilute) compartment, where urea was hydrolyzed and the products NH; and COg-/HCO, were removed simultaneously by electrodialysis. The system was studied both under constant current and under constant voltage. The effects of urea concentration and applied current or voltage on the removal of urea and ammonium ions from the dilute solution were investigated. The variations of the pH of dilute solution, the current or voltage of system, and current efficiency were also examined during reactionelectrodialysis. The removal of urea by enzymic reaction was not affected significantly by the applied electric field. The current efficiencies for removing ammonium ions from dilute solution were mostly within 40-80’41, and the removal percentage of ammonium ions was dependent on current density and current efficiency.
Key words: membrane reactor-separator, immobilized urease, electrodialysis, urea-removal electrodialyzer, urea hydrolysis, polyurethane foam.
1 INTRODUCTION
NOTATION
Membrane separation has been used in various chemical, biochemical, a n d biomedical processes.’-4 Recent interest has centered on the combination of membrane separation with b i o ~ a t a l y s i s . ~T.h~e so-called reactor-separator in which simultaneous catalysis and removal o f products from the resulting reaction mixtures has particular attractions.‘. Ton exchange membrane electrodialysis has been utilized effectively in the d e s a l i n a t i ~ n ~a n. ~d concentrationln of sea water, the removal o r recovery of heavy metal ions a n d toxic compounds in waste water,’’-l5 desalination in the food a n d pharmaceutical industries,3. 1 6 . 1 7 the separation of amino acids and proteins,”. ’* the isolation of trace components from blood plasma,’ and the production of acids and b a s e s 3 The electrodialyzer was formerly used mostly as a separation unit with few reports of its combination with biocatalysts in a reactor-separator. Notable examples were the urea-
Effective area of membrane (cm2) Current efficiency (%) Faraday’s constant (coulomb mol-’) Current density (mA cm-2) Current (mA) Mole flux of ammonium ions (mmolcm-2 min-’) Ammonium ion concentration (mmol dm-3) Ammonium ion concentration in the concentrate solution (mmol dm-’) Time (min) Voltage (volts) Volume of concentrate solution (dms)
’
* To whom correspondence should be addressed. 191 J . Chem. Tech. Biotechnol. 0268-2575/92/$05.00 0 1992 SCI. Printed in Great Britain 13-2
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Fig. 1. Urea-removal electrodialyzer and flow scheme. A : Anion exchange membrane (Selemion AMV); C : cation exchange membrane (Selemion CMV); P: five-head tubing pump; T: teflon frame; 1 : immobilized enzyme PU foam; 2 : P U foam; 3 : PVC spacer; 4:rubber gasket; 5: graphite (anode); 6 : stainless steel (cathode); 7 : platinum wire; 8 : adhesive foam sheet; 9: dilute solution (urea solution); 10: concentrate solution; 11 : electrode rinse; 12: constant-temperature water bath; 13: stir bar; 14: variable area
flowmeter.
removal dialyzer and electrodialyzer containing a comfermentation broths and the development of the dialyzate posite of immobilized urease membrane and anion regeneration system for artificial kidneys. Of course, it is exchange membrane.'g~20 However, it is now recognized also valuable to other similar chemical, biochemical, and that the product of urea hydrolysis by urease is biomedical processes. ammonium carbamate rather than ammonium carbona t e . 2 1 . 2 z Although ammonium carbamate rapidly breaks 2 EXPERIMENTAL down into ammonium ~ a r b o n a t e ,it' ~was found that the urea-removal dialyzer and electrodialyzer reported previously cannot remove ammonium-nitrogen from the 2.1 Materials feed solution of urea completely due to the backflow of carbamate ions across the anion exchange membrane.1g~20 Urease from Jack beans (Type 111) was purchased from In this study, a reactor-separator combining imSigma Chemical Co. (St Louis, Missouri). Urea and mobilized urease and electrodialysis was developed for bovine serum albumin were guaranteed reagents of E. Merck (Darmstadt). Glutaraldehyde (50 YOw/v in water) the removal of urea from aqueous solution. Polyurethane was the product of Fluka Chemie AG, Switzerland. (PU) foam was used as the support of immobilized Except those used for preparing PU foam, all other urease because it has a favorable hydrodynamic property owing to its quasi-spherical membrane structure.23 chemicals were extra pure grade or guaranteed reagents Urease was immobilized on the surface of the reticulated commercially available. PU foam with bovine serum albumin and glutaraldehyde. The flexible polyether-based PU foam used as the The performance of the reactor-separator at various urea support of immobilized urease was prepared by the oneconcentrations and at constant current or constant shot method.24All the raw materials: PC3010 (polyethervoltage was studied. Using the device, all ammoniumtype polyol), TDI-80 (toluene diisocyanate, 2,4-TDI/2,6nitrogen in the form of ammonium carbamate and TDI = 80/20), T9 (stannous octoate), DABCO-33 ammonium carbonate were removed from the urea(diazabicyclooctane, 33 % in dipropylglycol), L-580 containing feed solution. This newly developed urea(silicone oil), and methylene chloride, were those used in industry. The bulk density and porosity of the produced removal electrodialyzer is particularly important to both the development of the device for removing urea from PU foam were about 30 kg m-3 and 0.96 (v/v), re-
A study opt the removal of urea
193
TABLE 1 Experimental Conditions for Urea-Removal Electrodialyzer (A) Under constant current
Initial urea
Current
concentration (mmol dm-3)
(MA)
7 10
Weight of immobiIized urease (mg)
15
1.59 2.18 2.20
10 15 20
1.87 1.79 1.84
20 25 30 (B) Under constant voltage
1.97 1.88 1.23
5
10
50
Initial urea concentration (mmol d w 3 )
10
Voltage (volts)
Weight of immobilized urease (mg)
10
2.69 2.17 2.39
15
20
spectively. Prior to the immobilization of urease, PU foam was washed with water and cut into thin sheets of 5 cm in diameter with a thickness of 1 mm using an electrically-heated hot wire. Reagent-grade water produced by Milli-Q SP UltraPure-Water Purification System of Nihon Millipore Ltd, Tokyo, was used throughout this work.
2.2 Immobilization of urease on PU foam The enzyme solution for immobilization was prepared according to the method originally developed by Broun et ~ 2 1 by . ~ ~dissolving 0.6 g bovine serum albumin and 0.02g urease in 10 cm3 of 0.02 mol dm-3 potassium phosphate buffer solution containing 0.25 % (w/v) glutaraldehyde (pH 7.0) at 4°C. Urease was immobilized as follows: PU foam was first immersed in the enzyme solution and squeezed by a glass rod to remove any air trapped in the foam. The PU foam was then removed and brushed gently on the upper rim of the container in order to remove the excess solution. Finally it was air dried at 25°C. After drying (about 10 h), the PU foam was washed with 0.02 mol dm-3 cool potassium phosphate buffer solution (pH 7.0) until the washing solutions were free of glutaraldehyde and uncrosslinked urease and bovine serum albumin. Then the immobilized enzyme was dried and stored at 4°C prior to use. The thickness of the resulting enzyme film on PU foam was measured to be 7-15 pm microscopically. The
percentage crosslinking of enzyme film supported on PU foam was defined as the ratio of the dry weight of enzyme film after and before removal of the uncrosslinked portion. Since the crosslinking percentages were found to be above 90% for almost all the immobilized enzymes, the relative weight percentages of urease and bovine serum albumin in enzyme film were considered to be close to those in the free enzyme solution. Therefore, ignoring the weight of glutaraldehyde, the amount of urease immobilized was estimated to be : (weight of enzyme film) x (weight percentage of urease relative to the sum of urease and bovine serum albumin in the free enzyme solution). 2.3 Apparatus
The five-compartment electrodialyzer used in this work comprised three anion exchange membranes and a cation exchange membrane, forming one dilute compartment, two concentrate compartments and two electrode compartments (Fig. 1). The ion exchange membranes were Selemion AMV and CMV, from the Asahi Glass Co., Ltd, Japan. The electrodialyzer frames were made of teflon. The immobilized enzyme PU foam was placed in the central (dilute) compartment between an anion exchange membrane and a cation exchange membrane to form a fixed bed-like compartment through which urea solution flowed. On both sides of the central compartment two concentrate compartments received the products of urea hydrolysis, CO:-/HCO; and possibly NH, COO- on the anodic side and NH: on the cathodic side. Each concentrate compartment had a PU foam sheet (5 cm in diameter and 1 mm thick) to keep the same flow conditions as in the dilute compartment. The PU foam sheets were held in place with two polyvinyl chloride spacers (5 cm in diameter) against its two sides to keep the PU foam flat and to decrease the aqueous film resistances on the ion exchange membranes and on the PU foams. Both the anodic and the cathodic electrode compartments were separated from concentrate compartments by anion exchange membranes. For each electrode compartment, three rubber gaskets of 2 mm thick were interposed between two frames to allow the platinum wires connected to graphite anode or stainless steel cathode extend out of the compartment. Each of the dilute and the concentrate compartments has a diameter of 5 cm and a width of 5 mm. The effective area of each membrane and of each electrode were 20 cm2. 2.4 Experimental methods The flow scheme for the reaction-separation of urea in five-compartment electrodialyzer is illustrated in Fig. 1. The solutions in dilute and concentrate compartments initially were pure water and 0.005 mol dm-3 NaCI, respectively. The electrode rinse was 0.5 mol dm-3 NaCl. The electrode rinse, dilute solution, and concentrate
T.-C. Huang, D . - H . Chen
194
t
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(h)
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Fig. 2. Variation of ammonium ion concentration with time: -, dilute solution; ----,concentrate solution. (A) Under constant current; initial urea concentration: 5 mmol dm-3; (0): I = 7 mA, immobilized urease 1.59 mg; (0): I = 10 mA, immobilized urease 2.18 mg; I = 15 mA, immobilized urease 2.20 mg. (B) Under constant current; initial urea concentration: 10 mmol dm-3;(0): I = 10 mA, immobilized urease 1.87 mg; (0): I = 15 mA, immobilized urease 1.79 mg; I = 20 mA, immobilized urease 1.84 mg. (C) Under constant current; initial urea concentration: 50 mmol dm-3; (0): I = 20 mA, immobilized urease 1.97 mg; (0): I = 25 mA, immobilized urease 1.88 mg; (A): I = 30 mA, immobilized urease 1.23 mg. (D) Under constant voltage; initial urea concentration: 10 mmol dm-3;(0): V = 10 volts, immobilized urease 2.69 mg; ( 0 ) V : = 15 volts, immobilized urease 2.17 mg; V = 20 volts, immobilized urease 2.39 mg.
(a):
(a):
(a):
solutions, kept in the same constant temperature bath, were pumped into the electrodialyzer from their solution bottles and were circulated by a five-head MasterFlex tubing pump (Cole-Parmer) at a flow rate of 60 cm3 min-’. When the flow of solutions reached steady state, the concentrated urea solution was added to the dilute solution to start the urease-catalyzed hydrolysis of urea. The electric field was applied after 10 min in order to avoid the water splitting because the dilute solution initially was free of salt. The volumes of the ureacontaining dilute solution, concentrate solution, and electrode rinse initially were 100, 200, and 200 cm3, respectively. The temperature was kept at 25°C. At each pre-set time interval, 0.1 cm3 dilute solution and 0.1 cm3 concentrate solution were removed for the analysis of ammonium ion concentrations. The system was studied under various urea concentrations and constant currents or constant voltages. The experimental conditions are listed in Table 1. The power was supplied by an ELBA D6012 dual-digital-power
supply, from which the applied voltage also could be read. The current was measured by an Escort EDM-2116 digital multimeter. The ammonium ion concentration was measured by the phenol-nitroprusside colorimetric method.2s The pH value of the dilute solution was monitored continuously by TOA AUT-211 pH STAT.
2.5
Calculation of current efficiency
Although the present reactor-separator was developed to remove all ammonium-nitrogen in the form of ammonium carbonate and ammonium carbamate, we found that all carbamate ions had been decomposed into ammonium and bicarbonate ions before they were transported into the anion exchange membrane in our other work with the same device.27 In that work, two concentrate solution streams were not mixed, and their ammonium-nitrogen concentrations were analyzed separately. This phenomenon was attributed to the fact that carbamate ions were produced not near enough to the
195
A study on the removal of urea
t
(-1
t
(mfn) Fig. 3. Removal percentage of ammonium ions and retention percentage of urea : -, ammonium-removal ; ----, urea-retention. I = 7 mA, immobilized urease 1.59 mg; (0): I = 10 mA, (A) Under constant current; initial urea concentration : 5 mmol dm-3; (0): I = 15 mA, immobilized urease 2.20 mg. (B) Under constant current; initial urea concentration: immobilized urease 2.18 mg; (A): 10 mmol dm-3; (0): I = 10 mA, immobilized urease 1.87 mg; (0): I = 15 mA, immobilized urease 1.79 mg; (A): I = 20 mA, I = 20 mA, immobilized immobilized urease 1.84 mg. (C) Under constant current; initial urea concentration: 50 mmol dm-3; (0): urease 1.97 mg; (0): I = 25 mA, immobilized urease 1.88 mg; (A): I = 30 mA. immobilized urease 1.23 mg. (D) Under constant voltage; initial urea concentration: 10 mmol dm-3; (0): V = 10 volts, immobilized urease 2.69 mg; (0): V = 15 volts, immobilized urease 2.17 mg; (A): V = 20 volts, immobilized urease 2.39 mg.
surface of the anion exchange membrane a n d their halflife was too short. Therefore, the current efficiency (CE) for removing ammonium ions can be defined as C E (YO)= 100 N F / i
(1)
where i is current density (= Z/An,), A, is the effective area of membrane, F is Faraday’s constant, N is the mole flux of ammonium ions a n d can be represented as N
= ( y , / A n , )d[NH;],./dt
(2)
where y, is the total volume of the concentrate solutions, “Hi],, is the ammonium ion concentration in the concentrate solution, and t is the time. In this work, the value of d[NHi],./dt a t each time was obtained by differentiating the polynomial equation best fitting the time course of ammonium ion concentration. Since the current a t each time was measured, the current efficiency could be calculated according to eqns ( I ) and (2).
3 RESULTS AND DISCUSSION 3.1 Reaction-separation of urea by combining immobilized urease and electrodialysis The variations of ammonium ion concentrations in the dilute and the concentrate solutions during reactionelectrodialysis under various urea concentrations a n d constant currents o r constant voltages are shown in Fig. 2 . F o r each case, it was obviously observed that the concentration of ammonium ions in the concentrate solution increased both with time a n d with increasing current density. The concentration of ammonium ions in the dilute solution initially increased with time, and then decreased after reaching a maximum concentration under sufficiently high current density or after sufficiently long operation time. The maximum ammonium ion concentration was lower a n d occurred earlier under higher constant current or voltage. This is due to the
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variation of the difference between the hydrolysis rate of urea by immobilized urease and the removal rate of ammonium ions by electrodialysis. According to Fig. 2, both the removal of ammonium ions from the dilute solution and the retention of urea in the dilute solution were calculated. The results are shown in Fig. 3. It can be seen clearly that in each case the removal of ammonium ions increased both with time and with increasing current density. Also, ammonium ions were removed from the dilute solution almost completely by using a high current density or by operating for a sufficiently long time. Figure 3 also shows that the retention of urea in the dilute solution was not significantly affected by the intensity of the electric field. For the cases with the same initial urea concentration, the slight differences in urea retention at different current density could be attributed to the differences of enzyme activity. This indicated that the rate of urea hydrolysis by immobilized urease was not affected by the application of electric field. Furthermore, Fig. 3 shows that the retention of urea in the
dilute solution increased with increasing the initial urea concentration in spite of the fact that the amount of urea hydrolyzed by urease was increased. This is because the hydrolysis of urea by immobilized urease essentially followed a reaction scheme of the Michaelis-Menten equation.
3.2 Variation of pH of dilute solution with time The variations of pH of dilute solution during reactionelectrodialysis are shown in Fig. 4.For each case, it was found that the pH of dilute solution increased rapidly up to a constant value of about 9 within a few minutes. The constant pH value for the case under larger current density was smaller than that under lower current density. This is due to the buffer action of ammonium carbonate and the fact that the concentration of ammonium ions in the dilute solution was lower under larger current density as shown in Fig. 2. It was also found that the pH of dilute solution decreased gradually down to below 7 with time when
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3.3 Variations of current or voltage with time
The variations of the voltages at constant currents and the currents at constant voltages during reactionelectrodialysis are shown in Fig. 5. Under lower constant current, the voltage applied to the system was decreased slightly during reactionelectrodialysis because more ammonium ions were
produced and the overall electric resistance of the system was hence reduced. Under larger constant current, the voltage applied to the system increased rapidly after operating for a period of time. This is due to the fact that high voltage was needed for the decomposition of water into H' and OH- ions in order to maintain the constant current when ammonium ions in the dilute solution were removed to a quite low concentration level. Under constant voltage of 10 volts, the current increased with time. Under constant voltages of 15 and 20 volts, the current initially increased with time, and then decreased after reaching a maximum value. These phenomena are obviously due to the variations of the ammonium ion concentrations in the dilute and the concentrate solutions, which resulted in the variation of the overall electric resistance of the system.
3.4 Variation of current efficiency with time According to eqns (1) and (2), the variations of current efficiencies with time for removing ammonium ions from
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4 CONCLUSIONS A urea-removal electrodialyzer with immobilized urease was developed. The system was studied both under constant current and under constant voltage. The effects of urea concentration and applied current density on the removal of urea and ammonium ions from the dilute solution were investigated. The variations of the pH of dilute solution, the current or voltage of system, and current efficiency during reaction-electrodialysis were
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the dilute solution were calculated and are shown in Fig. 6. The current efficiencies for various cases were found to vary from 20 to loo%, and mainly within 40-80%. It was also found that the current efficiency decreased with time after reaching a maximum value under large current density. The reduction of current efficiency is due to the H' and OH- ions resulting from water splitting beginning to participate in the current-carrying process.
.
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also examined. Although the system was not finally formulated because of its complexity, the experimental results showed that it is feasible to remove urea and ammonium ions from aqueous solution simultaneously using the newly developed reactor-separator. ACKNOWLEDGEMENT
This work was performed under the auspices of the National Science Council of the Republic of China, under contract number NSC 80-0402-E006-05, to which the authors wish to express their thanks. REFERENCES Hwang, S. T. & Kammermeyer, K., Membrunrs in Separations. John Wiley & Sons, New York, 1975. 2. Meares, P., Membrune Sepurution Processes. Elsevier, Amsterdam. 1976. 1.
A study on the renzocial of urea 3. Bungay, P. M., Lonsdale, H. K. & de Pinho, M. N., Synthetic Membranes : Science, Engineering and Applications. NATO, AS1 Series, Vol. 181, Reidel, 1986. 4. McGregor, W. C., Membrane Separations in Biotechnology. Marcel Dekker, New York and Basel, 1986. 5. Michaels, A. S. & Matson, S. L., Membranes in biotechnology: state of the art. DesaZination, 53 (1985) 231-58. 6. Barker, S. A. & Burns, R. F., Reactor separators incorporating membrane bound enzymes. Chem. & Ind., (1973) 801-2. 7. Matson, S. L. & Quinn, J. A., Membrane reactors in bioprocessing. Biocltem. Eng., 6 ( I 986) 1 5 2 4 5 . 8. Itoi, S., Ionic membrane electrodialyzer for marine use. Desalination, 2 (1 967) 378-86. 9. Seto, T., Ehara, L., Komori, R., Yamaguchi, A. & Miwa, T., Seawater desalination by electrodialysis. Desalination, 25 (1978) 1-7. 10. Yamane, R., Ichikawa, M., Mizutani, Y. & Onoue, Y., Concentrated brine production from sea water by electrodialysis using ion exchange membranes. Ind. Eng. Chem. Process Des. Dev., 8 (1 969) 159-65. 11. Korngold, E., Kock, K. & Strathmann, H., Electrodialysis in advanced waste water treatment. Desalination, 24 (1978) 129-39. 12. Itoi, S., Nakamura, I. & Kawahara, T., Electrodialytic recovery process of metal finishing waste water. Desalination, 32 (1 980) 393-9. 13. Nott, B. R., Electrodialysis for recovering acid and caustic from ion-exchange regeneration wastes. Ind. Eng. Chem. Prod. Res. Deu., 20 (1981) 170-7. 14. Huang, T. C., Yu, I. Y. & Lin, S. B., Ionic mass transfer rate of CuSO, in electrodialysis. Chem. Eng. Sci., 38 (1983) 1871-6. 15. Huang, T. C. & Juang, R. S., Recovery of sulfuric acid with multi-compartment electrodialysis. Ind. Eng. Chem. Process Des. Dev., 25 (1986) 53742. 16. Zang, J. A., Moshy, R. J. & Smith, R. N., Electrodialysis
199 in food processing. Chem. Eng. Progr. Syrn. Ser., 62 (1966) 105-10.
17. Leitz, F. B. & Eisenmann, J. L., Electrodialysis as a separation process. AIChE Syrn. Ser., 77 (1981) 204-12. 18. Hara, Y., The separation of amino acids with an ionexchange membrane. Bull. Chem. Soc. Jpn, 36 (1963) 1373-6. 19. Tanny, G. B., Kedem, 0. & Bohak, Z . , Coupling of transport and enzymic reaction in a membrane composed of an anion exchanger and immobilized urease. J . Membrane Sci., 4 (1979) 363-77. 20. Ohshima, Y., Shirane, K. & Funakubo, H., Fundamental studies on a urea-removal electrodialyzer for wearable artificial kidney. Ann. Rep. Eng. Res. Inst., Fac. Eng., Univ. Tokyo, 39 ( I 980) 8 1-6. 21. Blakeley, R. L., Hinds, J. A., Webb, E. C. & Zerner, B., Jack Beans urease (EC 3.5.1.5). Determination of a carbamoyl-transfer reaction and inhibition by hydroxamic acids. Biochemistry, 8 (1969) 1991-2000. 22. Jespersen, N. D., A thermochemical study of the hydrolysis of urea by urease. J . Am. Chem. Soc., 97 (1975) 1662-7. 23. Braun, T., Navratil, J. D. & Farag, A. B., Polyurethane Foam Sorbents in Separation Science. CRC, Boca Raton, Florida, 1985. 24. Oertel, G., Polyurethane Handbook. Carl Hanser Verlag, Munich, 1985. 25. Broun, G., Thomas, D., Gellf, G., Domurado, D., Berjonneau, A. M . & Guillon, C., New methods for binding enzyme molecules into a water-insoluble matrix : properties after insolubilization. Biotechnol. Bioeng., 15 (1973) 359-75. 26. Chaney, A. L. & Marbach, E. P., Modified reagents for determination of urea and ammonia. Clin. Chem., 8 (1962) 130-2. 27. Huang, T. C. & Chen, D. H., Coupling of urea hydrolysis and ammonium removal in an electrodialyzer with immobilized urease. Chem. Eng. Commun. (in press).
