Urea removal from agricultural waste waters by means of urease immobilized on nylon membranes grafted with cyclohexyl-methacrylate

Applied Catalysis B: Environmental 46 (2003) 613–629

Urea removal from agricultural waste waters by means of urease immobilized on nylon membranes grafted with cyclohexyl-methacrylate S. Di Martino a , H. El-Sheriff a,1 , N. Diano a , A. De Maio a,b , V. Grano a , S. Rossi a , U. Bencivenga a , A. Mattei b,2 , D.G. Mita a,b,∗ b

a Institute of Genetics and Biophysics of CNR, Via G. Marconi 12, 80125 Naples, Italy Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli 16, 80138 Naples, Italy

Received 28 January 2003; received in revised form 14 June 2003; accepted 22 July 2003

Abstract In view of the treatment of agricultural waste waters, urea polluted, a catalytic and hydrophobic membrane was constructed by immobilizing urease on a nylon sheet grafted with cyclohexyl methacrylate (CHMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and crosslinking agent, respectively. With reference to the soluble counterpart immobilized urease was found to exhibit (i) a shift of the optimum pH towards more acidic values; (ii) a shift of the optimum temperature towards higher temperatures; (iii) higher values of Km . The latter result indicated an apparent loss of affinity of immobilized urease towards urea. To recovery this affinity loss, the catalytic membranes were employed in a bioreactor operating under non-isothermal conditions. Under these conditions the catalytic membranes exhibited reaction rates higher and apparent Km smaller than those measured under comparable isothermal conditions. As a consequence, percentage increases of enzyme activity and reduction of the production times, proportional to the magnitude of the applied temperature difference were observed. Results have been discussed in the frame of reference of the process of thermodialysis. The technology of the non-isothermal bioreactors confirmed its usefulness also in the reduction of urea concentration in aqueous solutions. © 2003 Elsevier B.V. All rights reserved. Keywords: Urease; Catalytic membranes; Grafting process; Membrane bioreactors; Urea

1. Introduction Enzyme immobilization techniques and the exploitation of enzyme derivatives in industrial biotech∗ Corresponding author. Tel.: +39-081-2395887; fax: +39-081-2395887. E-mail address: [email protected] (D.G. Mita). 1 Permanent address: Department of Polymers and Pigments, National Research Center, Dokki, Cairo, Egypt. 2 Permanent address: Dipartimento di Scienze Chirurgiche ed Anestesiologia, Second University of Naples, Naples, Italy.

nological processes are one of the major growing points of the research activity in academic and industrial laboratories [1–3]. Advantages in the employment of immobilized enzymes are their reusability, the possibility of batch or continuous operational modes, the rapid termination of reactions, the controlled product formation, a greater variety of engineering designs for continuous processes. Methods for immobilizing enzymes are physical or chemical. Physical methods are based on enzyme adsorption on carrier by means of physical forces (electrostatic

0926-3373/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0926-3373(03)00323-0

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interactions, formation of ionic bonds, protein–protein interactions) or on enzyme entrapment within particular spongy structure or gelatinous polymers. Enzyme immobilization by this technique is reversible. Chemical methods include formation of at least one covalent bond between the aminoacid residues of the enzyme and the functionalized water-insoluble carrier. Chemical methods are generally irreversible, since the original enzymes cannot be regenerated or recovered. Both methods, however, in some cases offer the disadvantage of a loss of enzyme activity since equal amounts of soluble and insoluble enzyme exhibit different activity, that of the soluble being higher. When present, the activity loss is mainly due either to changes in enzyme structure or to diffusional restrictions induced by the functionalized carrier to the movement of substrate and reaction products towards or away from the catalytic site. The latter effect is present when the catalyst is immobilized on or into the pores of a hydrophobic membrane. It is well known, indeed, that across a hydrophobic porous membrane the diffusion coefficients of solutes undergo at least a two order decrease. The synergism of these two effects results in an increase of the value of the constant Km of the immobilized enzyme in comparison with the value of the soluble counterpart. The changes in enzyme structure can be minimized by involving in the attachment aminoacidic residues far from the catalytic site. The diffusional limitations can be reduced by immobilizing the enzymes onto soluble–insoluble matrices [4–6], in thermally reversible hydrogels [7–9] or in pressure-sensitive gels [10]. Recently to overcome the diffusional restrictions across a hydrophobic and catalytic porous membrane, such as teflon or nylon membranes, it has been proposed a new technology based on the employment of the membranes in non-isothermal bioreactors [11–19]. Under these conditions an increase of the enzyme activity of the catalytic membrane in respect to that measured under comparable isothermal conditions is observed. Indeed, when the catalytic membrane separates two substrate solutions kept at different temperatures, thermodiffusive substrate fluxes, driven by the process of thermodialysis [20], take place across the membrane. These fluxes add to the diffusive ones, so that in the unit of time the enzymes immobilized onto the membrane pores or on its surfaces encounter

a substrate concentration higher than that of the bulk solution. It follows than the enzyme reaction proceeds at a rate higher than that occurring under comparable isothermal conditions, where only diffusive substrate fluxes are responsible for the reaction. The increases of the reaction rates are proportional to the macroscopic temperature difference across the membrane. Interesting enough it is the circumstance that under non-isothermal conditions the apparent kinetic constants Km for the enzymes immobilized on hydrophobic membranes take on a value intermediate between those measured under isothermal condition for the soluble and insoluble enzyme. The apparent recovery of the catalytic activity for the enzyme derivatives was dependent on the membrane hydrophobicity, on the immobilization method, on the spacer length, and on the magnitude of the applied transmembrane temperature difference. Membrane hydrophobicity is the main prerequisite for the increase of the enzyme reaction rate. In this paper we will discuss the results obtained under isothermal and non-isothermal conditions with a new type of catalytic membrane. The carrier was a nylon membrane grafted with cyclohexyl methacrylate (CHMA). Hexamethylenediamine (HMDA) and glutaraldehyde (GA) were used as spacer and crosslinking agent, respectively. The immobilized enzyme was urease. Urease occupies a unique place in enzymology, in that it was the first enzyme to be crystallized [21]. Besides its ecological employment, since urea has been recognized as a pollutant agent [22] of agricultural sewage, urease finds further applications in blood detoxification and urea removal from beverages and food [23]. Immobilized urease has also been employed as sensing element of biosensors since 1965 [24]. The broad range of application of this enzyme promoted intensive work on the preparation and characterization of urease derivatives [25–33]. The present study was undertaken in view of the employment of these new membranes in the treatment of urea polluted agricultural waste waters by means of bioreactors operating under isothermal or non-isothermal conditions. It will be demostrated the increase of the efficiency of the catalytic process under non-isothermal conditions. Results will be discussed in terms of different substrate fluxes across the catalytic membrane. When possible, comparison will be done with the behaviour of the soluble urease, to

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understand the modifications introduced in the enzyme activity by the immobilization process.

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2. Apparatus, materials and methods

couples, placed at 1.5 mm from each of the membrane surfaces, were used to measure the temperatures inside each half-cell. The temperatures were programmed by means of circulation in the external jackets of the half-cells of water coming from thermostatic baths.

2.1. The bioreactor

2.2. Materials

A three-dimensional picture, not to scale, of the core of the bioreactor is represented in Fig. 1. The bioreactor consisted of two cylindrical half-cells, 2.5 mm in depth and 35 mm in diameter, filled with the substrate solution and separated by the catalytic membrane. Solutions were recirculated in each half-cell at a rate of 3.5 ml min−1 by means of a peristaltic pump through hydraulic circuits starting and ending in a common container C, not reported in figure. In this way the whole solution reacted with the two faces of the catalytic membrane and the catalytic power of both faces was in fact averaged. Thermo-

Type III urease (E.C. 3.5.1.5) from Jack beans was used as catalyst. Urease hydrolizes urea into ammonia and carbon dioxide. As solid support to be grafted we used Nylon Hydrolon membranes from Pall Italia (Pall Italia s.r.l., Milan, Italy). Membrane thickness was 150 ␮m. The diameter of membrane pores was 0.2 ␮m. Pore size is defined as the diameter of the smallest particles that the membrane retains, since in the membrane there are no classical pores, but irregular cavities, crossing the entire membrane thickness, costituted by the interstices between the nylon fibers.

Fig. 1. Three-dimensional picture, not to scale, of the core of the bioreactor. Under non-isothermal conditions the two half-cells are thermostatted by means of two thermostatic baths, kept at different temperatures. The hydraulic circuits, through which the substrate solutions are recirculated, have been omitted.

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As monomer to be grafted CHMA was used, while HMDA was employed as spacer between the grafted membrane and the enzyme. The presence of the spacer was required to minimise the effect of the electric charges of the nylon support on the enzyme structure and on the microenvironment in which the catalytic macromolecule was operating. Glutaraldehyde was used as bifunctional coupling agent. Coupling of urease to GA occurs through the amine groups of the enzyme, thus forming amide. The –SH groups of urease may also crosslink with glutaraldehyde after amine groups have been used. It is important to notice that –NH2 groups are more susceptible than –SH groups to react with glutaraldehyde [34]. We have chosen glutaraldehyde to covalently attach the enzyme to the graft nylon support, since in a previous work [18] we found that the greatest activity increases under non-isothermal conditions occurred when covalent urease immobilization was carried out through condensation by using glutaraldehyde than, for instance, through hydrozinolysis. All chemicals, including the enzyme, were purchased from Sigma (Milan, Italy) and used without further purification.

HMDA aqueous solution for 10 min at 60 ◦ C. The amine excess was removed by means of several washings with double distilled water, then the membranes were activated with a 2% (v/v) glutaraldehyde aqueous solution, pH 6.0, for one hour at room temperature. (c) Enzyme immobilization: Enzyme immobilization was obtained by allowing the preactivated membranes to interact, for 16 h at 4 ◦ C, with a 0.1 M phosphate buffer solution, pH 6.5, containing urease at a concentration of 1 mg/ml. At the end of this treatment, after further washings with 0.1 M phosphate buffer, pH 6.5, the enzyme derivatives were ready to be used. When not in use, the enzyme membranes were stored at 4 ◦ C in the same phosphate buffer solution.

(1)

The processes of grafting, membrane activation, and enzyme immobilization are reported in graphical form in Fig. 2. We choosed to bind the enzyme to the membrane by covalent attachment instead of by physical adsorption, since by using the latter method the enzyme could be easy removed by the flux of water induced by the process of thermodialysis [20] under non-isothermal conditions. Of course we cannot exclude that a certain amount of enzyme is initially attached also by adsorption, but this is, probably, the enzyme amount lost during the first days after the immobilization process (see Section 2.3.3). The experimental conditions to obtain catalytic membranes with the highest enzyme activity were found to be 156 mM CHMA and 15.6 mM K2 S2 O8 in an 80% (v/v) water–ethanol solution for the grafting process. Under these conditions a grafting value of 17 ± 1% was obtained. The immobilization yield was 6 ± 2%, while the activity retention was about 23 ± 4%. Immobilized yield is given by the ratio of the amount (g) of immobilized enzymes to the amount (g) of enzyme used for the immobilization. The activity retention is given by the ratio of the activity of the immobilized enzymes to that of an equal amount of soluble enzyme.

where wb and wa are the membrane mass before and after the grafting process, respectively. (b) Membrane activation: The grafted membranes were partially hydrolized by using a 10% (v/v)

2.3.2. Membrane activity measurements Membrane activity was determined by sampling, at regular time intervals, into the common cylinder C, the urea solution interacting with the catalytic

2.3. Methods 2.3.1. Catalytic membranes preparation The preparation of the catalytic membranes was carried out by three steps: (a) membrane grafting; (b) membrane activation; (c) enzyme immobilization. (a) Membrane grafting: The nylon membranes were immersed in a CHMA alcoholic aqueous solution containing potassium persulphate as initiator. The reaction is allowed to go on for 40 min at 50 ◦ C, after then the membranes were washed with acetone to remove the formed homopolymers. Later on, the membranes were put into a stove and weighted until a constant weight was reached. The grafting percentage was measured as: G (%) =

wa − wb × 100 wa

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Fig. 2. Schematic representation of grafting, membrane activation and enzyme immobilization. RT is used for room temperature, and NH2 -urease indicates an amine group in the amminoacid sequence of urease.

membrane, and by measuring the ammonia concentration resulting from the reaction. Ammonia concentration was spectrophotometrically determined at 625 nm, by using a calibration curve by means of the phenol–hypochlorite method [35] through which a blue colored solution is developed. By this method two different solutions were prepared according to the following procedure. Solution A was prepared by dissolving 10 g of phenol and 0.05 g of sodium nitroprusside in 1 l of water. Solution B was prepared by dissolving 5 g of sodium hydroxide and 0.42 ml of sodium hypochlorite in 1 l of water. To measure the ammonia concentration 100 ␮l of sample extracted by the solution reacted with the catalytic membrane were added to 2.5 ml of reagent A and 2.5 ml of reagent B. The mixture was kept for 20 min at 50 ◦ C under agi-

tation. A colored solution was obtained. The intensity of the solution color is proportional to the ammonia concentration. Membrane activity, indicated also as enzyme reaction rate, is expressed in ␮mol min−1 , and is given by the slope of the linear plot of the ammonia production (␮mol) as a function of time (min). 2.3.3. Time stability of the catalytic membranes Urease contains several –SH groups per molecule. The presence of these groups affects the structure of the enzyme and, consequently, its activity. Sulfhydryl groups at the active site may be in ionized form, which is prone to oxidation. Normally, inside the cell the reducing medium and the presence of other sulfhydryl-containing molecules protect these groups

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on urease. To stabilize urease [36] lactose and dithiothreithol (5 mM) were used in the reaction mixtures and in the buffer solutions. Time stability of the catalytic membranes was assessed by measuring their activity every day, under the same experimental conditions, i.e. 15 mM urea in 0.1 M citrate buffer, at pH 5.0 and 25 ◦ C. After few days, during which the membranes lost part of their activity, a stable condition was reached for more than 1 month. Only these stabilized membranes were used for our study. 2.3.4. Experimental data treatment The points reported in the figures are each the average of five independent experiments performed under the same conditions. The experimental errors never exceeded 4.5%.

3. Results and discussion Preliminarily to the biochemical characterization of our enzyme membrane, it is important to report the results which allowed to establish the experimental conditions giving the most active derivatives. In Fig. 3a the measured grafting percentages have been reported as a function of the composition of the water–ethanol solution used for the grafting, keeping constant at 156 mM the CHMA concentration. Results in Fig. 3a clearly indicate that the CHMA grafting on the membrane increases by increasing the water percentage. This means that the water concentration is a fundamental parameter controlling the grafting. In Fig. 3b the relative activities of the catalytic membranes are reported as a function of the CHMA grafting percentage. The experimental conditions were: 15 mM urea in 0.1 M citrate buffer, pH 5.0 and T = 25 ◦ C. Results in Fig. 3b show an optimum grafting percentage where the maximum enzyme activity occurs. At values of grafting percentage higher than the value of optimum, the relative enzyme activity decreases, since the increase of the density of immobilized enzyme induces protein–protein interactions, which reduce the number of active molecules. This is a well known effect and we have already found this result with a different enzyme, a different carrier and a different immobilization method [37].

Considering these results all the experiments reported in the following, have been carried out with membranes exhibiting a 17% of CHMA grafting, obtained with a 80% (v/v) water–ethanol solution. 3.1. Isothermal characterization of the catalytic membrane 3.1.1. pH dependence It is well known that the pH–activity profile of an immobilized enzyme is characteristic of the nature of the enzyme, of the carrier and of the immobilization method. The pH–activity profile of an immobilized enzyme, indeed, can be affected by the synergetic action of the partitioning effect [38,39] and by the increased stability of the catalyst, particularly when multipoint attachment is obtained. Partitioning effect affects the optimum pH value, while the modified stability affects the shape of the pH–activity profile. Through the partitioning effect the support (and the graft monomer) can change the pH value into the microenvironment in which the enzyme is operating owing to the electrostatic or hydrophobic interactions between the matrix and the low molecular species present in solution. In this way charged species, such as hydrogen and hydroxyl ions, exhibit different concentrations between the microenvironment around the catalytic site and the bulk solution. The position of the optimum activity, consequently, can be displaced to higher or lower pH values or not at all. With reference to the stability of the catalyst, the shape of the pH–activity curve of the immobilized enzyme can be broader, narrower, more asymmetrical than or identical to that of the soluble enzyme. To know how in our case the pH–activity profile is affected by the immobilization procedure, we have studied the activity of the free and immobilized urease in the pH range between 4.0 and 7.0. The results of this investigation are illustrated in Fig. 4a, where the relative activities of soluble and insoluble urease are reported as a function of pH. The experimental conditions were 15 mM urea and T = 25 ◦ C. From Fig. 4a it is easy to see that the position of the optimum pH of the insoluble enzyme is shifted towards more acidic values, from pH 6.0 for the free enzyme to pH 5.7 for the insoluble enzyme. This shift is a clear indication of the presence of a negative charge density on the activated nylon

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Fig. 3. (a) CHMA grafting percentage as a function of water content (v/v, %) in the water–ethanol mixture. (b) Relative activity of the catalytic membrane as a function of CHMA grafting percentage.

membrane. Similar results have been obtained by us with the same nylon membranes loaded with urease through different spacers [18] or with ␤-galactosidase [40–43]. Fig. 4a also shows different “optimum pH range” for the free and soluble enzyme. The “optimum pH range” is the range in which the relative activity is higher than 95%. The optimum pH range is between 5.29 and 6.12 for the immobilized urease and between

5.86 and 6.22 for the soluble enzyme. The existence of an optimum pH range more than twice large for the immobilized urease in respect to that of the soluble counterpart is indicative of the increased stability of the catalyst as a consequence of the immobilization process. From Fig. 4 it is also evident that immobilized urease is more resistant than the free enzyme to acidic solutions. At pH 4.5, indeed, the enzyme derivative retains about 45% of its maximum activity, while at

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Also in the temperature–activity profile it is possible to observe a different extent of the “optimum temperature range”, this range being between 74.2 and 85.7 ◦ C in the case of the insoluble urease and between 66.3 and 79.5 ◦ C for the soluble form. From Fig. 4b it is also evident that at temperatures lower than that of the optimum small differences occur between the relative activities of the immobilized and the free form of urease, while high differences appear at temperature higher than that of the optimum. On the basis of this observation, one can conclude that our membranes can be usefully employed in biotechnological processes occurring at high temperatures. So, for example, at 90 ◦ C the immobilized urease still retains almost 80% of its maximum activity, while the free urease at the same temperature retains only 15% of its maximum activity. When the results of Fig. 4b are reported in forms of Arrhenius plots, relatively to the temperature range from 25 ◦ C to the optimum temperature, one obtains the values of the activation energies of the enzymatic process. These values are equal to 5.1 and 6.7 kcal mol−1 for the free and immobilized urease, respectively. This result suggests that, at the urea concentration of 15 mM, the enzyme reaction is diffusion-controlled. Fig. 4. Relative activity of free (䊐) and immobilized (䊊) urease as a function of (a) pH and (b) temperature.

the same pH value the activity retention of the free urease is about 5%. 3.1.2. Temperature dependence In Fig. 4b the relative activities of the soluble and insoluble urease have been reported as a function of temperature. The experimental conditions were 15 mM urea in 0.1 M buffer citrate, pH 5.0. Free urease shows an optimum temperature at about 75 ◦ C, while the optimum of the immobilized urease was shifted to about 81 ◦ C. This means that the immobilization procedures preserve the active structure of the macromolecule at temperatures higher than those denaturating the soluble form. The same behavior has been found by us with the same nylon membranes loaded with urease through different spacers [18] or with ␤-galactosidase [40–43].

3.1.3. Substrate concentration dependence Having ascertained the influence of the immobilization process on the pH and temperature activity profiles for our immobilized urease, it is reasonable to expect also differences between the values of the kinetic constants for the soluble and insoluble urease. To this aim we have studied for both enzyme forms the activity dependence on substrate concentration in a 0.1 M buffer citrate, pH 5.0. In Fig. 5 the enzyme activities are reported as a function of substrate concentration for the soluble (Fig. 5a) and insoluble (Fig. 5b) urease. In both figures the curve parameter is the temperature of the reaction mixture. Results in Fig. 5 show that both soluble and insoluble forms of urease display a Michaelis–Menten behavior and that at each substrate concentration the enzyme activity increases with the temperature. When the results of Fig. 5 are reported in forms of Hanes plots one obtains the values of the Michaelis–Menten constant for the free (Km ) and immobilized (Km,app ) urease as well as the values of the maximum reaction rates Vmax and Vmax,app .

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Fig. 5. Soluble (a) and insoluble (b) urease activity as function of urea concentration. Symbols: (䊊) = 25 ◦ C; (䊐) = 35 ◦ C; (䉫) = 45 ◦ C.

The values of these constants are listed in Table 1. Results in table show that: (i) the Km values of the free urease are smaller than those of the insoluble form; (ii) the values of the Michaelis–Menten constant for both forms of urease decrease with the temperature increase. The increase of the Km,app values clearly indicates a lower affinity towards the urea for the immobilized urease than that of the soluble urease. This result can be attributed to the changes induced in the enzyme structure by the interaction of the catalyst with the support and to the increased diffusional resistance encountered by the substrate in its approach to the cat-

alytic site and by the reaction products in their removal from the same site. Both these effects add to those induced by the partitioning effect. The decrease of the Michaelis–Menten constants with the temperature increase is the consequence of the corresponding increases of the diffusional fluxes of substrate and reaction products with temperature. 3.2. Non-isothermal characterization of the catalytic membrane From the above, it clearly emerges that the immobilization procedures not only affect the pH and

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Jvolume = A

P x

(2)

therm Jvolume =B

T x

(3)

hydr

Table 1 Kinetics parameters of soluble and insoluble urease Enzyme system

Thermal conditions (◦ C)

Km (mM)

Vmax (␮mol min−1 )

Soluble urease

Tav = 25, T = 0 Tav = 35, T = 0 Tav = 45, T = 0

18.1 11.7 5.0

11.5 14.9 16.7

Insoluble urease

Tav = 25, T = 0 Tav = 35, T = 0 Tav = 45, T = 0

25.8 18.9 13.9

8.7 9.9 10.8

Insoluble urease

Tav = 25, T = 35

16.7

9.5

the temperature–activity profiles, but also the affinity of urease for urea. In the last case the immobilized urease shows an apparent loss of affinity towards urea. In previous works [11–19] it has been shown that is possible to recovery the affinity loss of enzyme derivates by employing the catalytic membranes in non-isothermal bioreactors, provided the membranes were hydrophobic. It was interesting at this point to verify if also the present enzyme membrane was able to recovery under non-isothermal conditions the affinity loss, since it was to be expected that grafting procedures affected some physical properties of the treated membranes. In particular, grafting procedures could affect two parameters controlling the volume (water) transport through the membranes: the hydraulic permeability coefficient, A, and the thermoosmotic coefficient, B. The former couples the isothermal volume flux across a membrane to its conjugate thermodynamic force, i.e. the pressure gradient (P/x = 0 and T/x = 0); while the latter couples the non-isothermal volume flux to its conjugate thermodynamic force, i.e. the temperature gradient (T/x = 0 and P/x = 0). The A (N−1 m4 s−1 ) and B (m2 s−1 K−1 ) coefficients are given by the equations:

where J = V/St (in ms−1 ) is calculated from the water volume V (m3 ) transported across the membrane of surface area S (m2 ), in the time interval t (s). P/x (N m−3 ) and T/x (K m−1 ) are the pressure and the temperature gradient, respectively. x (m) is the membrane thickness. Eqs. (2) and (3) are particular cases of a more general set of phenomenological equations of the thermodynamic of irreversible processes [44,45]. These equations, in the case of a system constituted by a membrane separating two volumes of pure water, are JH2 O = L11 X1 + L12 X2

(4)

JH2 O = L21 X1 + L22 X2

(5)

where JH2 O are the mass fluxes, Lij are the phenomenological coefficients, and Xi are the generalized forces. In our case, X1 = P/x, X2 = T/x, and L12 = L21 , according to Onsager [46]. When X2 = 0, Eq. (4) reduces to Eq. (2), and L11 is equal to A. When X1 = 0, Eq. (5) reduces to Eq. (3) and L22 became equal to B. To know the effect of the grafting on membrane hydrophobicity, we have studied the transmembrane volume transport across the untreated and the catalytic membrane, in the presence of pressure or temperature gradients. The hydraulic fluxes were obtained at T = 25 ◦ C with a constant pressure of P = 35 mbar, while the experimental conditions for the thermoosmotic fluxes were T = 20 ◦ C with a Tav = 25 ◦ C. The results are reported in Table 2. From these data it is possible to observe how the catalytic membrane has a larger A and a smaller B in comparison to the corresponding values of the untreated membrane. This implies that the chemical treatment, such as grafting

Table 2 Physical characterization of the untreated and catalytic membrane Nylon membrane

Jhyd × 107 (m s−1 )

Jtherm × 106 (m s−1 )

A × 1014 (m4 s−1 N−1 )

B × 1013 (m2 s−1 K−1 )

C = B/A (N m−2 K−1 )

Untreated Catalytic

0.16 6.60

4.20 1.23

0.04 1.65

24.0 7.0

6000 42

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and activation, as well as enzyme immobilization, reduces the hydrophobicity of the membrane in comparison to its original state. The membrane, however, remains hydrophobic in as much as it still gives rise to thermodialysis. Results in the Table 2 also show that the chemical treatment has a greater effect on the hydraulic than on the thermoosmotic permeability, indicating that these two coefficients are affected by a different extent. Recently we have related the hydrophobicity of a membrane to a parameter C (N m−2 K−1 ) given by C=

B A

(6)

When C > 0, a thermoosmotic water flow increasing with the C value, is observed. For C = 0, no thermoosmotic flow occurs. It has been also shown that the extent of the non-isothermal mass transport across the membrane depends on membrane hydrophobicity. Therefore, the coefficient C can be seen as a parameter able to foresee the behavior of a catalytic membrane operating in non-isothermal bioreactors. High C values imply greater mass (substrate/products) transport, and thus a higher catalytic activity under non-isothermal conditions. The C values for our catalytic or untreated membrane are reported in Table 2. The values in table show a C value for the catalytic membrane much lower than that for the untreated membrane. In any

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case, being the C value of the catalytic membrane different from zero, one can expect an increase of enzyme activity in the presence of temperature gradients. In Fig. 6 the ammonia productions under isothermal (T = 0) and non-isothermal (T = 0) conditions are reported as a function of time. The curve parameter is the T value. The experimental conditions were 15 mM urea in 0.1 M buffer citrate, pH 5.0 and Tav = 25 ◦ C. Tav is (Tw + Tc )/2, where Tw and Tc are the temperatures read at the position of the thermocouples in the warm (W) and cold (C) half-cell, respectively. T is equal to Tw − Tc . Under isothermal conditions Tav results equal to Tisoth . Results in Fig. 6 indicate that at each time the ammonia production increases with the increase of the applied T. This is an interesting result when the exploitation of non-isothermal bioreactors in biotechnological processes of industrial interest is considered. From Fig. 6, indeed, it is possible to calculate the reduction of the production times, τ r , defined as: τiso − τnon-iso τr (%) = (7) τiso where τ iso and τ non-iso are the times to obtain the same amount of a reaction product under isothermal (τ iso ) and non-isothermal (τ non-iso ) conditions. When Eq. (7) is applied to the results of Fig. 6 one obtains the results in Fig. 7a, where it is possible to

Fig. 6. Ammonia production as a function of time. (䊊) Tav = 25 and 0 ◦ C; (䊐) Tav = 25 and 10 ◦ C; (䉫) Tav = 25 and 20 ◦ C; (䉭) Tav = 25 and 30 ◦ C.

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Fig. 7. (a) Reduction of the production times (τ) as a function of temperature difference read at the thermocouple positions. (b) PAI as a function of temperature difference read at the thermocouple positions.

see that the reductions of the production times of our enzyme reaction increase with the applied T. In view of the application of the technology of the non-isothermal bioreactors in industrial processes it is interesting to calculate another parameter, which has been called by us percentage activity increase (PAI). The PAI represents the percentage activity increase under non-isothermal conditions and it is defined by means of the expression: PAI|T =

C R|C T =0 − R|T =0

R|C T =0

(8)

C where R|C T =0 and R|T =0 are the values of the reaction rate, at a substrate concentration C, under isothermal and non-isothermal conditions, respectively. As a matter of the fact, the PAI |T value indicates the percentage activity increase when a temperature difference T is read in the bioreactor at the thermocouple positions. In Fig. 7b the PAI values relative to the results of Fig. 6 are reported as a function of T. Inspection of Fig. 7b shows that the PAI is a linear function of the applied T. The linearity of results in Fig. 7b allows us to define another interesting parameter concerning the exploitation of non-isothermal

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bioreactors in industrial processes. This new parameter, α (%, ◦ C−1 ), is related to the PAI value through the expression: α=

PAI T

(9)

where α is a measure of the percentage activity increase when a temperature difference T = 1 ◦ C is read at the thermocouple positions. In the case of Fig. 7b the α value is 1.8. Having ascertained that the non-isothermal conditions increase the reaction rate of the immobilized urease, it is interesting to investigate if also the kinetic constants are affected by the presence of a temperature difference across the catalytic membrane. To this aim membrane activity has been studied under non-isothermal conditions (T = 30 ◦ C and Tav = 25 ◦ C) as a function of urea concentration. The results of this investigation are reported in Fig. 8, where, to allow a direct comparison, also the results obtained under isothermal conditions (T = 0 ◦ C and Tisoth = Tav = 25 ◦ C) have been added. Results in Fig. 8 show that under non-isothermal conditions the enzyme activity still exhibits a Michaelis–Menten behaviour and that at each urea concentration the non-isothermal reaction rate is higher than that measured under comparable isothermal conditions. When the results in Fig. 8 are displayed in form of Hanes plots one obtains the

625

values of the apparent Km reported in Table 2. The Km values in Table 2 show that under non-isothermal conditions the immobilized urease recovers part of the affinity towards the substrate lost under isothermal conditions. In Fig. 9 the PAI values relative to the experimental results of in Fig. 8 are reported as a function of the urea concentration. Calculation has been done taking at each substrate concentration the values of reaction rate on the curve interpolating the experimental points of Fig. 8. Results in Fig. 9 show that the PAI values decrease with the increase of substrate concentration. Similar results have been obtained by us with different enzyme derivatives [11–19]. The PAI values in Fig. 9 confirm the usefulness of employing catalytic and hydrophobic membranes in non-isothermal bioreactors. The significance of the results in Fig. 9 becomes more impressive by considering the actual value of the temperature difference across the catalytic membrane. We have demonstrated that under our experimental conditions the motion of the substrate solutions in the two half-cells is laminar [11–19]. It follows that, by applying the Fourier’s law, the temperature difference T read at the thermocouple positions corresponds to an actual temperature difference T∗ across the catalytic membrane given by T ∗ = γ T

Fig. 8. Membrane activity as a function of substrate concentration under isothermal (䊐) and non-isothermal (䊊).

(10)

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Fig. 9. Percentage activity increase (PAI) (left scale) and α∗ (right scale) as a function of urea concentration.

where γ is a constant depending on the thermal conductivities and thicknesses of the liquid solutions in the half-cells and of the membrane. γ in the case of our membranes has been found to be about 0.1. In these calculations we assumed the thermal conductivity of our solutions equal to that of the pure water [47], while the thermal conductivity of the membrane was taken from Touloukian [48]. This means that, by assuming γ = 0.1, the PAI values on Fig. 9 can be attributed to an actual temperature difference T∗ equal to 3 ◦ C across the membrane. It follows that it is possible to define now a new parameter α∗ (%, ◦ C) by means of the expression: α∗ =

PAI T ∗

(11)

where α∗ is the percentage activity increase when an actual temperature difference of 1 ◦ C is applied across the catalytic and hydrophobic membrane. In Fig. 9 the α∗ values relative to the results of the Fig. 8 have been reported. Inspection of the α∗ values confirms the usefulness of the technology of non-isothermal bioreactors. To understand the molecular mechanism underlying the increase of the activity of a catalytic membrane in the presence of a temperature difference, it is opportune to known the substrate concentration profiles within the catalytic membrane under isothermal and

non-isothermal conditions. To do this a mass balance for substrate into an elementary volume, x thick, of the catalytic membrane must to be carried out, according to the general equation Rate of input − rate of output −rate of consumption by enzyme reaction = rate of accumulation

(12)

Each member of Eq. (12) is measured in mol s−1 , and the rate of input and output are the substrate fluxes (J, mol cm−2 s−1 ) multiplied for the surface area (A, cm2 ) of the membrane through which the mass flow occurs. To identify the substrate fluxes crossing the membrane reference must be done to Fig. 10, representing a catalytic membrane separating two substrate solutions at concentrations C0 , kept at temperatures T2 and T1 , respectively. The elementary volume between x and x+∆x is also represented. Fig. 10 a refers to the isothermal case, Fig. 10b to the non-isothermal one. Under isothermal conditions only diffusive flux of substrate take places across the membrane. Under non-isothermal conditions a substrate flux dragged by the flow of water and a specific thermodiffusive substrate flux add to the diffusive one (Fig. 10b). Considering Fig. 10a it follows that under isothermal conditions the general Eq. (12), when expressed

S. Di Martino et al. / Applied Catalysis B: Environmental 46 (2003) 613–629

627

Fig. 11. Substrate concentration profiles into the catalytic membrane under isothermal (—) and non-isothermal (- - -) conditions.

rameters Vm and Km measured in mol cm−3 s−1 and mol cm−3 , respectively. C(x, t)/t indicates the rate of substrate accumulation (mol cm−3 s−1 ). Upon division by A x, for x → 0 and t → 0, Eq. (13) becomes the second order differential equation for the variable C(x, t): D∗

Fig. 10. Schematic representation of substrate fluxes into the membrane and, hence, into the elementary volume of thickness x, under isothermal (a) and non-isothermal (b) conditions.

C(x, t) = A x t

(14)

By solving Eq. (14) under the condition that ∂C(x, t)/∂t ≤ 5 × 10−6 , one obtains a substrate concentration profile into the catalytic membrane under isothermal conditions similar to that reported in form of full line in Fig. 11. Similarly, considering Fig. 10b, it follows that, under non-isothermal conditions the mass balance Eq. (12) can be written as: Jdiff |x A − Jdiff |x+x A + JS,drag |x A − JS,drag |x+x A V ∗ C(x, t) A x + JS,td |x+x A − JS,td |x A − ∗ m Km + C(x, t)

in analytic form, becomes Jdiff |x A − Jdiff |x+x A −

∂2 C(x, t) ∂C(x, t) Vm C(x, t) = − Km + C(x, t) ∂t ∂x2

Vm C(x, t) A x Km + C(x, t)

= (13)

where Jdiff are the substrate diffusive fluxes given by −D∗ C/x; and Vm C(x, t)/Km + C(x, t) mol cm−3 s−1 represents the substrate consumption by the enzyme reaction characterized by the kinetic pa-

C(x, t) A x t

(15)

∗ now are the values of the kinetic where Vm∗ and Km parameters under non-isothermal conditions, JS,th = D0∗ T ∗ /x is the thermodiffusive flux from cold to the warm side, and JS,drag = VH2 O C is the a drag substrate flux in the opposite direction.

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Upon division by A x and substitution of Jdiff , Jdrag , JS,th with the respective expressions, for x → 0 and t → 0, one obtains the following second order differential equation:
2 ∗  ∂C(x, t) ∗ ∂ C(x, t) ∗ T D − vH2 O − Dtd ∂x x ∂x2 Vm∗ C(x, t) ∂C(x, t) − ∗ = (16) Km + C(x, t) ∂t ∗ T ∗ /x) = K, Eq. (16) becomes Putting (vH2 O −Dtd

D∗

∂2 C(x, t) ∂C(x, t) −K 2 ∂x ∂x Vm∗ C(x, t) ∂C(x, t) − ∗ = Km + C(x, t) ∂t

(17)

When K = 0, i.e. in the absence of thermodialysis, Eq. (17) reduces to Eq. (14), i.e. to the equation under isothermal conditions. By solving Eq. (17) one obtains a substrate profile concentration into the catalytic membrane under non-isothermal conditions similar to that reported in form of dotted line in Fig. 11. Since the areas delimited by the substrate concentration profiles and the horizontal axis (giving the initial substrate concentration) represent the substrate consumption due to the enzyme activity it is evident that under non-isothermal conditions the substrate consumption (and hence the ammonia production) is higher than that under isothermal conditions. The curves of Fig. 11 are similar to those published in reference [49], obtained by computer simulation and by using numerical values from separate experiments of diffusion and thermodialysis of lactose and from experiments of lactose catalysis by means of immobilized ␤-galactosidase. In reference [49] also the theoretical dependence of the percentage increase of the reaction rate on substrate concentration has been calculated. Calculation confirmed the experimental data.

4. Conclusions From all the above results it can be concluded that the activity of urease immobilized by means of HDMA and GA on a nylon membrane, grafted with CHMA, shows, under isothermal conditions, pH and temperature-profiles different from those of the solu-

ble form. In particular the insoluble urease exhibits an optimum pH value displaced towards more acidic values, while the optimum temperature is shifted towards higher temperatures. This means that our membranes can be usefully employed in processes carried out at temperatures and pH values inactivating the soluble urease. When the catalytic membrane is employed in a bioreactor operating under non-isothermal conditions, urease exhibits activity increases proportional to the applied temperature difference and decreasing with the increase of urea concentration. The percentage activity increases, comparable to those obtained by us with other enzyme derivatives, confirm the usefulness of employing the nylon–CHMA–HMDA–GA–urease membranes in non-isothermal bioreactors. Significant reduction of the production times are also obtained under non-isothermal conditions.

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